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ANLISISDEALIMENTOSCONEL
USODENANOPARTCULAS
NEWMETHODOLOGIESINFOOD
ANALYSISUSINGNANOPARTICLES
TesisDoctoral
JuanGodoyNavajas
Marzo2014
3. PartedeladefensadelaMemoriaserealizarenlalengua
oficialdeotropaseuropeo.
deTesisDoctoral,TinayMariPaz.
GraciasTinapordarmelaoportunidaddetrabajarcontigodurante
estosaosyporverenmialgoquenadiehabavistoanteriormente.
MariPaz,graciasporvolcartodostusconocimientosconmigo,por
sacarlomejordemyporensearmelofascinantequepuedellegaraserel
mundodelainvestigacin.
Aambasosestareternamenteagradecido.
cuandolohenecesitado.
Graciasamiscompaerasdelaboratorio.Todashabisconseguido
Analtica (Paco, Mara, Isa Montesinos, Mara Jos, Jose, Noelia Luque,
Noelia Caballero, Antoito, Carmen, Ana Ballesteros, Laura Soriano,
Guille, Lola, Nani, Isa Mrquez) pero especialmente a mi grandsimo
amigo JuanMa Jimnez. A veces la vida pone en mitad de tu camino
pruebas muy difciles de superar. Pero en otras ocasiones, coloca a gente
maravillosaqueteayudaasuperarestaspruebas.Muchasgraciasportodo
ynuncaosolvidar.
Nopuedoolvidarmedevosotros,demiscompaerosdepisoyde
carrera.Estecaminolocomenzamosjuntoshaceyamsdeunadcadaylo
Graciasamifamilia,especialmenteamishermanos:Puri,Carmen
yJose.Fuielpequeodeunafamilianumerosapero,sobretodo,mesent
siempre el ms querido. Hemos vivido muchos momentos buenos y
algunosmuymalos,perosiemprehemosestadotodosunidos.Muchsimas
graciasportodovuestroapoyoincondicional.
Hedejadoparaelfinalalaspersonasmsimportantesenmivida.
Mispadres.Vosotroshabissidolospilaresdemivida,demieducaciny
formacin.Graciasavosotros,hoysoyloquesoy.Vosotroshabisluchado
durante toda vuestra vida para que pudisemos llegar a lo ms. No hay
palabras para agradecer todo lo que habis hecho por m y por mis
hermanos.EstaTesisDoctoralesvuestra.
IwouldliketothankProf.TeroSoukkaforofferingmethe
opportunity to work with him in the Department of
BiotechnologyoftheUniversityofTurku(Finland).
INDICE
NDICE
Pgina
Objeto/Aim
Introduccin
Captulo1
HerramientasAnalticas
Materialesyreactivos
Instrumentacin
Programasinformticos
77
80
85
86
Captulo2
87
95
123
145
Captulo3
Longwavelength
fluorimetric 197
determination of food antioxidant
capacitybyusingnileblueasreagent
Automatic
determination
of
polyphenols in wines using laccase 219
andterbiumoxidenanoparticles
Captulo4
Captulo5
Discusinderesultados
Introduccin
1 Nanopartculas de slice en anlisis
dealimentos
2 Nuevas
estrategias
para
la
determinacin de antioxidantes en
alimentos
3 Nuevas investigaciones en el uso de
upconverting
phosphors
en
sistemas de transferencia de energa
resonanteluminiscente
Discussionoftheresults
Introduction
1 Silicananoparticlesinfoodanalysis
2 Newstrategiesforthedetermination
ofantioxidants
3 New investigations in the use of
upconverting
phosphors
in
luminescence resonance energy
transfer
Conclusiones /Conclusions
Anexo/Annex
283
285
285
306
324
343
343
343
362
378
397
407
OBJETO
AIM
Objeto
Desarrollo
de
nuevas
metodologas
analticas
para
la
Estudiosistemticodelautilidaddelfenmenodetransferenciade
energa de resonancia luminiscente (LRET) entre nanocristales de
iones lantnidos, que presentan luminiscencia antiStokes
(upconverting phosphors), y fluorforos orgnicos para la
determinacin de biotina mediante ensayos de afinidad en medio
homogneo.
Aim
ThegeneralaimoftheinvestigationsincludedinthisDissertation
hasbeenthedevelopmentoffastmethodsforfoodanalysismainlyusing
theespecialpropertiesofnanomaterials.Thestudiesperformedhavetried
to expand the application field of Nanotechnology and open new
possibilitiesbydevelopinganalyticalmethodsalternativetothosealready
established in order to improve food quality control processes. The
investigationsperformedtoachievethisgoalaredescribedbelow:
Studyofthepotentialusefulnessofluminescentresonanceenergy
transfer(LRET)betweennanocrystalsoflanthanideionsthatshow
antiStokes luminescence (upconverting phosphors) and organic
fluorophorestodevelophomogeneousaffinityassaysforbiotin.
INTRODUCCIN
Introduccin
1. Nanopartculasdeslice
Las numerosas investigaciones que se estn desarrollando en el
campo de la Nanotecnologa, y los procesos involucrados en la sntesis,
manipulacinydesarrollodenanomateriales,estndandolugaranuevas
herramientasmetodolgicaseinstrumentalesconaplicacionesendistintas
reas analticas [1]. Entre la variedad de los nanomateriales actualmente
disponibles, el uso de nanopartculas de slice (SiO2NPs) constituye una
opcinmuytilcomosedescribeacontinuacin.
El inters que presenta la utilizacin de SiO2NPs con fines
analticos es atribuible a sus especiales caractersticas. No solo muestran
Introduccin
sintetizado
otros
tipos
de
SiO2NPs
usando
materiales
1.1 Sntesisdenanopartculasdeslice
mtodoStberyelmtododemicroemulsindemicelasinversas.
10
Introduccin
queseobtienenSiO2NPsdedimetromedioinferiora100nm.Elproceso
implicalahidrlisisdeunprecursoralcxidodeslice(comopuedeserel
tetraetoxisilano, TEOS) en una mezcla de etanol e hdroxido amnico.
Duranteestahidrlisistienelugarlageneracindecidosilcicoy,cuando
la concentracin de ste supera su solubilidad en etanol, se produce la
nucleacin, dando lugar a la formacin de las NPs. El dimetro de estas
partculas puede ser controlado mediante modificacin de diversas
variables experimentales como las concentraciones de los diferentes
reactivos y la temperatura de la reaccin [4]. El dimetro de las NPs
obtenidaspuedeoscilarentre10nmy1m,loquedalugaraunconjunto
bastante heterogneo. La incorporacin de fluorforos orgnicos a estas
NPs puede realizarse mediante enlace covalente para lo que es necesario
modificarlosfluorforoscongruposfuncionalescomoelisocianato,elcual
seuneagruposaminosprocedentesdealgnprecursordeslice,comoel
3aminopropiltrietoxisilano, APTES, por lo que los reactivos TEOS y
APTESseincluyensimultneamenteenelprocesodesntesis.
El mtodo de microemulsin de micelas inversas, tambin
conocidocomomtododemicroemulsindeaguaenaceite(W/O),origina
agregados termodinmicamente estables a partir de un surfactante
anfiflico.Lascabezashidroflicasseorientandeformaqueaslanmultitud
de gotas de agua de tamao nanomtrico, mientras que las colas
hidrofbicas quedan orientadas hacia el disolvente orgnico. Estas
nanogotasaisladas del medioorgnico actancomo nanoreactores donde
sellevaacabolaformacindelasNPs.AligualqueenelmtodoStber,
11
Introduccin
1.2 Nanopartculasdopadas
Nanopartculasdopadasconfluorforosorgnicos
12
Introduccin
Porelcontrario,lasSiO2NPsdopadasconmolculasfluorescentes
la
utilizacin
de
SiO2NPs
dopadas
con
tris(2,2
13
Introduccin
determinacindeozonobasadoenelfenmenodetransferenciadeenerga
resonante electroquimioluminiscente (ECRET). En este caso, las NPs
dopadasconRu(phen)32+(RuSiNPs)transfierenlaenergaaotrofluorforo
queactacomoaceptor[15].
1.2.2
Nanopartculasdeslicedopadasconquelatosdeiones
lantnidos
Nanopartculasdeslicedopadasconquantumdots(QDs)
ejemplo,
se
ha
descrito
un
nuevo
inmunosensor
14
Introduccin
sistemaparaladeterminacindeionesmercurio[19]odemelamina[20].
Otra combinacin de los QDs con las SiO2NPs ha sido la unin de estos
nanocristales a la superficie de la slice mediante enlaces covalentes para
serutilizadosenladeterminacindeclulasymarcadorestumorales[21
23], anticuerpos y otras protenas [24, 25], as como molculas de menor
tamaocomoeltrinitrotolueno[26].
1.2.4
NanopartculasdesliceactivasalaradiacinRaman
15
Introduccin
Nanopartculasmagnticas
Lasnanopartculasmagnticas(MNPs)tienenungranatractivoen
16
Introduccin
1.3 Mtodosparamodificarlasuperficiedelasnanopartculasdeslice
El uso de SiO2NPs en aplicaciones analticas se ha extendido
gracias a la relativa facilidad para modificar su superficie. Existen
precursores de slice que permiten introducir grupos funcionales como
aminas, carboxilos, y tioles, creando as puntos de unin a biomolculas.
Porejemplo,losoligonucletidospuedenserinmovilizadosenlasuperficie
de las SiO2NPs usando la reaccin de acoplamiento del disulfuro, donde
las SiO2NPs se funcionalizan con 3mercaptopropiltrimetoxisilano. Una
reaccin de intercambio tioldisulfuro permite la conjugacin de las
17
Introduccin
18
Introduccin
elcidofosfnico,paraladeterminacindeglucosaenmuestrasrealesen
presenciadeglucosaoxidasa,midindoselacorrienteelctrica[41].
Las biomolculas ms utilizadas para inmovilizarlas en la
superficie de las SiO2NPs son las enzimas y los anticuerpos, debido a su
elevadaselectividad,peroesimprescindiblelageneracindealgntipode
sealquepuedamedirseyrelacionarseconlaconcentracindeanalito.Por
ejemplo, se han utilizado enzimas unidas a la superficie de SiO2NPs que
puedencatalizarreaccionesquimioluminiscentes.Sehadescritoelusode
SiO2NPs marcadas con anticuerpos antistaphylococcal enterotoxin B y
peroxidasa de rbano (HRP) para obtener una seal luminiscente en
presencia de perxido de hidrgeno y luminol [42]. Tambin se han
utilizadoespeciesmagnticascomomtododeseparacin.Porejemplo,en
una separacin basada en una inmunoreaccin tipo sndwich para
microcistinaLR, se ha marcado uno de los anticuerpos con NPs
ferromagnticas, mientras que el segundo anticuerpo se ha enlazado a
SiO2NPs sobre las que se ha inmovilizado previamente la HRP. En
presencia del analito, ambos conjugados quedan unidos y se separan del
resto de la matriz de la muestra utilizando un imn, debido a las
propiedades magnticas de uno de los conjugados. A continuacin, se
adiciona un precursor quimioluminiscente que acta como sustrato de la
enzimayseregistralasealquimioluminiscente[43].
SiO2NPs,porejemplo,medianteatrapamientoenunamatrizporosadeeste
material, puede ser una alternativa til en ciertas tcnicas biomdicas,
biotecnolgicas y bioanalticas. Utilizando las caractersticas de la matriz
19
Introduccin
de
tertbutilhidroquinona
utilizando
un
sensor
electroqumicodeimpresinmolecular[46].
1.4.Usodenanopartculasdesliceenanlisisdealimentos
SiO2NPs,sehandescritodiversasaplicacionesinteresantes,aunquesuuso
no est generalizado. Estos nanomateriales se han utilizado como nuevos
soportes slidos en mtodos de extraccin y separacin del analito de la
muestra, previos a la etapa de determinacin. Una opcin descrita en la
queseutilizanSiO2NPsparalaseparacinhaconsistidoensudopajecon
un ncleo magntico y su posterior funcionalizacin con un anticuerpo.
Utilizando estas NPs como medio de extraccin, se ha determinado
Salmonella en muestras de zumo de limn y leche pasteurizada va PCR
[47].
20
Introduccin
2. Nanomaterialesbasadosenlantnidos
Esta seccin se centrar en la descripcin de las principales
caractersticas y aplicaciones de los nanomateriales basados en lantnidos
utilizadosenlasinvestigacionesincluidasenestaMemoria,talescomolos
upconverting phosphors y las nanopartculas de xidos de lantnidos.
Aunqueexistenotrosmaterialesutilizadosconfinesanalticos,talescomo
lasNPsdeslicedopadasconquelatosdeioneslantnidos,sudescripcin
se realiz en el apartado anterior de esta Introduccin por lo que no se
incluyeenestaseccin.
21
Introduccin
2.1 Upconvertingphosphors
Debido a que en el Captulo IV de esta Memoria se describen las
investigaciones realizadas con un tipo especial de nanomateriales como
son los upconverting phosphors, se dedica una parte de esta
Introduccin a la descripcin de sus caractersticas y de algunas
aplicacionesanalticas.
Elfenmenodeupconversionesunprocesoenelcualsegenera
unaintensaemisindeenergaapartirdeunaradiacinmenosenergtica.
Este incremento de energa se debe a la absorcin de varios fotones,
normalmente dos o tres, por cada fotn emitido. La transicin del estado
electrnico excitado superior al estado electrnico ms bajo, o a otros
niveles menos energticos, da lugar a una emisin luminiscente a
longitudesdeondamscortasquelalongituddeondadeexcitacin.Este
proceso ptico no lineal, tambin conocido como fotoluminiscencia anti
Stokes,implicaestadosdeenergaintermedios,imprescindiblesparallevar
acaboelsaltoelectrnicoylaemisindeenerga[50].
A diferencia de los fenmenos de fluorescencia basados en
desplazamientoStokes,dondesellevaacabolaexcitacinalongitudesde
ondamscortasylaemisintienelugaraunalongituddeondamayor,el
fenmeno de upconversion requiere una menor energa de excitacin
que los fenmenos Stokes (Figura 1). Por lo tanto, este proceso permite
irradiar la muestra sin el riesgo de descomposicin fotoqumica de la
misma. Adems, se minimizan las interferencias espectrales procedentes
dedichamatriz,comosecomentarposteriormente.
22
Introduccin
Figura1.Representacinesquemticadelosfenmenosdefluorescencia
condesplazamientoStokes(a)yantiStokes(b).
23
Introduccin
con
desplazamiento
antiStokes,
estos
UCPs
son
24
Introduccin
UCPs
son
cristales
inorgnicos
hidrofbicos
que,
25
Introduccin
LacombinacindelosUCPsconbiomolculasselectivas,comoson
las implicadas en las interacciones antgenoanticuerpo o biotina
estreptavidina, constituyen una excelente alternativa para su uso en
aplicaciones bioanalticas. Tanto la biomolcula como el UCP, deben
presentar grupos funcionales adecuados para poder unirse mediante
enlaces covalentes. La adsorcin fsica de las biomolculas sobre la
superficie de los UCPs ha sido estudiada aunque la inestabilidad que
presentanlashaceinapropiadasparalosbioensayos[55].
Sehandesarrolladovariasaplicacionesbioanalticasutilizandolos
UCPs como marcadores en ensayos homogneos [56 59] y heterogneos
[60 62], en ensayos de flujo lateral [63 66] y como sensores [67, 68]
basndose algunas de estas aplicaciones, especialmente los ensayos
homogneos, en sistemas basados en transferencia de energa resonante
luminiscente (LRET). En el captulo IV correspondiente a las
investigaciones realizadas con el uso de upconverting phosphors se
abordar con ms profundidad la utilidad de estos nanocristales en el
desarrollodesistemasLRET.
2.2.Nanopartculasdexidoslantnidos
Lasnanopartculasdexidoslantnidos,ymsespecficamentelas
nanopartculas de xido de terbio y de europio, a pesar de haberse
utilizado en numerosas aplicaciones industriales, tales como en el
desarrollodedispositivosdeiluminacinenestadoslido,hanpresentado
hastalafechaescasasaplicacionesanalticas.Sehadescritorecientemente
elusodelasnanopartculasdeEu2O3paraladeterminacindelantibitico
26
Introduccin
tetraciclinaenmuestrasdeorinaanimalymiel[69].Elmtodoestbasado
en la interaccin directa de dicho antibitico con las Eu2O3NPs, para
producir una intensa luminiscencia sensibilizada. Esta emisin
luminiscentepresentacaractersticassimilaresalaluminiscenciaobservada
conquelatosdeioneseuropio,talescomoestrechasbandasdeemisin,un
amplio desplazamiento Stokes y una larga duracin de la luminiscencia
[70]. Esta ltima caracterstica posibilita la realizacin de medidas en el
mododetiemporesuelto,quepermiteeliminarlainterferenciadeseales
fluorescentes de ms corta duracin. Las nanopartculas de Eu2O3 se han
utilizadotambinparaeldesarrollodeinmunoensayosenfaseslida[71,
72]. Estas NPs no tienen grupos funcionales en su superficie, por lo que
sta ha de modificarse para poder ser utilizadas como marcador, lo que
puede conducir a la inhibicin de su luminiscencia por la accin de los
reactivos requeridos para la activacin y conjugacin. Para evitar esto, se
recurreasuencapsulamientoenmatricesdesliceoalmina,comoseha
descrito para un inmunoensayo para la determinacin de atrazina, en el
quelasEu2O3NPssehanrecubiertodesliceparaformarelmarcador[72].
La luminiscencia sensibilizada de las nanopartculas de xido de
terbio(III,IV),tambindenominadoterbia(Tb4O7),porsalicilatoylasalocid
se ha estudiado de forma sistemtica, desarrollndose finalmente un
mtodoparaladeterminacindelasalocidenmuestrasdeagua,depienso
y de huevos [73]. La selectividad del mtodo propuesto ha permitido la
determinacin de dicho antibitico con un tratamiento sencillo de las
muestras.EnlasinvestigacionesincluidasenestaMemorianoseabordar
elusodelaluminiscenciasensibilizadadeestasnanopartculas,sinoquese
27
Introduccin
describirunanuevapropiedadcomoactivadordelaenzimalaccasapara
ladeterminacindepolifenolesenvinos.
3. Fluorforosdelargalongituddeonda
Elusodefluorforosdelargalongituddeonda(LWFs)enQumica
Analtica es una alternativa til para mejorar la selectividad espectral de
lasmedidasfluorescentesfrentealosfluorforostradicionales.Laemisin
de fluorescencia a larga longitud de onda tiene lugar en una zona del
espectroelectromagntico(>600nm)dondeprcticamentenoseproducela
absorcin o emisin de posibles interferentes presentes en la matriz de la
muestra. Adems, debido a la baja energa utilizada para excitar el
fluorforo,elriesgodedegradacindelamuestraesbajomientrasquelas
interferencias que originan seales Raman tambin se reducen
considerablemente [74]. Por otro lado, la posibilidad de sufrir fenmenos
de inhibicin es muy reducida ya que los LWFs muestran un tiempo de
vida corto. La utilidad de este tipo de fluorforos se ha demostrado
ampliamente, especialmente en anlisis biolgico, donde la seal
procedente de la matriz de la muestra puede ser una fuente de
interferenciasmuyimportante.
Laversatilidaddeestoscompuestossehapuestodemanifiestoen
eldesarrollodenuevossustratosenzimticos,actuandocomomarcadores
eninmunoensayos,ensecuenciacindecidosnucleicosycomoreactivos
derivatizantes en electroforesis capilar (CE) y cromatografa de lquidos
(LC). Adems, estos fluorforos se han utilizado para el desarrollo de
28
Introduccin
metodologasbasadasensistemasFRETydenuevossensores,ascomoen
metodologascinticas[75].
3.1 Tiposypropiedadesdelosfluorforosdelargalongituddeonda
Enlosltimosaossehanutilizadodiferentestiposdefluorforos
delargalongituddeondacomoreactivos,entrelosquesepuedendestacar
tres grupos bien diferenciados: materiales inorgnicos, fluorforos
orgnicosycompuestosorganometlicos(Figura2).
Figura2.Clasificacindelosfluorforosdelargalongituddeonda.
29
Introduccin
Dentrodelosmaterialesinorgnicos,sepuedendestacardostipos,
los quantum dots (QDs) [76] y los upconverting phosphors (UCPs) [77],
estos ltimos descritos en el apartado anterior. Los QDs suelen estar
formados por una estructura sintetizada a partir de materiales
semiconductores como CdSe, CdTe, PbS, o materiales similares, y cuya
composicin y tamao inciden en las longitudes de onda mximas de
excitacinyemisinquepresentanestosmateriales.
Aunque el nmero de fluorforos orgnicos de larga longitud de
ondaesmenorqueeldelosfluorforosconvencionales,sehasintetizado
una gran variedad de compuestos de este tipo en los ltimos aos, los
cuales se han utilizado en muchos casos en el diseo de lseres de
colorantes. Un LWF debe presentar una estructura rgida con enlaces
conjugadosoanillosaromticoscondensados.Estesistemadeconjugacin
puede favorecer la inestabilidad del reactivo as como procesos de foto
descomposicin cuando es excitado. Tambin pueden mostrar otros
inconvenientes tales como estrechos desplazamientos Stokes, baja
solubilidad, y procesos de fotooxidacin. Sin embargo, las ventajas
anteriormente indicadas justifican su aplicacin en Qumica Analtica [78,
79],describindoseacontinuacinalgunascaractersticasdelosLWFsms
utilizados.
Losfluorforosdelafamiliadelascianinasconstituyenunodelos
principalesgruposdeLWFs.Suestructurapresentadosanillosaromticos
y heterocclicos unidos mediante una cadena de polimetina con dobles
enlaces carbonocarbono conjugados (Figura 3). Estos fluorforos tienen
mximos de excitacin en el intervalo 600900 nm, obtenindose amplios
30
Introduccin
desplazamientosespectralesbatocrmicosconlaadicindegruposviniloa
la cadena de polimetina. Las cianinas presentan algunas limitaciones: no
tienen grupos reactivos para unirlos a los analitos, su vida media en el
estado excitado es muy corta, bajo rendimiento cuntico y tienden a
agregarseendisolucin,loqueoriginaunrpidodescensodelaintensidad
defluorescencia[80].Sinembargo,lafotofsicadeestoscompuestospuede
mejorarseaadiendomacromolculasodisolventesorgnicosalmedio.La
adicindegrupossulfonatoalaestructuradelfluorforopuedemejorarsu
solubilidadenagua,elrendimientocunticoysuestabilidadfotoqumica.
Adems, la adicin de grupos funcionales, como el isotiocianato, permite
su uso como marcador ya que puede unirse a otras molculas. Los
derivados indolio de estos colorantes muestran una buena estabilidad
fotoqumica, la cual puede incluso mejorarse incluyendo una estructura
anularalacadenadepolimetina[81].
Dosfluorforoscianinaampliamenteutilizadosconfinesanalticos
sonCy5yverdedeindocianina(ICG),denominadotambinIR125(Figura
3). Se han descrito numerosas aplicaciones analticas para Cy5,
principalmenteenCE[82]ysensores[83].ElICGesunfluorforocargado
negativamente,solubleenagua,queseutilizinicialmenteparatcnicasde
diagnstico mdico ya que no es txico para el organismo humano [84].
AunqueelICGporssolopresentaunabajaintensidaddefluorescenciaen
disolucin acuosa, sta aumenta despus de su unin a algunos
compuestos,talescomoprotenas[85].
31
Introduccin
+
N
a)
H3C
CH3
HO3S
SO3H
H3C
CH3
+
N
N
(CH2)5COOH
CH2CH3
b)
CH3
H3C
CH3
H3C
+
N
CH2
CH2
(CH2)3
(CH2)3
SO3Na
SO3Na
c)
Figura3.Estructuradelosfluorforoscianina:a)Estructurabsica,b)Cy5,
c)verdedeindocianina.
32
Introduccin
N
+
H2N
a)
C2H5HN
N
R
b)
Figura4.Estructuradeloscolorantesoxacina:a)violetadecresilo(R:H2)
yazulnilo(R:C2H5),b)Oxacina750.
33
Introduccin
SO2Cl
SO3
CN
a)
N Cl
b)
Figura5.Estructurade:a)Rodamina800,b)RojoTexas.
34
Introduccin
7
6
N
5
1
2
N
F
35
Introduccin
36
Introduccin
4. Tcnicasdeinmunoensayoysuusoenanlisisdealimentos
Los inmunoensayos son tcnicas analticas basadas en la elevada
selectividad que presentan las reacciones antgenoanticuerpo, siendo
ampliamente utilizados en anlisis clnico, ambiental y agroalimentario.
Desdeunpuntodevistageneral,losinmunoensayospuedendividirseen
dosgrandesgrupos,directoseindirectosoconreactivosmarcados.
Los inmunoensayos directos se basan en la medida de una
propiedadfsicoqumicadelmedioquesemodificaalreaccionarelanalito
con el inmunoreactivo. Dentro de los inmunoensayos directos, los ms
utilizadoshansidolainmunoturbidimetraylainmunonefelometra[115,
116],basadasenlamedidadeladispersindelaradiacinalformarseel
inmunocomplejo. Estas tcnicas se utilizan para la determinacin de
macromolculas, principalmente protenas. Su principal limitacin es la
posiblesealdefondodebidaalacapacidaddispersantedecomponentes
de la muestra, como pueden ser lipoprotenas y agregados de protenas,
dandolugaralmitesdedeteccinrelativamentealtos.
Los inmunoensayos indirectos ofrecen mayor versatilidad que los
anteriores y se basan en el uso de un reactivo adicional, denominado
marcadorotrazador.Estereactivoesunantgenoounanticuerpo,segnel
tipodeinmunoensayo,unidoaunasustancia,denominadalabel(L)en
laterminologainglesa,quepresentaunapropiedadcomoradioactividad,
37
Introduccin
fluorescenciaoactividadenzimtica,entreotras,cuyamedidaserelaciona
conlaconcentracindelanalito.Estosinmunoensayosseclasificanendos
grandes grupos en funcin del formato que utilicen, homogneos y
heterogneos[117].
En el inmunoensayo homogneo, la unin del antgeno y el
anticuerpo da lugar a un cambio en la propiedad del marcador que se
relaciona con la concentracin de analito. En este caso no es necesario
eliminar la matriz de la muestra para realizar la medida, por lo que el
ensayo es muy rpido, pero la presencia de la seal de la matriz de la
muestra da lugar a lmites de deteccin mayores que el inmunoensayo
heterogneo.
En el inmunoensayo heterogneo el marcador no modifica su
propiedad al intervenir en la inmunoreaccin, por lo que es necesario
inmovilizar algn reactante en un soporte slido para conseguir la
separacin de las fracciones enlazada y libre del marcador. Este
inmunoensayo es ms verstil que el homogneo, obtenindose adems,
como se ha indicado, mejores lmites de deteccin. Sin embargo, se
requiere un mayor nmero de etapas por lo que los ensayos son ms
lentos. A continuacin se describensucintamente algunos de los distintos
tiposincluidosencadaformato.
4.1 Inmunoensayohomogneo
Normalmente es competitivo, es decir, se considera que el
anticuerpo no distingue entre el analito y el marcador y ambos compiten
38
Introduccin
EMIT(Enzymemultipliedimmunoassaytechnique):Fueelprimer
CEDIA(Clonedenzymedonorimmunoassay):Esuntipoespecial
deensayohomogneo,patentadoporMicrogenics.Comosemuestraenel
Figura 7, utiliza dos fragmentos inactivos de la enzima galactosidasa, a
los que se les denomina dador (ED) y aceptor (EA) enzimticos, y que al
unirse forman la enzima activa. El ensayo se realiza en una etapa
39
Introduccin
mezclandolamuestraquecontieneelanalitocondosreactivos:1)Reactivo
1,formadoporelanticuerpoyelfragmentoEAdelaenzima,y2)Reactivo
2,quecontienealmarcadorformadoporelfragmentoEDunidoalhapteno
y el sustrato de la enzima. Despus de incubar, slo el fragmento ED del
marcador libre puede unirse al fragmento EA formando la enzima,
mientrasquestanoseformaconelfragmentoEDdelmarcadorunidoal
anticuerpo. Por tanto, la seal originada por el producto de la reaccin
enzimticaserdebidaalmarcadorlibreydirectamenteproporcionalala
concentracindeanalito[119].
AceptorEnzimtico
Antgeno()
(Analito)
Anticuerpo
FormaInactiva
Antgeno(+)
(Analito)
FormaActiva
DadorEnzimtico
Figura7.EsquemadelformatoCEDIA.
40
Introduccin
FPIA(Fluorescencepolarizationimmunoassay):Enestesistemase
41
Introduccin
hacambiado.Portanto,sisehaexcitadoconradiacinpolarizada
verticalmente,emitirradiacinpolarizadaenlamismadireccin.
Figura8.EsquemadelformatoFPIA.
4.2 Inmunoensayoheterogneo
Estos inmunoensayos tienen mayor versatilidad ya que mediante
formatoscompetitivosynocompetitivososndwichpuedendeterminarse
haptenos,antgenosmacromolecularesyanticuerpos.
Puesto que, como se ha indicado anteriormente, la propiedad del
marcador no se modifica al producirse la reaccin inmunoqumica, en
cualquier inmunoensayo heterogneo es imprescindible la inmovilizacin
dealgnreactanteenunsoporteslidoparaconseguirlaseparacindelas
fracciones enlazada y libre del marcador. Este tipo de inmunoensayo ha
sufridounacontinuaevolucinalolargodeltiempo.Latendenciaactual
42
Introduccin
secentraenelusodenanomateriales,yaqueofrecenunagransuperficie
til para la inmovilizacin, aumentando la eficacia de las interacciones
entre analitos y reactivos, y, con ello, la sensibilidad de los diferentes
mtodos. Adems, la utilizacin de nanopartculas magnticas facilita su
separacinmedianteunimn.
Losdiferentesinmunoensayosheterogneospuedenclasificarsea
su vez en dos formatos bsicos en funcin del diseo del ensayo,
competitivoynocompetitivoosndwich:
43
Introduccin
44
Introduccin
individuo de otra especie animal, por ejemplo una oveja, con suero de
ratn. Este marcador puede utilizarse como reactivo general para
determinardistintosanalitossiemprequeloscorrespondientesanticuerpos
primarios procedan de la misma especie animal. Como alternativa a este
marcador, podra usarse directamente el anticuerpo del analito marcado,
reduciendo as el nmero de etapas del ensayo, pero se necesitara un
marcador distinto para cada determinacin, aumentado su coste. Este
formato se aplica preferentemente a analitos macromoleculares, ya que
deben disponer de grupos adecuados para enlazarse al soporte y al
anticuerpo.
45
Introduccin
Figura10.Formatosnocompetitivos,A)directoyB)indirecto,concaptura
deantgeno.
El formato sndwich implica que el analito debe tener, al menos,
dosdeterminantesantignicosparaqueseenlacenaloscorrespondientes
anticuerpos.Teniendoencuentaquelosanticuerpossonmacromolculas,
espreferiblequeelanalitotambinseamacromolecular,deformaqueno
exista impedimento estrico y puedan enlazarse adecuadamente los dos
anticuerpos. Este formato, utilizado normalmente para determinar
protenas antignicas, ofrece mayor selectividad que el formato
competitivo,yaqueseusandosanticuerposfrentealanalito.
Elensayosndwichtambinseutilizaparadeterminaranticuerpos
frente a un antgeno y, al igual que en el caso anterior, la determinacin
puede realizarse de dos formas. Como muestra la Figura 11, se puede
46
Introduccin
llevaracaboinmovilizandoelantgenoounanticuerpoinespecficofrente
al anticuerpo a determinar. En el primer caso, si la muestra contiene el
anticuerpo que se va a determinar, ste quedar retenido en la superficie
slida. Despus de lavar, se adiciona el marcador formado por otro
anticuerpo.Enelsegundocaso,seinmovilizaunanticuerposecundarioen
la superficie slida y se adiciona la muestra. Tras incubar y lavar, se
adiciona el antgeno especfico frente al anticuerpo a determinar y,
finalmente, se aade el marcador, formado por un anticuerpo especfico
frentealantgeno.
Figura11.Formatosnocompetitivosparaladeterminacindeanticuerpos:
A)conantgenoinmovilizadoyB)conanticuerpoinmovilizado.
47
Introduccin
medianteelusodeistoposradiactivosunidosabiomolculasparaformar
el marcador. Este ensayo presenta gran versatilidad, bajos lmites de
deteccin,elevadaprecisinyausenciadeinterferenciasbienambientales,
talescomolasdebidasalpH,temperaturaofuerzainica,obiendebidasa
la matriz de la muestra. Sin embargo, tambin presenta limitaciones,
siendoelprincipalinconvenienteelusodematerialesradioactivosconsus
posibles riesgos para la salud, necesidad de permisos y recintos
exclusivamente preparados para ello, as como la gestin especial de los
residuos, lo que encarece el ensayo y limita su uso a laboratorios de
referencia[123125].
48
Introduccin
Figura12.EsquemadelsistemaDELFIA.
ELISA(Enzymelinkedimmunosorbentassay):Puedeconsiderarse
elinmunoensayoheterogneomsutilizadoenanlisisclnico,ambientaly
agroalimentario. En este caso, el marcador es sintetizado a partir de una
enzima que, en presencia del sustrato correspondiente, cataliza una
reaccin, dando lugar a cambios medibles normalmente mediante
fotometra o fluorimetra. Las enzimas ms utilizadas para formar el
marcador son galactosidasa, fosfatasa alcalina y peroxidasa de rbano,
las cuales necesitan su correspondiente sustrato para llevar a cabo la
deteccin[128131].
49
Introduccin
basadosenlainmovilizacindeunantgenoounanticuerpoenlazonade
reconomiento del sensor. Hasta hace unos aos, estos inmunosensores
presentabanproblemasdesensibilidad,yaqueloscambiosdesealapenas
podan ser recogidos por el transductor. En cambio, la utilizacin de los
nanomateriales ha provocado una revolucin en estos dispositivos. La
inmovilizacin de NPs sobre la superficie del sensor proporciona un
aumento de la superficie especfica del mismo y, en consecuencia, de la
cantidad de reactivo inmovilizado, consiguiendo mayores cambios de
sealyunamejoradelasensibilidaddelmtodo.
Los inmunosensores pueden ser clasificados en diferentes grupos
en funcin de la seal originada al unirse el analito a la superficie del
sensor(Figura13).
Potenciomtricos
Amperomtricos
Impedimtricos
Conductimtricos
Capacitivos
ELECTROQUMICOS
INMUNOSENSORES
PTICOS
PIEZOELCTRICOS
Quimioluminiscentes
Electroquimioluminiscentes
Resonanciadelplasmn superficial
Figura13.Clasificacindelosinmunosensores.
50
Introduccin
Acontinuacin,sedescribensucintamentelosdistintostipos:
Los inmunosensores electroqumicos se caracterizan por presentar
buenasensibilidad(desdelainclusindelosnanomateriales),bajocostey
fcil automatizacin. Como se muestra en la clasificacin, los
inmunosensores electroqumicos estn divididos en varios subgrupos, en
funcindeltipodesealqueseorigina.As,lospotenciomtricosregistran
el cambio de potencial en la superficie del sensor, los amperomtricos
recogen la corriente elctrica generada, los conductimtricos miden los
cambios en la conductividad, los sensores de impedancia miden la
resistenciaalpasodecorrienteelctricaylossensoresdecapacitanciase
basan en los cambios de la constante dielctrica del sensor al unirse el
analitoasusuperficie[132146].
Losinmunosensorespticossebasanenlamedidadelaabsorcin
oemisinproducidatrasllevarseacabolainmunoreaccinenlasuperficie
delsensor.Estosinmunosensoressonmuyutilizadosenbioanlisisgracias
a las ventajas que presentan: modo de trabajo no destructivo y rpida
generacin de la seal. Al igual que ocurre con los inmunosensores
electroqumicos, los inmunosensores pticos han experimentado una
revolucingraciasalusodenanopartculas.Losnanomaterialesutilizados
vandesdeSiO2NPsdopadasconfluorforos,AuNPs,QDs,CNTs,hastalos
recientemente utilizados UCPs. Estos inmunosensores pticos, como
muestra el esquema anterior, se pueden dividir en sensores
quimioluminiscentes (se recoge la intensidad luminiscente generada por
unareaccinqumica),electroquimioluminiscentes(implicanlaformacin
de especies que, tras una transferencia electrnica, emiten radiacin
51
Introduccin
Otro
tipo
de
inmunosensores
son
los
inmunosensores
52
Introduccin
marcadorunidoalanalito,indicandolapresenciadelanalitoenlamuestra
[156160].
Figura14.Esquemadeensayodeflujolateral.
4.3.Usodelinmunoensayoenanlisisdealimentos
Aunque las tcnicas de inmunoensayo tienen su principal campo
deaplicacinenanlisisclnico,suusosehaidoconsolidandotambinen
anlisisdealimentos,comolodemuestraelnmerorelativamenteelevado
de kits de ensayos comerciales actualmente disponibles. La Figura 15
muestra la distribucin de los artculos publicados en los ltimos aos
sobre esta rea. Puede observarse que, aunque la mayora de las tcnicas
anteriormentedescritassehanaplicadoalanlisisdealimentos,ELISAha
sido y actualmente sigue siendo la ms utilizada. Algunas de las razones
quejustificansueleccinincidenenlosbajoslmitesdedeteccin,fciluso
y coste relativamente bajo que presentan los mtodos basados en esta
tcnica.
53
Introduccin
EnlaTabla1serecogenalgunosejemplosdelosmtodosdescritos
utilizando distintos inmunoensayos [161 171]. Como puede observarse,
se han desarrollado determinaciones para diversos analitos tales como
micotoxinas,plaguicidas,residuosdeantibiticosyhormonasaplicablesal
anlisisdealimentosdestinadosalconsumohumanoydealimentospara
animales. Cabe destacar que, aunque ELISA sigue siendo la tcnica ms
extendida en esta rea, existe una clara tendencia al desarrollo de
inmunosensores, principalmente electroqumicos y pticos [172], aunque
sucomercializacinhastalafechahasidoprcticamentenula.
54
Introduccin
Tabla1.Ejemplosrepresentativosdeinmunoensayosaplicadosalanlisis
dealimentos
Tipode
Fundamento
Analito
Muestra
Ref.
FREThomogneo
Micotoxinas
Cebada
[161]
[162]
Inmunoensayo
Homogneo
porfluorescencia
intrnsecadelos
anticuerpos
Homogneo
Dispersindela
Protenasde
Zumode
radiacincon
soja
frutasyyogur
consoja
AuNPs
Homogneo
FPIA
Micotoxinas
Grano
[163]
Homogneo
FPIA
Plaguicidas
Vegetablesy
[164]
rgano
agua
fosforados
ambiental
Heterogneo
Heterogneo
ELISA
ELISA
Fluoro
Pescadosy
quinolonas
mariscos
[165]
Dodecilciclo
Ternera
[166]
[167]
butanona
Heterogneo
ELISA
Heterogneo
DELFIA
Heterogneo
Ensayoflujolateral
Fenil
Piensosy
etanolaminaA
carne
Estradiol
Suerobovino
[168]
Residuosde
Orinadecerdo
[169]
Leche
[170]
Carnedecerdo
[171]
clembuteroly
raptomina
Inmunosensor
Sensor
Residuosde
amperomtrico
sulfonamiday
tetraciclina
Inmunosensor
Sensor
Clembuterol
electroquimio
luminiscente
55
Introduccin
Bibliografa
(1)
D.Knopp,D.Tang,R.Niessner.Review:Bioanalyticalapplications
of biomoleculefunctionalized nanometersized doped silica
particles.Anal.Chim.Acta(2009)647,1430.
(2)
(3)
(4)
(5)
(6)
N.Gan,J.Hou,F.Hu,Y.Cao,T.Li,Z.Guo,J.Wang.Arenewable
and ultrasensitive electrochemiluminescence immunosensor based
on magnetic RuL @ SiO2Au~RuLAb2 sandwichtype nano
immunocomplexes.Sensors(2011)11,77497762.
(7)
(8)
(9)
(10)
Q.Sun,X.Zhang.ElectrochemiluminescenceDNAsensorbasedon
Ru(bpy)32+ doped silica nanoparticles labeling and proximity
dependent surface hybridization assay. J. Solid State Electr. (2012)
16,247252.
56
Introduccin
(11)
(12)
(13)
(14)
(15)
(16)
detection
assay
for
human
thyroid
stimulatinghormone.Anal.Chim.Acta(2012)722,9599.
(17)
(18)
J.Wang,H.Han,X.Jiang,L.Huang,L.Chen,N.Li.Quantumdot
based nearinfrared electrochemiluminescent immunosensor with
gold nanoparticlegraphene nanosheet hybrids and silica
nanospheres doubleassisted signal amplification. Anal. Chem.
(2012)84,48934899.
(19)
B.Liu,F.Zeng,G.Wu,S.Wu.NanoparticlesasscaffoldsforFRET
based ratiometric detection of mercury ions in water with QDs as
donors.Analyst(2012)137,37173724.
57
Introduccin
(20)
(21)
(22)
nanosheets
based
electrochemiluminescence
Y.Li,L.Deng,C.Deng,Z.Nie,M.Yang,S.Si.Simpleandsensitive
aptasensor based on quantum dotcoated silica nanospheres and
thegoldscreenprintedelectrode.Talanta(2012)99,637642.
(24)
(25)
(26)
K.Zhang,H.Zhou,Q.Mei,S.Wang,G.Guan,R.Liu,J.Zhang,Z.
Zhang. Instant visual detection of trinitrotoluene particulates on
various surfaces by ratiometric fluorescence of dualemission
quantumdotshybrid.J.Am.Chem.Soc.(2011)133,84248427.
(27)
X.Kong,Q.Yu,X.Zhang,X.Du,H.Gong,H.Jiang.Synthesisand
application of surface enhanced Raman scattering (SERS) tags of
Ag@SiO2 core/shell nanoparticles in protein detection. J. Mater.
Chem.(2012)22,77677774.
(28)
F.Wang,R.G.Widejko,Z.Yang,K.V.T.Nguyen,H.Chen,L.P.
Fernando,K.A.Christensen,J.N.Anker.Surfaceenhancedraman
scattering
detection
of
pH
58
with
silicaencapsulated
Introduccin
P.Devi,P.Reddy,S.Arora,S.Singh,C.Ghanshyam,M.L.Singla.
SensingbehaviorstudyofsilicacoatedAgnanoparticlesdeposited
onglassycarbontowardnitrobenzene.J.Nanopart.Res.(2012)14,
1172.
(30)
(31)
(32)
(33)
(34)
(35)
B.Liu,B.Zhang,Y.Cui,H.Chen,Z.Gao,D.Tang.Multifunctional
goldsilica nanostructures for ultrasensitive electrochemical
immunoassayofstreptomycinresidues.ACSAppl.Mater.Interfaces
(2011)3,46684676.
(36)
nanotubegraphene
composite
and
functionalized
mesoporousmaterials.Biosens.Bioelectron.(2012)33,2935.
(37)
Introduccin
acetylcholinesterase
on
the
solgel/multiwalled
carbon
nanotubes/cholineoxidasecompositemodifiedplatinumelectrode.
Biosens.Bioelectron.(2012)33,4449.
(38)
(39)
J.Zhou,N.Gan,T.Li,H.Zhou,X.Li,Y.Cao,L.Wang,W.Sang,F.
Hu. Ultratrace detection of Creactive protein by a piezoelectric
immunosensorbasedonFe3O4@SiO2magneticcapturenanoprobes
and HRPantibody coimmobilized nano gold as signal tags. Sens.
Actuat.B:Chem(2013)178,494500.
(40)
High
sensitivity
carbon
nanotube
based
W.Zhao,Y.Fang,Q.Zhu,K.Wang,M.Liu,X.Huang,J.Shen.A
novel glucose biosensor based on phosphonic acidfunctionalized
silica nanoparticles for sensitive detection of glucose in real
samples.Electrochim.Acta(2013)89,278283.
(42)
(43)
J.Lu,W.Wie,L.Yin,Y.Pu,S.Liu.Flowinjectionchemiluminescent
immunoassay of microcystinLR by using PEImodified magnetic
beads as capturer and HRPfunctionalized silica nanoparticles as
signalamplifier.Analyst(2013)138,14831489.
(44)
(45)
Introduccin
phatalatebyhydrolyzingbasedonbiologicalrecognitionmaterials.
J.Pharm.Biomed.Anal.(2013)75,123129.
(46)
electrochemical
sensor
based
on
coreshell
nanoparticles.FoodChem.(2013)139,10011007.
(47)
P.Bakthavathsalam,V.K.Rajendran,U.Saran,S.Chatterjee,B.M.
J. Ali. Immunomagnetic nanoparticle based quantitative PCR for
rapid detection of Salmonella. Microchim. Acta (2013) 180, 1241
1248.
(48)
(49)
Ru
(phen)32+
doped
silica
nanoparticles
based
(51)
(52)
(53)
J.F.Suyver,D.Biner,P.Gerner,J.Grimm,S.Heer,K.Krmer,C.
Reinhard, H. U. Gdel. Novel materials doped with trivalent
lanthanides and transition metal ions showing nearinfrared to
visiblephotonupconversion.Opt.Mater.(2005)27,11111130.
(54)
J.F.Suyver,J.Grimm,M.K.vanVeen,D.Biner,K.W.Krmer,H.
U. Gdel. Upconversion spectroscopy and properties of NaYF4
dopedwithEr3+,Tm3+and/orYb3+.J.Luminesc.(2006)117,112.
(55)
61
Introduccin
lanthanidedopedupconvertingnanoparticles.NanoLett.(2011)11,
835840.
(56)
(57)
fluorescence
resonance
energy
transfer
in
homogeneousassays.Anal.Chem.(2007)79,63126318.
(58)
M.Wang,W.Hou,C.C.Mi,W.X.Wang,Z.R.Xu,H.H.Teng,C.
B.
Mao,
S.K.
Xu.
Immunoassay
of
goat
antihuman
(60)
(61)
P.L.Corstjens,S.Li,M.Zuiderwijk,K.Kardos,W.R.Abrams,R.S.
Niedbala, H. J. Tanke. Infrared upconverting phosphors for
bioassays.IEEProceedingsNanotech.(2005)152,6472.
(62)
(63)
L.Li,L.Zhou,Y.Yu,Z.Zhu,C.Lin,C.Lu,R.Yang.Development
of upconverting phosphors technologybased lateralflow assay
62
Introduccin
(65)
(66)
(67)
D.E.Achatz,R.J.Meier,L.H.Fischer,O.S.Wolfbeis.Luminescent
sensing of oxygen using a quenchable probe and upconverting
nanoparticle.Ang.Chem.Int.Ed.(2011)50,260263.
(68)
(69)
(70)
(71)
(72)
63
Introduccin
(74)
(75)
(76)
V.J.Pansare,S.Hejazi,W.J.Faenza,R.K.Prudhomme.Reviewof
longwavelength optical and NIR imaging materials: contrast
agents,fluorophores,andmultifunctionalnanocarriers.Chem.Mat.
(2012)24,812827.
(77)
M.Haase,H.Schfer.Upconvertingnanoparticles.Ang.Chem.Int.
Ed.(2011)50,58085829.
(78)
T.B.Deligeorgiev,N.I.Gadjev,I.I.Timtcheva,V.A.Maximova,H.
E. Katerinopoulos, E. Foukaraki. Synthesis of homodimeric
monomethinecyaninedyesasnoncovalentnucleicacidlabelsand
their absorption and fluorescence spectral characteristics. Dyes
Pigments(2000)44,131136.
(79)
(80)
(81)
64
Introduccin
(82)
J.M.Serrano,M.Silva.Traceanalysisofaminoglycosideantibiotics
in bovine milk by MEKC with LIF detection. Electrophoresis (2006)
27,47034710.
(83)
K.E.Sapsford,C.R.Taitt,S.Fertig,M.H.Moore,M.E.Lassman,
C. M. Maragos, L. C. ShriverLake. Indirect competitive
immunoassayfordetectionofaflatoxinB1incornandnutproducts
using the array biosensors. Biosens. Bioelectron. (2006) 21, 2298
2305.
(84)
M.Tadatsu,S.Ito,N.Muguruma,Y.Kusaka,T.Bando,Y.Tadatsu,
K. Okamoto, K. Li, Y. Nagao, S. Sano, H. Taue. A new infrared
fluorescentlabeling agent and labeled antibody for diagnosing
microcancers.Bioorg.Med.Chem.(2003)11,32893294.
(85)
(86)
JP.Song,YJ.Guo,Q.Zhao,SM.Shuang,C.Dong,M.M.F.Choi.
Assemblies of brilliant cresyl violet to DNA in the presence of
cyclodextrin.Talanta(2010)82,681686.
(87)
(88)
(89)
(90)
65
Introduccin
bloodserumsamplesbykineticspectrophotometricmethod.Biol.
TraceEl.Res.(2011)144,14301436.
(91)
X.Wang,C.Boschetti,M.J.RuedasRama,A.Tunnacliffe,E.A.H.
Hall.RatiometricpHdotANSors.Analyst(2010)135,15851591.
(92)
(93)
(94)
WD. Chen, WT. Gong, ZQ. Ye, Y. Lin, GL. Ning. FRETbased
ratiometric fluorescent probes for selective Fe3+ sensing and their
applicationsinmitochondria.DaltonTrans.(2013)42,1009310096.
(95)
(96)
HS. Lv, SY. Huang, BX. Zhao, JY. Miao. A new rhodamine B
based lysosomal pH fluorescent indicator. Anal. Chim. Acta (2013)
788,177182.
(97)
(98)
(99)
Introduccin
(100)
B.T.Glaser,J.P.Malerich,S.J.Duellman,J.Fong,C.Hutson,R.M.
Fine, B. Keblansky, M. J. Tanga, P. B. Madrid. A highthroughput
fluorescencepolarizationassayforinhibitorsofgyraseB.J.Biomol.
Screen.(2011)16,230238.
(101)
(102)
(103)
(104)
(105)
S.Ding,X.Qiao,J.Suryadi,G.S.Marrs,G.L.Kucera,U.Bierbach.
Usingfluorescentpostlabelingtoprobethesubcellularlocalization
of DNAtargeted platinum anticancer agents. Ang. Chem. Int. Ed.
(2013)52,33503354.
(106)
(107)
(108)
Introduccin
(109)
(110)
(111)
immunoextraction
and
dynamic
longwavelength
fluorometry.Microchim.Acta(2013)180,12791286.
(112)
(113)
(114)
Y.Li,P.Wang,X.Wang,M.Cao,Y.Xia,C.Cao,M.Liu,C.Zhu.An
immediate luminescence enhancement method for determination
ofvitaminB1,usinglongwavelengthemittingwatersolubleCdTe
nanorods.Microchim.Acta(2010)169,6571.
(115)
(116)
(117)
(118)
Introduccin
(120)
(121)
J.Tian,L.Zhou,Y.Zhao,Y.Wang,Y.Peng,X.Hong,S.Zhao.The
application of CdTe/CdS in the detection of carcinoembryonic
antigen by fluorescence polarization immunoassay. J. Fluoresc.
(2012)22,15711579.
(122)
J.Tian,L.Zhou,Y.Zhao,Y.Wang,Y.Peng,S.Zhao.Multiplexed
detectionoftumormarkerswithmulticolorquantumdotsbasedon
fluorescencepolarizationimmunoassay.Talanta(2012)92,7277.
(123)
(124)
J.H.Sloan,B.J.Ackermann,M.ODell,R.R.Bowsher,R.A.Dean,
R.J.Konrad.Developmentofanovelradioimmunoassaytodetect
autoantibodiestoamyloidbetapeptidesinthepresenceofacross
reactive therapeutic antibody. J. Pharm. Biomed. Anal. (2011) 56,
10291034.
(125)
(126)
H.Siitari,I.Hemmil,E.Soini,T.Lvgren.DetectionofhepatitisB
surface antigen using timeresolved fluoroimmunoassay. Nature
(1983)301,258260.
(127)
Introduccin
M.M.Vdovenko,A.S.Stepanova,S.A.Eremin,N.V.Cuong,N.A.
Uskova,
I.
Y.
Sakharov.
Quantification
of
2,4
Z.Chao,M.Tan,M.K.Paudel,S.Sakamoto,L.Ma,K.S.Tabata,H.
Tanaka, Y. Shoyama, L. Xuan, S. Morimoto. Development of an
indirect
competitive
enzymelinked
immunosorbent
assay
(131)
(132)
(133)
of
potentiometric
immunosensor
based
on
(135)
Introduccin
(137)
(138)
K.Mao,D.Wu,Y.Li,H.Ma,Z.Ni,H.Yu,C.Luo,Q.Wei,B.Du.
Labelfree
electrochemical
immunosensor
based
on
T.Yu,W.Cheng,Q.Li,C.Luo,L.Yan,D.Zhang,Y.Yin,S.Ding,
H. Ju. Electrochemical immunosensor for competitive detection of
neuron specific enolase using functional carbon nanotubes and
goldnanoprobe.Talanta(2012)93,433438.
(140)
Q.Gao,J.Han,Z.Ma.Polyamidoaminedendrimerscappedcarbn
dots/Au nanocrystal nanocomposites and its application for
electrochemicalimmunosensor.Biosens.Bioelectron.(2013)49,323
328.
(141)
(142)
X.Wang,L.Chen,X.Su,S.Ai.Electrochemicalimmunosensorwith
graphene quantum dots and apoferritinencapsulated Cu
nanoparticles doubleassisted signal amplification for detection of
avianleucosisvirussubgroupJ.Biosens.Bioelectron.(2013)47,171
177.
(143)
Introduccin
integratedwithredoxprobesastracermatrixes.Biosens.Bioelectron.
(2013)43,440445.
(144)
(145)
(146)
(147)
(148)
Y.Liebes,L.Amir,R.S.Marks,M.Banai.Immobilizationstrategies
ofBrucellaparticlesonopticalfibersforuseinchemiluminescence
immunosensors.Talanta(2009)80,338345.
(149)
luminol
cathodic
electrochemiluminescence
Z.Guo,T.Hao,S.Du,B.Chen,Z.Wang,X.Li,S.Wang.Multiplex
electrochemiluminescence immunoassay of two tumor markers
using multicolor quantum dots as labels and grapheme as
conductingbridge.Biosens.Bioelectron.(2013)44,101107.
(151)
72
Introduccin
(152)
D.E.P.Souto,J.V.Silva,H.R.Martins,A.B.Reis,R.C.S.Luz,L.
T.Kubota,F.S.Damos.Developmentofalabelfreeimmunosensor
basedonsurfaceplasmonresonancetechniqueforthedetectionof
antiLeishmania infantum antibodies in canine serum. Biosens.
Bioelectron.(2013)46,2229.
(153)
(154)
(155)
(156)
C.C.Liu,C.Y.Yeung,P.H.Chen,M.K.Yeh,S.Y.Hou.Salmonella
detectionusing16SribosomalDNA/RNAprobegoldnanoparticles
andlateralflowimmunoassay.FoodChem.(2013)141,25262532.
(157)
(158)
(159)
C.S.Pantalen,J.Wichers,A.A.Somovilla,A.VanAmerongen,A.
A. Fuentes. Development of an immunochromatographic assay
based on carbon nanoparticles for the determination of the
phytoregulatorforchlorfenuron.Biosens.Bioelectron.(2013)42,170
176.
73
Introduccin
(160)
A.N.Berlina,N.A.Taranova,A.V.Zherdev,Y.Y.Vengerov,B.B.
Dzantiev. Quantum dotbased lateral flow immunoassay for
detection of chloramphenicol in milk. Anal. Bioanal. Chem. (2013)
405,49975000.
(161)
(162)
(163)
A.P.Bondarenko,S.A.Eremin.Determinationofzearalenoneand
ochratoxin A mycotoxins in grain by fluorescence polarization
immunoassay.J.Anal.Chem.(2012)67,790794.
(164)
ZL.Xu,Q.Wang,HT.Lei,S.A.Eremin,YD.Shen,H.Wang,R.
C. Beier, JY. Yang, K. A. Maksimova, YM. Sun. A simple, rapid
and highthroughput fluorescence polarization immunoassay for
simultaneous detection of organophosphorus pesticides in
vegetable and environmental water samples. Anal. Chim. Acta
(2011)708,123129.
(165)
Chemiluminescence
competitive
indirect
enzyme
enzymelinked
immunosorbent
assay
based
on
monoclonalantibodiesforthedetectionof2dodecylcyclobutanone
inirradiatebeef.J.Agric.FoodChem.(2013)61,77497753.
(167)
B.Cao,G.He,H.Yang,H.Chang,S.Li,A.Deng.Developmentofa
highlysensitiveandspecificenzymelinkedimmunosorbentassay
(ELISA) for the detection of phenylethanolamine A in tissue and
74
Introduccin
(169)
MZ.Zhang,MZ.Wang,ZL.Chen,JH.Fang,MM.Fang,J.Liu,
XP. Yu. Development of a colloidal goldbased lateralflow
immunoassay for the rapid simultaneous detection of clenbuterol
and raptopamine in swine urine. Anal. Bioanal. Chem (2009) 395,
25912599.
(170)
immunosensors
for
the
simultaneous
(172)
75
CAPTULO 1
Herramientas analticas
HerramientasAnalticas
EneldesarrolloexperimentaldelapresenteTesisDoctoralsehan
empleadodiferentesherramientasanalticasparallevaracabolasdistintas
investigaciones realizadas. En este captulo de la Memoria de la Tesis
Doctoral se enumeran dichas herramientas y se profundiza en sus
caractersticasyaspectosmsrelevantesdesuutilizacin.
79
Captulo1
Materialesyreactivos
1 Materialesnanoestructurados
EnlasiguienteTablaserelacionanlosdiferentesnanomaterialesno
sintetizados en el laboratorio y necesarios para desarrollar los trabajos
experimentales descritos en esta Memoria, sus dimetros medios y la
fuentedesuministro.
TipodeNanomaterial
NaYF4:Yb(III),Er(III)
Tamao
Distribuidor
1020nm
DepartamentodeQumicade
losMaterialesyAnlisis
Qumico.UniversidaddeTurku
(Finlandia)
NaYF4:Yb(III),Tm(III)
1020nm
DepartamentodeQumicade
losMaterialesyAnlisis
Qumico.UniversidaddeTurku
(Finlandia)
NaYF4:Yb(III),Ho(III)
1020nm
DepartamentodeQumicade
losMaterialesyAnlisis
Qumico.UniversidaddeTurku
(Finlandia)
Tb4O7
<100nm
Aldrich
Eu2O3
<150nm
Aldrich
Diamante
<10nm
Aldrich
10nm
Aldrich
Ag
80
HerramientasAnalticas
2 Precursoresdeslice
En la siguiente Tabla se relacionan los diferentes precursores de
slice utilizados para la sntesis de nanopartculas de slice y el
recubrimientodelosupconvertingphosphors,ascomolafuncionalizacin
delasuperficiedeambosmateriales.
Precursordeslice
Tetraetoxisilano(TEOS)
(3Aminopropil)trietoxisilano
Funcin
Distribuidor
Estructuradeslice
Aldrich
Introduccingrupos
Aldrich
(APS)
amino
3(Trihidroxisilil)propil
Introduccingrupos
metilfosfonato(THPMP)
fosfonato
(N(3(trimetoxisilil)propil)etilen
Introduccingrupos
diamino
Aldrich
Aldrich
amino
3 Fluorforos
En la siguiente Tabla se relacionan los diferentes fluorforos, as
como sus respectivas longitudes de onda de excitacin y emisin,
utilizadosenlasdiferentesinvestigacionespresentadasenestaMemoria.
81
Captulo1
excitacin
emisin
(nm)
(nm)
Violetadecresilo
585
620
Sigma
Azulnilo
620
680
Sigma
Fluorescenasdica
485
520
Sigma
cido8(hidroxipireno1,3,6
455
510
SigmaAldrich
AzureA
632
645
Sigma
AzureB
648
662
Sigma
2[4(Dimetilamino)styryl]1
465
505
Aldrich
Styryl7
565
620
SigmaAldrich
Azuldetoluidina
620
638
Sigma
ficoeritrina(BPE)
550
575
Cyanotech
Fluorforo
Distribuidor
trisulfnico(HPTS)
metilpiridinioyoduro
(2Di1ASP)
Corp
Rficoeritrina(RPE)
565
575
Cyanotech
Corp
AlexaFluor488
495
520
Molecular
Probes
Invitrogen
Paysley
AlexaFluor546
556
570
Molecular
Probes
Invitrogen
Paysley
AlexaFluor680
680
702
Molecular
Probes
Invitrogen
Paysley
ATTO495
495
82
525
SpaBioSpa
HerramientasAnalticas
4 Tensoactivos
Se han utilizado los siguientes tensoactivos neutros, aninicos y
catinicos:TritnX100(Sigma),Tween20(Sigma),dodecilsulfatosdico
(SDS) (SigmaAldrich) y bromuro de hexadeciltrimetilamonio (CTAB)
(Fluka).
5 Disolventesorgnicos
Se han utilizado diferentes disolventes orgnicos como medio de
reaccin,eliminacindereactantesypreparacindedisolucionesdeetanol
(Panreac), acetona (Panreac), ciclohexano (Panreac), 1hexanol (Merck),
metanol (Panreac), dimetilformamida (SigmaAldrich) y acetonitrilo
(Panreac).
6 Anticuerpos
Sehanutilizadovariostiposdeanticuerposparallevaracabolos
diferentes formatos de inmunoensayo de esta tesis: Anticuerpos
policlonales antiprotena de soja producidos en conejo (Sigma),
anticuerpos IgG anticonejo producidos en cabra (SigmaAldrich),
anticuerpos policlonales antimonensn producidos en oveja (Abcam) y
anticuerposIgGantiovejaproducidosenconejo(SigmaAldrich).
7 Estndares
EnlasiguienteTablaserelacionanloscompuestosqumicosutilizados
para preparar estndaresde los analitos y de lospotenciales interferentes
utilizados en las diferentes investigaciones experimentales recogidas en
83
Captulo1
esta Tesis Doctoral, su pureza y las casas comerciales que los han
suministrado.
Analito
Pureza
Distribuidor
Protenadesoja
90%
Doscadesa
9095%
Sigma
97%
SigmaAldrich
cidocafeico
98%
Sigma
cidomlico
99,5%
Merck
cidoglico
97,5102,5%
Sigma
99,8%
SigmaAldrich
cidoascrbico
99%
SigmaAldrich
Sacarosa
99%
Fluka
cidoctrico
99,5%
Sigma
Sulfitosdico
98%
Merck
Biotina
99%
SigmaAldrich
Monensn
Trolox
Glucosa
8 Otrosreactivos
Para llevar a cabo las investigaciones realizadas a lo largo de esta
Memoriasehanusadodisolucionesreguladoraspreparadasapartirdesus
correspondientes sales, cidos y bases, as como otros reactivos los cuales
se relacionan a continuacin junto con la casa comercial donde fueron
adquiridos: Hidrxido amnico (Panreac), albmina de suero bovino
(SigmaAldrich), borohidruro sdico (SigmaAldrich), acetato sdico
(SigmaAldrich), cido sulfrico (Panreac), additol (Surface Specialities),
carbonato sdico (SigmaAldrich), tetraborato sdico (Panreac), anhdrido
84
HerramientasAnalticas
(EDAC)
(SigmaAldrich),
diclorhidrato2,2azobis(2metilpropionamida)(AAPH)(Aldrich),piridina
(Sigma),
sulfoNhidroxisuccinimida
(sulfoNHS)
(SigmaAldrich),
Instrumentacin
siguienteinstrumentacin:
85
Captulo1
Programasinformticos
estadsticoyelaboracindelasdiferentesrepresentacionesgrficas:Origin
7,0(ORIGINLAB),Statgraphics5.1,MicrosoftExcelySigmaPlot7.0.
86
CAPTULO 2
CHAPTER 2
Sntesis y caracterizacin de
nanopartculas de slice con
fluorescencia a larga longitud de
onda y su aplicacin al anlisis de
alimentos
Synthesis and characterization of
longwavelength fluorescent silica
nanoparticles and their application
to food analysis
Sntesisdenanopartculasdesliceysuaplicacin
Estecaptulorecogelasinvestigacionesrealizadasparadesarrollar
nuevosmtodosdeanlisisutilizandoconjuntamentelasprestacionesque
ofrecen la nanotecnologa y la fluorimetra de larga longitud de onda. Se
describelasntesisycaracterizacindenanopartculasdeslice(SiO2NPs)
dopadasconoxazinasysedemuestrasuutilidadenanlisisdealimentos
mediante inmunoensayo heterogneo. Los estudios realizados han dado
lugaralossiguientesartculos:
-
Synthesis
and
characterization
of
oxazinedoped
silica
nanoparticlesfortheirpotentialuseasstablefluorescentreagents.
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens,Journal
ofFluorescence,20(2010)171180.
Determinationofmonensininmilksamplesbyfrontsurfacelong
wavelength fluoroimmunoassay using nile bluedoped silica
nanoparticles as labels. J. GodoyNavajas, M. P. AguilarCaballos,
A.GmezHens,Talanta,94(2012)195200.
89
Captulo2
inmunocromatografa
en
nitrocelulosa[3].
90
membranas
desechables
de
Sntesisdenanopartculasdesliceysuaplicacin
BIBLIOGRAFA
(1)
(2)
(3)
91
Chapter2
Thischaptercontainstheinvestigationsperformedtodevelopnew
analyticalmethodsbycombiningthebenefitsofnanotechnologyandlong
wavelength fluorometry. The synthesis and characterization of silica
nanoparticles (SiO2NPs) doped with oxazine dyes is described and their
usefulness in food analysis by the development of heterogeneous
immunoassays is shown. These investigations have given rise to the
followingarticles:
-
Synthesis
and
characterization
of
oxazinedoped
silica
nanoparticlesfortheirpotentialuseasstablefluorescentreagents.
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens,Journal
ofFluorescence,20(2010)171180.
Determinationofmonensininmilksamplesbyfrontsurfacelong
wavelength fluoroimmunoassay using nile bluedoped silica
nanoparticles as labels. J. GodoyNavajas, M. P. AguilarCaballos,
A.GmezHens,Talanta,94(2012)195200.
Theinvestigationsperformedhadtwomainobjectives:1)Tostudy
the potential versatility of the synthesized longwavelength fluorescent
92
Synthesisofsilicananoparticlesandtheirapplication
93
Chapter2
LITERATURE
(1)
(2)
(3)
94
Sntesisdenanopartculasdesliceysuaplicacin
JournalofFluorescence(2010)20:171180
Synthesisandcharacterizationofoxazinedopedsilica
nanoparticlesfortheirpotentialuseasstablefluorescent
reagents
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens
DepartmentofAnalyticalChemistry.UniversityofCrdoba.CampusofRabanales.
MarieCurieAnnexbuilding.14071Crdoba
Abstract
The synthesis process to obtain silica nanoparticles (NPs) dopedwith
two oxazine dyes, nile blue and cresyl violet, has been investigated using a
modification of the reverse micelle microemulsion method and a procedure
based on the Stber method. A micellar medium provided by the nonionic
surfactant Triton X100 in a hexanol:water mixture and an ethanol:water
mixture, have been used to provide the synthesis medium in each case.
Tetraethoxysilane has been used as the initiator of the polymerization and
condensationreactionsafteritshydrolysisinbasicmediumusingammonium
hydroxide. Dyesilane precursor NPs have been also synthesized in order to
compare their potential advantages against the NPs obtained by the direct
encapsulationoftheoxazinedyes.
SizedistributionandfluorescenceofthesynthesizedNPs,whichwere
monitored using Transmision Electron Microscopy (TEM) and a microplate
reader,respectively,dependonthemolarratioandtotalconcentrationofthe
reagents involved in the synthesis. NPs obtained using the developed
synthesis procedures had sizes below 400 nm in most instances and the best
luminescentpropertieswereobservedforNPswithsizesrangingfrom100to
300nm.Lowersizesresultinadecreaseinthefluorescenceintensitiesofthese
nanomaterials.ParametersrelatedwiththeluminescencefeaturesoftheseNPs
were calculated in order to compare the feasibility of both synthesis
approaches.Therepeatabilityofthereversemicellemicroemulsionprocedure
performedindifferentdaysgavearelativestandarddeviationof10%forthe
fluorescenceintensityvalues.
95
Captulo2
1. Introduction
Theuseofnanomaterialsishavingahugeimpactinthebioanalysis
field [19], being their composition and physicochemical properties the
mainfactorstochoosethedetectionsystembestsuitedforeachassay.The
useofsilicaNPsaslabelsisinterestinginbioassayswithopticaldetection
because of the transparency of silica to visible light. Other desirable
properties are: 1) silica polymerization chemistry is well known, 2) silica
material is not microbiologically degraded, and 3) porosity or swelling
changesofsilicaNPsdonothappenatmoderatepHvariations.However,
the porosity of these materials can originate drawbacks in their
performanceaslabelsduetolossesoftheencapsulatedsubstancesasitwill
bediscussedbelow.
Differentsilicaprecursors,suchastetramethoxysilane(TMOS)and
tetraethoxysilane (TEOS) are commonly used to synthesize silica NPs.
These precursors undergo hydrolysis and polycondensation reactions,
which result in the formation of monodisperse spherical silica particles
[10].TherearetwogeneralroutestosynthesizesilicaNPs,namelyreverse
micelle microemulsion and Stber methods. The reversemicelle
microemulsion method relies on the formation of a waterinoil
microemulsionformedbyasmallamountofwater,anorganicsolventand
asurfactant.Waternanodropletsinsidethereversemicellesactasreactors
inwhichthegrowthofsilicaNPstakesplace.TheStbermethodinvolves
the hydrolysis of the silica precursor in an alcohol/water mixture and the
silicic acid formed nucleates and condenses to give rise to spherical
monodisperseNPs.Thechoiceofeverysynthesisapproachdependsonthe
physicochemical properties of the species to be encapsulated [2, 4, 6]. In
96
Sntesisdenanopartculasdesliceysuaplicacin
97
Captulo2
signalsfromsamplematrix,whichusuallyhappensatshortwavelengths.
Analternativeistheuseoflongwavelengthfluorophores,suchasorganic
dyes (cyanines, oxazines, alexa dyes) and lanthanide and ruthenium
chelates[1618].
The work presented here encompasses different approaches to
synthesize longwavelength emitting silica NPs doped with cresyl violet
acetate and nile blue chloride for the first time. Two different solgel
methods, based on the modification of the reversemicelle microemulsion
and Stber approaches and on the use of the silane precursor TEOS and
ammonium hydroxide, have been developed for the direct encapsulation
of the fluorophore. The fluorescence of the NPs obtained by the first
methodhasbeenimprovedintheabsenceofcyclohexane,whichisusedin
the conventional procedure. The reversemicelle microemulsion method
has been also applied to the encapsulation of a newly synthesized silane
precursorfromnilebluechlorideusingglutaraldehydeand3aminopropyl
triethoxysilane (APS). The influence of the different reagents on the
featuresoftheNPsformedinbothsynthesisprocedureshasbeenstudied
andoptimizedbymeasuringthefluorescenceintensityusingamicroplate
reader and the NP size by Transmission Electron Microscopy (TEM). The
luminescent features of the NPs synthesized by both methods have been
comparedinordertoselectthesynthesisproceduremoresuitabletoobtain
nanomaterialsusefulasstablefluorescentreagentsforanalyticalpurposes,
such as labels in fluoroimmunoassays. Some properties of these long
wavelength emitting NPs, such as photodegradability, chemical stability
anddyeleakagearealsodiscussed.
98
Sntesisdenanopartculasdesliceysuaplicacin
2.Experimental
2.1.Instrumentation
A 1420 Multilabel counter Victor 3V microplate reader (Perkin
Elmer and Analytical Sciences, Wallac Oy, Turku, Finland) was used to
perform
fluorescence
measurements.
Different
filters
(nominal
2.2.Reagents
All
reagents
were
of
analytical
grade.
3Aminopropyl
99
Captulo2
2.3.Procedures
2.3.1. Synthesis of cresyl violet and nile bluedoped silica NPs by a modified
reversemicellemicroemulsionmethod
Thesynthesiswasperformedaccordingtothefollowingprocedure:
an amount of Triton X100 (510 530 mg or 0.79 0.82 mmol) was
dissolvedin9.6ml(0.53mol)ofdistilledwaterbystirringvigorouslythis
mixture for 5 min. Then, a volume of 100 l (0.44 mmol) of TEOS was
addedandthesolutionwasstirredfor5min.Avolume(1.8ml)of103M
(1.8 mol) cresyl violet or nile blue was added and the mixture stirred
againfor5min.Afterwards,3ml(0.024mol)ofhexanolwereaddedand
the microemulsion formed was stirred for 15 min. Concentrated
ammoniumhydroxide(70l,0.9mmol)wasthenaddedandthemixture
stirredfor5mintostarttheTEOShydrolysisandcondensationreactions.
Themixturewasthenplacedinathermostatedtankat35Cfor7.5hinthe
dark.
The reaction mixture of each fluorophoredoped NPs, which was
composedbytwophasesclearlydifferenciated,wascentrifugedfor5min
at 2.000 rpm to complete the separation of two phases. The upper phase,
which was strongly colored, was extracted and 5 7 ml of acetone were
100
Sntesisdenanopartculasdesliceysuaplicacin
2.3.2.SynthesisofcresylvioletdopedNPsusingaStbermethodbasedprocedure
Toavolumeof25mlofethanol(0.43mol)wereadded500lof103
M(0.5mol)cresylviolet.Then,1ml(4.4mmol)ofTEOSand1.5ml(19.5
mmol) of concentrated ammonium hydroxide solution were added. The
mixturewasstirredfor1houratroomtemperature.Afterthistime,itwas
centrifuged at 3.000 rpm for 10 minutes. The NPs synthesized were
purified by washing them with ethanol and water for several times as
mentionedaboveforthereversemicellemicroemulsionmethod.
2.3.3.SynthesisofNPsusingnileblueAPSprecursorasfluorophore
A volume (12 ml) of 8.9x 104 M (10 mol) nile blue solutionwas
mixedwith100lof1%glutaraldehyde(10mol)aqueoussolutionand,
immediately after, 20 l (85 mol) of APS were added and the mixture
stirred for 5 min before its use in the reversemicelle microemulsion
method above described. A volume (1.2 ml) of the reaction mixture,
101
Captulo2
withoutanyadditionalpurificationstep,wasaddedtothemixtureinstead
oftheunchangedfluorophore.
2.3.4.CharacterizationofcresylvioletandnilebluedopedsilicaNPs
intensitymeasurements.TEMexperimentswerecarriedoutbyspotting10
ldropsofNPsuspensionsinethanolontothecoppergrids,whichwere
placedaboveafilterpaperandlettodryatroomtemperatureforseveral
minutes.Fluorescencemeasurementswereobtainedbydispensingaliquots
of 200 l of NP suspensions onto microwells in triplicate and using the
filters above described to choose the adequate excitation and emission
wavelengths.
2.3.5.FluorescencestabilityofoxazinedopedsilicaNPs
SilicaNPsweredividedinfive1mlaliquotsinEppendorftubes.
All of them were washed several times with ethanol and water until the
supernatantspresentedafluorescenceintensitycorrespondingtotheblank
signaland,then,NPswerestoredat4C.Atthetimeoftheperformanceof
the fluorescence measurements, each aliquot was sonicated for 30 s and
thencentrifugedat10.000rpmfor5mintoremovethesupernatant.Then,
theywereagainreconstitutedandredispersedinwater.Avolume(200l)
oftheNPsuspensionwasaddedtoamicrowellplateandthefluorescence
intensitywasmeasuredintriplicate.
102
Sntesisdenanopartculasdesliceysuaplicacin
2.3.6.PhotobleachingstudyofoxazinedopedsilicaNPs
FreeorganicdyesolutionordyedopedNPdispersionwasplaced
in a quartz cuvette and the fluorescence intensity was measured at the
corresponding maximum excitation and emission wavelengths by
continuosly irradiating the cuvette with light from the 450 W xenon arc
lamp of the photon counting spectrofluorimeter. Fluorescence intensity
measurements were monitored at room temperature for 1 h, with an
integrationtimeof1.0min.
3.ResultsandDiscussion
3.1.SynthesisofcresylvioletandnilebluedopedsilicaNPs
3.1.1Optimizationofreversemicellemicroemulsionmethod
As indicated above, the formation ofmicroemulsions involves the
useofasurfactant,anaqueoussolutionandanorganicsolvent.Depending
on the relative amounts of the components, waterinoil microemulsions
(richinorganicsolvent)oroilinwatermicroemulsions(richinwater)can
be formed. In the first case, a cosurfactant, generally a medium or long
hydrocarbon chain alcohol, is often incorporated in the micelles.
Cyclohexane, nhexanol, Triton X100 and water were chosen to develop
the reversemicelle microemulsion method, according to the procedures
previously reported [2, 4, 6, 1214]. These approaches involve continuous
mechanical stirring to originate stable waterinoil microemulsions.
103
Captulo2
However,theuseoftheexperimentalconditionspreviouslydescribeddid
not give rise to satisfactory results for the synthesis of the oxazinedoped
silica NPs. The study of the stirring conditions showed that, after mixing
the reagents in the addition order described in the procedure and then
leavingthemixturetostand,twophasesappeared:anupperphase(richin
organicsolvent)andalowerphase(richinwater).Theupperphase,which
wasstronglycoloured,wasastablewaterinoilmicroemulsioncontaining
thebrightestNPs.
Studiescarriedoutaboutthemicroemulsioncompositionrevealed
that NPs can be synthesized in the absence of cyclohexane, yielding NPs
with similar features (number, size) but with slightly higher fluorescence
intensity than that obtained using cyclohexane. Figure 1 shows TEM
images of the NPs obtained in the presence of different volumes of
cyclohexane. The water volume was also modified to keep constant the
totalvolume.Ascanbeseen,theamountandsizesofNPssynthesizedin
theabsenceofcyclohexane(Figure1a)werecomparabletothoseachieved
using7.5mlofcyclohexane(Figure1c).However,theuseofintermediate
volumes of cyclohexane (Figure 1b) provided larger NPs with wider size
distributions. According to these results, the use of cyclohexane was
precluded and only water, Triton X100 and 1hexanol were the main
componentsofthemicroemulsion.
104
Sntesisdenanopartculasdesliceysuaplicacin
105
Captulo2
106
Sntesisdenanopartculasdesliceysuaplicacin
500000
400000
300000
200000
100000
0
0
100
200
300
400
Figure2.InfluenceofTritonX100/hexanolratioonthefluorescenceintensity
ofnilebluedopedNPs.Experimentalconditions:11.3ml(0.63mol)ofwater,
100l(0.44mmol)TEOS,0.2mlof1x103M(0.2mol)nileblue,3ml(0.024
mol)hexanol,70l(0.9mmol)NH4OH.
The fluorescence intensity and the size of the NPs were evaluated
modifying the temperature and the reaction time. Figure 3 shows the
influenceofthesevariablesinthefluorescenceintensityofnilebluedoped
NPs, finding that the fluorescence increased from 25 to 35 C, but it
decreasedat45C.Althoughthefluorescenceintensitywashigherafter9h
107
Captulo2
ofreaction,thesizeoftheNPsobtainedalsoincreasedso,atimeof7.5h
was chosen as optimum. The NP sizes slightly decreased with increasing
temperaturesintherangeof2545C.Thisstudywasalsoperformedfor
cresylvioletdopedNPsfindingsimilarresults.
700000
35 C
600000
500000
45 C
400000
25 C
300000
200000
100000
0
2
10
Time, h
ThefeaturesofnilebluedopedNPsobtainedafterareactiontime
of7.5handat35CarecomparedinTable1withthefeaturesofsimilar
NPs obtained after 24 h at 25 C. Some parameters were calculated in a
similar way as described elsewhere [19] using 200 l of 0.1 mg/ml NP
dispersionsineachwell.ThenumberofNPswasobtainedbydividingthe
amountofNPsintotheweightofoneparticle,whichwascalculatedfrom
108
Sntesisdenanopartculasdesliceysuaplicacin
Conditions
24h,25C
7.5h,35C
Concentration
0.1mg/ml
0.1mg/ml
Amount(mg)
0.02
0.02
Meandiameter,nm
323
170
Particlevolume,mm3
1.76x1011
2.57x1012
Particlenumber
4.92x108
3.38x109
Intensitya
180927
111708
Intensity/particleratio
3.67x104
3.3x105
Specificintensity
2.09x107
1.29x107
109
Captulo2
Theinfluenceofammoniumhydroxidewasstudiedintherangeof
35 140 l of a commercial concentrated solution (0.45 1.8 mmol). The
number of nile blueNPs formed was scarce at the limits of the assayed
interval, being also the fluorescence intensity obtained quite low (Figure
4a).ThesizeoftheNPsobtainedincreasedintherangeof70610nmof
diameter as NH4OH was increased. The decrease in the fluorescence
intensity at high NH4OH values would be ascribed to the partial
degradation of the fluorofore in the alkaline medium, as it has been
reportedelsewhereforacyaninedye[20].Thevolumechosenasoptimum
was70l(0.9mmol).
TheinfluenceofTEOSwasevaluatedfornileblueNPsintherange
of50800l(0.223.52mmol).IncreasingTEOSvolumes,thesizeofNPs
increasedfrom140to300nm,obtainingsizeslargerthan200nmabove100
l.Theseresultswereobtainedundertheoptimalexperimentalconditions,
exceptingforthefluorophoreamount,whichwastentimeslowerthanthe
optimum value. As it was found that the fluorescence intensity also
increased with the TEOS amount, a compromised solution was adopted
and 100 l (0.44 mmol) of TEOS was chosen as the optimum value to
obtainintermediatesizesandintensities.
Theamountoffluorophoreusedisacriticalvariable.Thevolume
of1x103Maqueousnilebluesolutionwasvariedfrom100lto7.2mlby
adjusting the total volume of the aqueous phase to 11.5 ml with distilled
water, in order to keep constant the initial concentrations of the other
reagents. The influence of this variable on the fluorescence intensity is
shown in Figure 4b, in which can be seen that the maximum value was
obtained using 1.8 mol of nile blue. The decrease in the fluorescence
110
Sntesisdenanopartculasdesliceysuaplicacin
500000
A)
400000
300000
200000
100000
0.0
0.4
0.8
1.2
1.6
2.0
mmol NH4OH
2400000
B)
2000000
1600000
1200000
800000
400000
0
0
Figure4.Influenceoftheamountofammoniumhydroxide(A)andnileblue
(B)addedonthefluorescenceintensityofnilebluedopedNPs.Experimental
conditions:510520mg(0.790.81mmol)ofTritonX100,11.3ml(0.63mol)of
water,100l(0.44mmol)TEOS,3ml(0.024mol)hexanol.InA)0.2mlof1x
103M(0.2mol)nileblue,inB)70l(0.9mmol)ofNH4OH.
111
Captulo2
thattheuseofequimolarratiosofnileblueandglutaraldehyde,andAPS
in an 8.5fold molar excess, gave the best results in terms of fluorescence
intensity of the NPs obtained. The time of reaction of the mixture was
studiedintheintervalof010min,and5minwerefoundtobeenough,
sincelargerirregularparticleswereobtainedatlongerreactiontimes.This
mixture was used for the NP synthesis without any further purification
step. The influence in the fluorescence intensity of the precursor volume
addedtothesynthesisprocedurewasstudiedintherangeof0.62.4ml,
providing1.2mlofprecursorthehighestvalue.ThemeansizeoftheNPs
synthesizedbyusing1.2mlofprecursormixturewas150nm,decreasing
thesizeathighervolumes.Figure1gshowstheaspectoftheNPsobtained
using 1.2 ml of precursor, which are not completely spherical, although
theyhaveauniformsizewitharelativestandarddeviationaround10%.
3.1.2.OptimizationofthesynthesisbytheStbermethod
112
Sntesisdenanopartculasdesliceysuaplicacin
113
Captulo2
Table2.ComparisonofthepropertiesofcresylvioletdopedNPsobtainedby
theproposedreversemicellemicroemulsionandStbermethods
Reversemicelle
microemulsionmethod
Stbermethod
Concentration,mg/ml
0.1
0.1
Particleamount,mg
0.02
0.02
Diameter,nm
178
133
2.94x109
7.06x109
125563
10138
Intensity/particleratio
4.27x105
1.44x106
Specificintensity
1.45x107
1.17x106
Particlenumber
Intensitya
Measurementswereperformedusingmicroplates(200l)andexcitationand
emissionfiltersof531/25and620/8nm,respectively
3.2.FluorescencespectraofcresylvioletandnilebluesilicaNPs
The spectral features of the synthesized NPs were compared to
those showed by pure dyes and by bare silica NPs. Figure 5 shows the
emission spectra obtained at the maximum excitation wavelength of each
fluorophore,inwhichcanbeseenthatthereisnotappreciablefluorescent
signalfrombaresilicaNPs.Theemissionbandsoftheoxazinedopedsilica
NPs show a slight shift of approximately 9 nm towards shorter
wavelengths.Thisbehaviourisinaccordancetotheresultsobtainedafter
encapsulating other fluorophores, such as fluorescein isothiocyanate or
ruthenium dyes [6], which experienced shifts towards shorter and longer
wavelengths,respectively.Thisfactcouldbeascribedtotheinteractionof
the dyes, which are cationic, with the negatively charged silanol groups
fromthesilicamatrix.Figure5alsoshowsthattheemissionspectrumfor
114
Sntesisdenanopartculasdesliceysuaplicacin
nile blueAPSdoped NPs is practically the same as that obtained for nile
bluedopedsilicaNPs.
800
A)
600
33
400
200
1
1
0
580
600
620
640
660
680
700
720
(nm)
2000
B)
22
1500
33
1000
500
11
0
640
660
680
700
720
(nm)
Figure 5. Emission spectra in distilled water of: bare silica NPs (A.1, B.1), 0.07 M
pure cresyl violet (A.2) and 0.5 M nile blue (B.2) dye solutions, and cresyl violet(A.3) and nile blue-doped (B.3) NPs, respectively.
3.3.FluorescencestabilityofNPs
ThesuspensionsofcresylvioletandnileblueNPsweredividedin
fivealiquotsof1mleach,whichwerestoredat4C.Attheassaytime,NPs
were washed, centrifuged and redispersed in water. The fluorescence
intensitywasmeasuredthesamedaythatNPsweresynthesizedand1,2,5
and9dayslater,usinginallinstancesthesamenumberofwashestoeach
aliquot (three times with 1 ml of ethanol and four times with distilled
water), until supernatant fluorescence close to blank signal. Under these
conditions,thefluorescenceintensityremainedalmostconstantforatleast
9dayswithoutappreciablechangesinthefluorescenceintensitycausedby
dyeleakage.ThefluorescenceintensityfromnileblueandnileblueAPS
dopedNPsdecreasedafterthesevenwashes,beingthedecreaseofthefirst
115
Captulo2
NPs two fold that of the second ones, which would be ascribed to the
covalentbindingofthefluorophoretothesilanereagent,whichminimizes
dyeleakage.
OncetheNPsweretotallypurified(supernatantfluorescenceclose
toblanksignal),theyweresubjectedtoadditionalsubsequentwasheswith
1mlofdistilledwatertostudythepotentialdyeleakagefromthecoreof
NPs, finding that the fluorescence of both nile blue and nile blueAPS
dopedNPsremainedalmostconstant.ThestabilityofnilebluedopedNPs
couldbeascribedtotheabovementionedfactthatnileblueisacationicdye
andwouldinteractwiththenegativesilanolgroupsfromsilicamatrix.In
thisway,dyeleakagewouldbelessfavouredthanforanionicdyes,suchas
Cy5,whicharemorepronetorepulsionforceswiththesilanolgroupsfrom
silicamatrix[12].
3.4.Photostabilityexperiments
ToinvestigatethepotentialphotobleachingofthesynthesizedNPs
in aqueous solution, they were irradiated for 1 h and the results were
comparedtothoseobtainedforpuredyesolutions(Figure6).Theintensity
ofpurecresylviolet(Figure6a,curve1)andnileblue(Figure6b,curve1)
dropped until the 72% and the 65%, respectively, of the initial intensity.
However, the fluorescence intensity of both cresyl violet NPs (Figure 6a,
curve 2) and nile bluedoped NPs (Figure 6b curve 2) remained almost
constant and the NPs prepared by using the nile blueAPS precursor
(Figure6b,curve3)experiencedadecreasebya10%oftheinitialintensity.
SimilarresultshavebeenreportedinrecentlysynthesizedNPscontaining
116
Sntesisdenanopartculasdesliceysuaplicacin
Cy5[12]andfluorescein[19],whichconfirmtheincreasedphotostabilityof
encapsulateddyesduetotheprotectionthatsilicamatrixconfersthem.
A)
1.0
0.8
0.6
0.4
1000
2000
B)
0.8
1
0.6
0.4
3000
TIME (s)
1.0
1000
2000
3000
TIME (s)
Figure6.Photobleachingexperimentsforpurecresylviolet(A.1)andnileblue
(B.1)solutions,respectively;cresylviolet(A.2)andnilebluedoped(B.2)NPs,
respectively;andnileblueAPSdopedNPs(B.3).
4.Conclusions
Theworkpresentedherereportsthesynthesisofcresylvioletand
nile bluedoped silica NPs for the first time. The emission at long
wavelengths of these NPs is a useful option to avoid the potential
interferences of static background signals from sample matrix. The
systematic study of the experimental variables involved in the synthesis
process has given rise to the achievement of NPs with homogeneous size
and high and stable fluorescence intensity. The modified reverse
microemulsion method proposed precludes the use of cyclohexane as
organicsolvent,whichisreplacedby1hexanol.Theuseoftheoxazinedye
and the APS precursor to obtain the NPs has shown that the second one
117
Captulo2
Acknowledgments
References
(1)
(2)
J.Yan,M.C.Estvez,J.E.Smith,K.Wang,X.Ho,L.Wang,W.Tan.
Dyedopednanoparticlesforbioanalysis.Nanotoday(2007)2,4450.
(3)
118
Sntesisdenanopartculasdesliceysuaplicacin
(4)
(5)
(6)
(7)
(8)
(9)
(10)
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(12) X.He,J.Chen,K.Wang,D.Qin,W.Tan.Preparationofluminescent
Cy5 doped coreshell SFNPs and its application as a nearinfrared
fluorescentmarker.Talanta(2007)72,15191526.
(13) X. L. Chen, J. L. Zou, T. T. Zhao, Z. B. Li. Preparation and fluoro
immunoassay application of new redregion fluorescent silica
nanoparticles.J.Fluoresc.(2007)17,235241.
(14) W. Yang, C. G. Zhang, H. Y. Qu, H. H. Yang, J. G. Xu. Novel
fluorescent silica nanoparticle probe for ultrasensitive immune
assays.Anal.Chim.Acta(2004)503,163169.
(15) S.R.Hu,J.M.Liu,T.L.Yang,H.Z.Lin,J.L.Huang,Q.W.Lin,G.H.
Zhu,X.M.Huang.Determinationofhumanalphafetoprotein(AFP)
bysolidsubstrateroomtemperaturephosphorescenceenzymelinked
immune response using luminescent nanoparticles. Microchim. Acta
(2005)152,5359.
(16) A. GmezHens, M. P. AguilarCaballos. Longwavelength fluoro
phores: new trends in their analytical use. Trends Anal. Chem. (2004)
23,127136.
(17) I.Hemmil,V.Laitala.Progressinlanthanidesasluminescentprobes.
J.Fluoresc.(2005)15,529542.
(18) A. GmezHens, M. P. AguilarCaballos. Terbiumsensitized
luminescence: a selective and versatile analytical approach. Trends
Anal.Chem.(2002)21,131141.
(19) N. Nakamura, M. Shono, K. Ishimura. Synthesis, characterization,
and biological applications of multifluorescent silica nanoparticles.
Anal.Chem.(2007)79,65076514.
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Sntesisdenanopartculasdesliceysuaplicacin
(20) J.F.Bringley,T.L.Penner,R.Wang,J.F.Harder,W.J.Harrison,L.
Buonemani.Silicananoparticlesencapsulatingnearinfraredemissive
cyaninedyes.J.ColloidInterf.Sci.(2008)320,132139.
(21) L. M. Rossi, L. Shi, F. H. Quina, Z. Rosenzweig. Stber synthesis of
monodispersed luminescent silica nanoparticles for bioanalytical
assays.Langmuir(2005)21,42774280.
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Sntesisdenanopartculasdesliceysuaplicacin
Heterogeneousimmunoassayforsoyproteindetermination
usingnilebluedopedsilicananoparticlesaslabelsandfront
surfacelongwavelengthfluorimetry
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens
DepartmentofAnalyticalChemistry,InstituteofFineChemistryandNanochemistry
(IAQFN).CampusofRabanales.MarieCurieBuilding(Annex).Universityof
Cordoba.14071Cordoba.Spain.
Abstract
A longwavelength fluoroimmunoassay for the determination of soy
proteinisreportedforthefirsttimeusingaconjugatecomposedofantisoyprotein
antibodies bound to nileblue doped silica nanoparticles (NPs). These NPs have
been synthesized by a reversemicelle microemulsion method and functionalized
by using 3(aminopropyl)triethoxysilane (APS) and 3(trihydroxysilyl)propyl
methylphosphonate (THPMP) to avoid NP aggregation. The tracer has been
obtained by linking the functionalized NPs with antisoy protein antibodies
previouslyoxidisedwithsodiumperiodate.Theimmunoassayhasbeendeveloped
in 96well microplates using a heterogeneous competitive format with antibody
capture.Soyproteinsareimmobilisedontothewellsandbovineserumalbuminis
added to block the surface, thus minimising nonspecific binding. After washing,
the microplates can be stored ready to use. At the analysis time, soy protein
standards or sample and tracer are added and incubated and, after the
corresponding washing and drying steps, the fluorescence is measured onto the
solidsurfaceatex620andem680nm.Themethodfeaturesadynamicrangeof0.1
10 mg L1 and a detection limit of 0.05 mg L1. The precision of the method has
been assayed at 0.5 and 5 mg L1 protein concentrations, obtaining the values of
relativestandarddeviationof9.6%and6.1%,respectively.Thisnewimmunoassay
has been applied to the analysis of food containing soy protein and the results
obtainedhavebeencomparedtothoseprovidedbyacommercialELISAkitwith
no statistically differing results. Also, a recovery study has been performed,
providingpercentagesintherangeof81.5111.0%.
123
Captulo2
1.Introduction
In recent years, the global consumption of soy foodstuffs has
increased because of their reported beneficial effects on nutrition and
health,suchasadecreaseoftheplasmacholesterol,preventionofcancer,
diabetesandobesity,andprotectionagainstbowelandkidneydisease[1].
Soybean proteins are used as additives in human foodstuffs, thus being
marketedasdifferentcommercialformulations,suchassoyflour,protein
concentrates and isolates. These formulations are used to obtain better
mechanical properties in soy milks, drinks, tofu and vegetarian meat
substitutes and a number of new food varieties is being developed.
However,soybeanscontainproteinsthatcanproduceallergicresponsesin
some people. The safe choice of processed foods free from soy proteins
may become more critical for these patients because these proteins and
their derivatives have been increasingly incorporated in a number of
processedfoods.Theallergenicityoftheseproteinslargelydependsonthe
foodprocessing,e.g.hightemperaturetreatmentsandconjugationtosome
organiccompounds,suchaspolysaccharides[2],amongothers.
Nativesoyproteinsareglobularproteins,mainlyconstitutedby7S
and 11S (glycinin and conglycinin) fractions [3], which have been
characterized
and
determined
by
liquid
chromatography
[4],
124
Sntesisdenanopartculasdesliceysuaplicacin
125
Captulo2
requiredforthedevelopmentofELISAassays,sincetheuseoftheenzyme
conjugateandsubstratesolutionsisavoided.Todate,alimitednumberof
immunoassays have been described which make use of biofunctionalized
dyedoped silica nanoparticles [16]. These assays mainly involve the
encapsulation of fluorescein and rhodamine derivatives, and ruthenium
and lanthanide chelates to obtain luminescent silica NPs. The results
obtained in these methods have shown that the sensitivity is greatly
increased when compared with the corresponding immunoassays
performedwithdirectfluorophorelabeling.
Tothebestofourknowledge,themethodreportedhereisthefirst
heterogeneous immunoassay described for soy protein determination in
food samples using NPs as labels. Two competitive formats involving
antibodyandantigencapturewereassayed,butonlythefirstone,inwhich
soy proteins were immobilised on the wells of a microplate, gave rise to
satisfactoryresults.
Themethodhasbeenappliedtotheanalysisoffruitandsoyjuice,
soy yoghourt and milk samples and the results obtained have been
compared to those provided by a commercial ELISA with no statistically
relevantdifferences.Also,thedetectionlimitusingthereportedmethodis
about10timeslowerthantheaffordedbythecommercialELISA.
2.Experimental
2.1.Instrumentation
A 1420 Multilabel counter Victor 3V microplate reader (Perkin
Elmer and Analytical Sciences, Wallac Oy, Turku, Finland) was used to
perform
fluorescence
measurements.
126
Two
filters
(nominal
Sntesisdenanopartculasdesliceysuaplicacin
2.2.Reagents
All the reagents were of analytical grade and used as supplied by
the manufacturer. Tetraethyl orthosilicate (TEOS) and Triton X100 were
obtained
from
Fluka
(Germany).
Nile
Blue
chloride,
127
Captulo2
5.5 and pH 4.8), and carbonate (0.05 M, pH 9.5) buffer solutions were
preparedbydissolvingtheappropriateamountofthesesaltsandadjusting
the pH with either hydrochloric acid or sodium hydroxide when
appropriate. A commercial ELISA kit (ELISA SYSTEMS, Windsor,
Australia)forthedeterminationofsoyprotein11wasusedforcomparative
purposes.
2.3.Procedures
2.3.1.SynthesisofaminofunctionalizedNBdopedsilicaNPs
TheprocedureusedtosynthesizeNBdopedsilicaNPsissimilarto
a reversemicelled microemulsion method previously reported [15], but it
has been modified in the last step to provide the doped silica NP surface
withaminogroupstobelinkedtoantisoyproteinantibodies,inorderto
giverisetotheconjugateusedastracer.
Briefly,anamountofTritonX100(510530mgor0.790.82mmol)
wasdissolvedin9.6mL(0.53mol)ofdistilledwaterbystirringvigorously
thismixturefor5min.Then,avolumeof100L(0.44mmol)ofTEOSwas
addedandthesolutionwasstirredfor5min.Avolume(1.8mL)of103M
(1.8mol)NBsolutionwasaddedandthemixturestirredagainfor5min.
Afterwards, 3 mL (0.024 mol) of hexanol were added and the
microemulsion formed was stirred for 15 min. Concentrated ammonium
hydroxide(70L,0.9mmol)wasthenaddedandthemixturestirredfor5
mintostarttheTEOShydrolysisandcondensationreactions.Themixture
wasthenplacedinathermostatedtankat25Cfor24hinthedarkand,
afterwards,itwascentrifugedfor5minat2000rpmtoseparatethephases
involved.Theupperphase(about3mL)wastransferredtoa10mLbeaker
128
Sntesisdenanopartculasdesliceysuaplicacin
129
Captulo2
2.3.3.Preparationofsoystandard
The procedure used involves a denaturationrenaturation process,
according to the procedure indicated by the antisoy protein antibody
supplier,asfollows:anamountofsoyproteinisolate(50mg)wasplacedin
a glass test tube and mixed with 3 g of urea, 1 mL of 0.001 M TRISHCl
buffersolutionofpH8.6,0.1mLof2mercaptoethanolandwater.Thetotal
volume of the reaction mixture did not exceed 5 mL. The test tube was
sealedwithacapandaluminumfoilandthetubewasplacedinaboiling
water bath for 2 h. After this time, the tube was cooled for 5 min in cold
waterandthereactionmixturewasdilutedto10mLwithdistilledwater.
This solution can be stored at 4 C in the fridge for a month. Working
standard solutions were daily prepared by diluting this stock solution in
phosphatebuffersolution.
2.3.4.Determinationofsoyprotein
A volume (80 L) of a 5 mg L1 soy protein solution prepared in
carbonatebuffersolutionwasaddedtoeachwell,andthemicroplatewas
incubated overnight at 4 C. Afterwards, wells were washed three times
withdistilledwaterandthen,80Lofa0.01%BSAsolutionpreparedin
phosphatebufferwereaddedtoeachwell.Themicroplatewasstirredfor1
130
Sntesisdenanopartculasdesliceysuaplicacin
2.3.5.Determinationofsoyproteininfoodsamples
and1.7goffruitandsoyjuice)offoodsamplewastreatedinthesameway
asdescribedaboveforsoyproteinstandards.
The sample extracts were diluted (1:2000) in phosphate buffer
solution and 225 L of this solution were analysed following the above
mentionedprocedureforsoyproteindetermination.
3.Resultsanddiscussion
3.1.
Choiceandoptimizationoftheimmunoassaysystem
The synthesis and optical properties of NBdoped silica NPs used
to obtain the tracer have been recently described [15]. These NPs feature
lowphotobleachingphenomenabecauseoftheprotectionthatsilicamatrix
confersthemandgoodstabilityowingtotheinteractionofthefluorophor
NBwithsilicamatrix.However,thesurfacefunctionalizationtointroduce
131
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132
Sntesisdenanopartculasdesliceysuaplicacin
development of the reaction. The reaction time was also studied from 30
minto2h,findingthat1.5hprovidedasilicashellincorporatingenough
amino groups to perform the further conjugation chemistry. Under these
conditions,theNBdopedsilicaNPsobtainedfeaturedameandiameterof
257nm,measuredbyTEM(Figure1).
Figure1.TEMimageoffunctionalizedNBdopedsilicaNPs.
133
Captulo2
134
Sntesisdenanopartculasdesliceysuaplicacin
135
Captulo2
30000
16000
B)
A)
1
14000
25000
1
2
12000
2
20000
10000
3
15000
8000
3
4
6000
10000
5
4000
5000
2000
4
5
0
0
-0.02 0.00
0.02
0.04
0.06
0.08
0.10
0.12
5
10
15
20
25
30
35
40
45
[BSA], (w/v%)
[tracer], nM
Figure2.InfluenceofBSA(A)andtracer(B)concentrationsontheassayinthe
presenceofdifferentsoyproteinconcentrations(mgL1):1)0.0;2)0.1;3)1.0;4)
5.0; 5) 10.0. [tracer] = 20 nM in (A). [BSA] = 0.01% in (B). Other experimental
conditionsinboth(A)and(B)are:[immobilizedsoyprotein]=5mgL1,assay
incubationtime=1.5h,temperature=37C.
3.2Analyticalfeatures
Fluorescenceintensitymeasurementswereperformedbyselecting
the most appropriate filters to fix the maximum excitation and emission
wavelengthsforthetracer,ex620andem680nm.Themethodpresenteda
dynamicrangeof0.110mgL1(Figure3).
136
Sntesisdenanopartculasdesliceysuaplicacin
16000
14000
12000
10000
8000
6000
4000
2000
0
0.001
0.01
0.1
10
-1
[soy protein], mg L
Fig.3.Calibrationcurveobtainedunderoptimumconditions.
y y0 (
1
a
( x x 0 )
e b
) ,
whereyisthefluorescenceintensityandxisthedecimallogarithmofsoy
protein concentration expressed in mg L1. The values of the regression
parametersa,b,x0andy0were(1.120.04)x104,0.170.02,0.130.02,
and (1.9 0.2) x 103, respectively. The value of the regression coefficient
was 0.9989. The detection limit, calculated according to IUPAC
recommendations [21], was 0.05 mg L1, which is about ten times lower
than that provided by the ELISA kit used as reference method [11]. The
precision, expressed as the percentage of relative standard deviation and
assayedattwodifferentsoyproteinconcentrations,0.5and5mgL1,gave
valuesof9.6%and6.1%,respectively.
137
Captulo2
3.3 Applications
Themethodwasappliedtotheanalysisofmilk,yoghourtandfruit
juice samples containing soy proteins. The sample treatment includes the
same procedure used for soy protein standard preparation to obtain soy
protein with a suitable conformation to be recognized by the antibody.
Similarprocedureshavebeenusedforthetreatmentofsomefoodsamples
in immunoassays intended for soy protein determination [22]. These
sampleswerealsoanalysedusingacommercialELISAkit[11],thusbeing
extracted using a commercial extraction solution for samples containing
polyphenols,accordingtotheprotocolgivenbythemanufacturer.
Table 1 shows the soy protein content found by the reported
immunoassayandtheELISAmethod,whichwerecomparedbyapairedt
test carried out at a 95% significance level, finding that there were not
statisticallyrelevantdifferencesbetweenthem.
138
Sntesisdenanopartculasdesliceysuaplicacin
Table1.Determinationofsoyproteincontentinfoodsamples
Sample
Reportedmethoda,b
CommercialELISAa,b
746
843
8.40.7
10.50.3
644
603
Soymilk
Fruitandsoyjuice
Soyyoghourt
MeanSD(n=3)
Units:fruitandsoyjuice,soyyoghourtareexpressedingkg1,soymilkunits
aregL1
Table2.Recoveryofsoyproteinaddedtofoodsamples
Addeda,b
Founda,b
Recovery(%)
Soymilk
24
72
123
272
694
11010
111.0
95.7
89.4
Fruitandsoyjuice
2.7
8.1
13.5
2.20.2
7.70.6
121
81.5
95.1
88.9
Sample
Soyyoghourt
23
212
91.3
65
614
93.2
113
957
84.1
aMeanSD(n=3)
bUnits:fruitandsoyjuice,soyyoghourtareexpressedingkg1,soymilkunits
aregL1
139
Captulo2
4. Conclusions
The reported method can be regarded as a useful alternative to
enzymelinkedimmunosorbentassays,sincetheuseofenzymeconjugate
and substrate solutions are avoided, thus decreasing the number of steps
required. In addition, the detection limit achieved with the reported
method is lower than that provided by some ELISA assays described for
thispurpose[11].Theuseofthe96wellformatensurestheautomationof
the assay together with a relativelyhigh sample throughputcompared to
othermethods,whichincludesequentialoperations.Proxyplatesallowthe
use of lower sample and tracer volumes than conventional 300L wells,
whichcontributetominimizethecostsassociatedtotheimmunoassay.
The results obtained for the analysis of real food samples using
the reported method are in accordance with those provided by an ELISA
methodusedasareference,whichindicatesthepracticalusefulnessofthe
developedmethod.
Acknowledgements
Authors gratefully acknowledge financial support from the
MICINN(GrantNo.CTQ200908621),fromtheJuntaofAndalucia(Grant
No. P09FQM4933) and from the FEDERFSE Program (Grant No. P09
FQM4933). J. Godoy Navajas thanks the Junta of Andalucia (Grant No.
P09FQM4933)forthefinancialsupportofhispredoctoralfellowship.
140
Sntesisdenanopartculasdesliceysuaplicacin
References
(1)
(2)
E.F.E.Babiker,A.Hiroyuki,N.Matsudomi,H.Iwata,T.Ogawa,N.
Bando, A. Kato. Effect of polysacchraride conjugation or
transflutaminase treatment on the allergenicity and functional
propertiesofsoyprotein.J.Agr.FoodChem.(1998)46,866871.
(3)
(4)
(5)
(6)
H.B.Krishnan,W.S.Kim,M.S.Kerley.Allthreesubunitsofsoybean
Conglycininarepotentialfoodallergens.J.Agr.FoodChem.(2009)
57,938943.
(7)
D.L.Brandon,M.Friedman.Immunoassaysofsoyproteins.J.Agr.
FoodChem.(2002)50,66356642.
(8)
(9)
141
Captulo2
(15) J.GodoyNavajas,M.P.AguilarCaballos,A.GomezHens.Synthesis
and characterization of oxazinedoped silica nanoparticles for their
potentialuseasstablefluorescentreagents.J.Fluoresc.(2010)20,171
180.
142
Sntesisdenanopartculasdesliceysuaplicacin
(17) V.S.Prisyaznoy,M.Fusek,Y.B.Alakhow.Synthesisofhighcapacity
immunoaffinity
sorbents
with
oriented
immobilized
(18) J.Yan,M.C.Estvez,J.E.Smith,K.Wang,X.He,L.WangandW.
Tan. Dyedoped nanoparticles for bioanalysis. NanoToday (2007) 2,
4450.
(21) G.L. Long, J.D. Winefordner. Limitof detection: a closer lookat the
IUPACdefinition.Anal.Chem.(1983)55,712A724A.
143
Sntesisdenanopartculasdesliceysuaplicacin
Determinationofmonensininmilksamplesbyfrontsurface
longwavelengthfluoroimmunoassayusingnilebluedoped
silicananoparticlesaslabels
JuanGodoyNavajas,MariaPazAguilarCaballos,AgustinaGmezHens
DepartmentofAnalyticalChemistry.InstituteofFineChemistryandNanochemistry
(IQFN).CampusofRabanales.AnnextoMarieCurie(C3)Building.Universityof
Cordoba.14071Cordoba.Spain.
Abstract
A heterogeneous immunoassay for monensin determination in milk
samplesusingatracerformedbyantimonensinantibodiesboundtonileblue
(NB)doped silica nanoparticles (NPs), 96well microplates as solid supports
and longwavelength fluorescence measurements is described for the first
time. The assay relies on the competition of the monensin present in the
samples with a monensinbovine serum albumin conjugate, which was
immobilized onto the well surface, for the active sites of antimonensin
antibodies.Aftersubsequentincubationandwashingsteps,thefluorescenceof
the bound tracer fraction is measured onto the dry surface of the well. An
antigen capture format was also assayed by immobilizing antisheep IgG
previously to the incubation of sheep antimonensin antibodies and using a
tracer formed by monensin bound to nile bluedoped silica NPs, which
competeswiththeanalyteforbindingtheimmobilizedantibody.Althoughthe
fluorescence signal obtained in both formats can be correlated to the analyte
concentration,betterresultswereobtainedusingtheantibodycaptureformat.
Aftertheoptimizationofthesystemusingthisformat,themethodfeaturesa
detectionlimitof0.015gL1andadynamicrangefrom0.05to5gL1.The
precision,assayedattwodifferentanalyteconcentrations,0.2and1gL1,and
expressed as relative standard deviation, gave values of 5.9% and 4.0%,
respectively. The method was satisfactorily applied to the analysis of milk
samples,whichonlyrequiredasimpleextractionstepinordertoremovethe
proteinsfromsamples,givingrecoveriesintherange83.3107.5%.
145
Captulo2
1.Introduction
146
Sntesisdenanopartculasdesliceysuaplicacin
methodsrequiretheavailabilityofasophisticatedandexpensivetechnique
and, sometimes, are timeconsuming owing to the extraction and further
cleanup procedures involved. For this reason, the use of screening
methods plays two essential roles: in one hand, the number of samples
subjectedtoconfirmatoryanalysisislowerand,ontheother,thecostofthe
analysis is reduced. Immunoassays, whenever used in a quantitative or
semiquantitativeway,haveshowntheirusefulnessasscreeningmethods
for antimicrobial residues in foodstuffs [14 21]. Several commercial and
noncommercial enzymelinked immunosorbent assays (ELISAs) have
been described for the determination of monensin in biological [14 18]
andenvironmental[19]samples.Mostofthesemethodsinvolvetheuseof
photometric measurements, reaching detection limits close to 1 ng mL1,
but a lower detection limit (0.06 ng mL1) has been reported using
chemiluminescence
detection
[18].
Also,
timeresolved
fluoroimmunoassaysinvolvingtheuseofaneuropium(III)chelateaslabel
havebeendescribedformonensindetermination[20,21],butthedetection
limit reached is very similar to those obtained using ELISA with
photometricdetection.
The use of functionalized inorganic matrix nanoparticles (NPs) as
alternative labels in immunoassay is a relatively new trend justified by
theirversatilephysicochemicalpropertieswhichallowtheimprovementof
thefeaturesoftheseassays[2225].AmongtheseNPs,dopedsilicaNPs
are a useful option owing to their chemical and thermal stability, fine
dispersion in aqueous solution, transparency to visible light, capability to
encapsulate a wide variety of compounds and relatively inert
environmental behavior, in addition to their large surface area and easy
147
Captulo2
148
Sntesisdenanopartculasdesliceysuaplicacin
2.Experimental
2.1.Instrumentation
A 1420 Multilabel counter Victor 3V microplate reader (Perkin
Elmer and Analytical Sciences, WallacOy, Turku, Finland) was used to
perform
fluorescence
measurements.
Two
filters
(nominal
2.2.Reagentsandsolutions
Allthereagentswereofanalyticalgradeandwereusedassupplied
by the manufacturer. Triton X100, NB chloride, sodium acetate, sodium
chloride, sodium borohydride, monensin sodium salt, anhydrous
Ndimethylformamide (DMF), Nhydroxysulfosuccinimide sodium salt
(sulfoNHS), N(3dimethylaminopropyl)Nethylcarbodiimide hydro
chloride(EDAC),bovineserumalbumin(BSA)andsodiumcarbonatewere
obtained from SigmaAldrich (USA). Tetraethyl orthosilicate (TEOS), (3
aminopropyl)triethoxysilane
(APS),
3(trihydroxysilyl)propylmethyl
149
Captulo2
2.3.Procedures
2.3.1.SynthesisandfunctionalizationofNPs
TheprocedureusedtosynthesizeNBdopedsilicaNPsissimilarto
a reversemicelle microemulsion method previously reported [26, 27].
Briefly:anamountofTritonX100(510530mgor0.790.82mmol)was
dissolvedin9.6mL(0.53mol)ofdistilledwaterbystirringvigorouslythis
mixture for 5 min. Then, a volume of 100 L (0.44 mmol) of TEOS was
150
Sntesisdenanopartculasdesliceysuaplicacin
addedandthesolutionwasstirredfor5min.Avolume(1.8mL)of103M
(1.8 mol) NB was added and the mixture stirred again for 5 min.
Afterwards, 3 mL (0.024 mol) of hexanol were added and the
microemulsion formed was stirred for 15 min. Concentrated ammonium
hydroxide(70L,0.9mmol)wasthenaddedandthemixturestirredfor5
mintostarttheTEOShydrolysisandcondensationreactions.Themixture
wasthenplacedinathermostatedtankat25Cfor24hinthedarkand,
afterwards, it was centrifuged for 5min at 537 x g to separate the phases
involved.Theupperphase(about3mL)wastransferredtoa10mLbeaker
and 40 L of APS and 80 L of THPMP were added to introduce amino
groupsontoNPsurface.Thismixturewasmagneticallystirredduring1.5
hinawaterbathat25C.Afterwards,themicroemulsionwasbrokenby
adding 10 mL of acetone and stirring for 5 min. The supernatant was
discarded and NPs were recovered from the bottom with 8 10 mL of
distilled water. The mixture was centrifuged for 5 min at 9300 x g to
separate the NPs from unreacted reagents. Then, the NP precipitate was
washed with ethanol and water until the fluorescence intensity of
supernatants was similar to the blank and, finally, the NPs were re
dispersedin1mLofphosphatebuffersolution.
2.3.2.PreparationoftracersusingnilebluedopedsilicaNPs
antibodiesormonensintoobtainthetracersrequiredtostudythepotential
determination of this drug using antibody or antigen capture format,
respectively. The synthesis of the NPsantimonensin antibodies tracer
involves an oxidation step of carbohydrate moieties of the antibody
151
Captulo2
152
Sntesisdenanopartculasdesliceysuaplicacin
and the mixture was left to stand for 10 min. An appropriate volume (50
L)ofthismixturewasmixedtogetherwiththeaminofunctionalizedNB
doped silica NPs and 950 L of phosphate saline buffer were added and
theresultingmixturewasincubatedfor2hatroomtemperature.Afterthis
time, the synthesized tracer was washed using absolute ethanol and
phosphate buffer solution, reconstituted in 1 mL of the same phosphate
molarconcentrationsolutionandstoredat4Cuntiluse.
2.3.3.SynthesisofBSAmonensinconjugate
TheuseofaBSAmonensinconjugatewasrequiredtoimmobilize
monensinontothewells.ThecouplingofmonensintoBSAmoleculewas
performedviaacarbodiimidereaction[29]usingthefollowingprocedure:
an amount of monensin (0.2 mmol) was dissolved in 1 mL of dry DMF,
thensulfoNHS(23.0mg,0.1mmol)andEDAC(41.2mg,0.2mmol)were
added in order to activate the carboxylic acid group of monensin. The
mixturewasstirredatroomtemperaturefor4h,andthenwascentrifuged
to remove the precipitate from the acylisourea derivative formed. An
aliquot(250L)oftheactivatedhaptensolutionwasaddeddropwisetoa
stirredBSAsolutionpreparedbydissolvingBSA(50mg)in5mLof0.05M
boratebuffer(pH7.8)andDMF(1.05mL).Thesynthesizedconjugatewas
purified using a HiTrap Desalting column (GE Healthcare) by a similar
procedure to that described above for the synthesis of NPanti monensin
conjugate.
153
Captulo2
methanolto1mLofKomarowskyreagentinglasstesttubes.Thesetubes
weresealedandplacedinawaterbathat80Cfor5min.Theredpurple
color indicative of monensin presence was measured at 520 nm
immediately after cooling the tubes to room temperature. The BSA
concentration in the synthesized conjugate was calculated from the
absorbance of the protein measured at 280 nm. As a convention, the
concentration of the conjugate will be given in terms of the monensin
concentrationfoundbyusingtheabovementionedKomarowskyreaction.
2.3.4.Determinationofmonensin
Toperformtheimmunoassay,avolume(60L)ofapreincubated
154
Sntesisdenanopartculasdesliceysuaplicacin
2.3.5.Analysisofmilksamples
3.Resultsanddiscussion
3.1.Choiceandoptimizationoftheimmunoassaysystem
Two
heterogeneous
competitive
immunoassays,
involving
antibodyorantigencaptureformat,wereassayedtochoosethebestoption
formonensindeterminationusingNBdopedsilicaNPsaslabels.Thefirst
formatwasbasedontheimmobilizationofaBSAmonensinconjugateand
theuseofanantimonensinantibodyNPtracer,whichwassynthesizedby
a similar procedure to that described in an immunoassay for soy protein
determination [27]. The second assay needed a monensinNP tracer,
155
Captulo2
156
Sntesisdenanopartculasdesliceysuaplicacin
25000
IF
10 mg L-1 BSA-monensin
0 mg L-1 BSA monensin
20000
15000
10000
5000
0
0.0001
0.001
0.01
0.1
10
[monensin], g L
-1
157
Captulo2
24000
IF
24000
A)
B)
IF
20000
20000
16000
16000
12000
12000
8000
8000
4000
4000
20
40
60
10
12
14
[BSA-monensin], mg L
[tracer], nM
158
Sntesisdenanopartculasdesliceysuaplicacin
3.2.Analyticalfeatures
Figure3showsthecalibrationcurveobtainedformonensinunder
optimalexperimentalconditionsandmeasuringthefluorescencesignalsat
ex620andem680nm.
22000
IF
20000
18000
16000
14000
12000
0.001
0.01
0.1
10
[monensin], g L
-1
Figure3.Calibrationcurveobtainedunderoptimumconditions.
159
Captulo2
160
Sntesisdenanopartculasdesliceysuaplicacin
3.3.Applications
skimmedandwholemilksamples.Thesampletreatmentwasquitesimple
and consisted in a deproteinization step after which the solvent was
evaporated owing to the lack of compatibility of the organic solvent with
the immunoassay performance. This compatibility was studied using
methanolandethanolandtheyprovedtodecreasethefluorescencesignal
atpercentagesabove3%ofeachsolvent.Theseextractionandevaporation
steps are common to those required by immunoassays for monensin
determination in sample extracts of feedstuff [15, 16] and foodstuff [21]
samples.Themonensinconcentrationinthemilksampleswasdetermined
according to the procedure above described. A recovery study was also
carriedouttovalidatethemethod(Table1),obtainingvaluesintherange
of83.3107.5%.
161
Captulo2
Table1.Recoveriesobtainedformonensinaddedtomilksamples
Monensin
Added/
Founda/
Recovery
Sample
gkg1
gkg1
(%)
Skimmedmilk
1.5
1.60.1
106.7
1.960.09
98.0
4.10.2
102.5
1.5
1.50.1
100.0
2.00.1
100.0
3.40.3
85.0
1.250.07
83.3
1.70.1
85.0
4.30.3
107.5
Semiskimmedmilk
Wholemilk
1.5
MeanSD(n=3)
4.Conclusions
TheresultsobtainedhaveshowntheusefulnessofNBdopedsilica
162
Sntesisdenanopartculasdesliceysuaplicacin
Acknowledgments
Authors gratefully acknowledge financial support from the
MICINN (grant no. CTQ200908621), from the Junta of Andalucia (grant
no. P09FQM4933) and from the FEDERFSE Program (grant no.P09
FQM4933).J.GodoyNavajasthankstheJuntaofAndalucia(grantno.P09
FQM4933)forthefinancialsupportofhispredoctoralfellowship.
References
(1)
OpinionoftheScientificPanelonContaminantsintheFoodChain.
EFSAJ(2008)592,140.
(2)
(3)
(4)
163
Captulo2
(5)
M.Sokolic,M.Pokorny.Comparativedeterminationofsalinomycin
by highperformance liquid chromatography, microbiological and
colorimetric methods in testing production processes and animal
feedpreparations.J.Pharm.Biomed.Anal.(1991)9,10471053.
(6)
and
validation
of
multiresidue
liquid
(8)
U.Vincent,Z.Ezerskis,M.Chedin,C.vonHolst.Determinationof
ionophorecoccidiostatsinfeedingstuffsbyliquidchromatography
tandem mass spectrometry. Part II. Application to cross
contamination levels and nontargeted fee. J. Pharm. Biomed. Anal.
(2011)54,526534.
(9)
(10)
164
Sntesisdenanopartculasdesliceysuaplicacin
(11)
(12)
(13)
drug
residues
in
feedingstuffs
by
liquid
chromatographytandemmassspectrometry.J.Chromatogr.A(2010)
1217,63946404.
(14)
H.Watanabe,A.Satake,M.Matsumoto,Y.Kido,A.Tsuji,K.Ito,M.
Maeda.Monoclonalbasedenzymelinkedimmnosorbentassayand
immunochromatographic rapid assay for monensin. Analyst (1998)
123,25732578.
(15)
MonensinELISAkit(Cat#KA1422V.01),http://www.abnova.com/.
(16)
165
Captulo2
(17)
(18)
M.A.J.Godfrey,M.F.Luckey,P.Kwasowski.IAC/cELISAdetection
of monensin elimination from chicken tissues, following oral
therapeuticdosing.FoodAddit.Contam.(1997)14,281286.
(19)
H.Dolliver,K.Kumar,S.Gupta,A.Singh.Applicationofenzyme
linked immunosorbent assay analysis for determination of
monensin in environmental samples. J. Environ. Qual. (2008) 37,
12201226.
(20)
(21)
V.Hagren,P.Peippo,M.Tuomola,T.Lvgren.Rapidtimeresolved
fluoroimmunoassayforthescreeningofmonensinresiduesineggs.
Anal.Chim.Acta(2006)557,164168.
(22)
(23)
(24)
166
Sntesisdenanopartculasdesliceysuaplicacin
(25)
D.Knopp,D.Tang,R.Niessner.Review:Bioanalyticalapplications
of biomoleculefunctionalized nanometersized doped silica
particles.Anal.Chim.Acta(2009)647,1430.
(26)
(27)
(28)
(29)
(30)
167
CAPTULO 3
CHAPTER 3
Determinacindeantioxidantesenalimentos
fluorimetric
determination
of
food
171
Captulo3
sustratooxidable,retrasasignificativamenteoinhibelaoxidacindedicho
sustrato[2].Estadefinicinsemodificmstarde,en2007,parapasara
definirlos como cualquier sustancia que retrasa, evita o elimina el dao
oxidativoaunamolculadiana[3].Surelacinconlasespeciesreactivas
de oxgeno (ROS) se estableci ese mismo ao mediante la definicin de
Khlebnikov y colaboradores [4], en la que se definen los antioxidantes
como cualquier especie que atrapa directamente ROS o indirectamente
actapararegularlasdefensasantioxidantesoparainhibirlaproduccin
de ROS. Despus de atrapar las ROS, las sustancias antioxidantes deben
ser capaces de formar un nuevo radical estable mediante la formacin de
puentesdehidrgenointramoleculares[5].Losantioxidantesrealizanesta
actividad de diversas formas: 1) como inhibidores de las reacciones de
oxidacin que transcurren mediante radicales libres, en las que actan
inhibiendolaformacinderadicaleslibresdelpidos;2)interrumpiendola
propagacin de las reacciones de autoxidacin; 3) como inhibidores del
oxgeno singlete; 4) mediante accin sinrgica con otros antioxidantes; 5)
como agentes reductores que convierten los hidroperxidos en otras
sustancias ms estables; 6) como agentes quelatantes de metales que
transforman metales prooxidantes (derivados de hierro y cobre) en
productosestablesy,porltimo,7)actuandocomoinhibidoresdeenzimas
prooxidantes (p.ej. lipooxigenasas). Actualmente se est trabajando en la
determinacin de la actividad de los antioxidantes a nivel celular para
expandirladefinicindesustanciasantioxidantesaaquellassustanciasque
activan factores de transcripcin capaces de inducir la expresin de
enzimasconactividadantioxidante[1].
172
Determinacindeantioxidantesenalimentos
o Enzimasprimarias
o Enzimassecundarias
ENZIMTICOS
ANTIOXIDANTES
NATURALES
NOENZIMTICOS
o Cofactores
(coenzimaQ10)
o Vitaminas (A:retinol,C:cidoascrbico,Etocoferol)
o Especiesinorgnicas
(zinc,selenio)
o Carotenoides (caroteno,licopeno,lutena,zeaxantina)
o Compuestosnitrogenados (noprotecos,cidorico)
o Compuestosorganosulfurados (indoles,glutatin)
o Flavonoides (flavonoles,flavanoles,flavonas,flavanonas,isoflavonoides,antocianinas)
o cidosfenlicos (cidoshidroxicinmicos,cidoshidroxibenzoicos)
Figura1.Clasificacindelassustanciasantioxidantesnaturales.
reacciones
de
regeneracin
de
sustancias
antioxidantes,
173
Captulo3
174
Determinacindeantioxidantesenalimentos
OH
OH
OH
OH
butilhidroxianisol
(BHA)
terbutilhidroquinona
(TBHQ)
butilhidroxitolueno
(BHT)
OH
OH
HO
HO
HO
O
O
galatodeoctilo
(GO)
galatodepropilo
(GP)
HO
OH
HO
4hexilresorcinol
Figura2.Estructurasqumicasdealgunosantioxidantessintticos.
175
Captulo3
Ensayosbasadosentransferenciadeelectrones(ET)
176
Determinacindeantioxidantesenalimentos
LosensayosbasadosenreaccionesHATmidenlacapacidaddeun
antioxidante para inhibir los radicales libres (generalmente radicales
perxido)mediantelacesindetomosdehidrgeno.Elmecanismoporel
que se interpreta la accin antioxidante de un compuesto fenlico que
transfiereunprotnaunradicaleselsiguiente:
177
Captulo3
reaccionessonelquemidelacapacidadantioxidantetotalequivalentede
Trolox (TEAC), el ensayo DPPH (que utiliza radicales 2,2
difenilpicrilhidracilo), el mtodo de FolinCiocalteu, el del potencial
antioxidantereductordeionesfrricos(FRAP)ylacapacidadantioxidante
reductoradeionescpricos(CUPRAC).
LasinvestigacionesdeestaMemoriaqueabordaneldesarrollode
178
Determinacindeantioxidantesenalimentos
Desplazarelfocodeatencindesdeelscreeningalestudiodelos
mecanismosdereaccindelassustanciasantioxidantes.
Analizarpotencialesinterferenciasmedianteelanlisisdemuestras
blanco en las que se analice la contribucin de las muestras sin
antioxidantes junto con el fluorforo, en presencia y ausencia del
reactivogeneradorderadicales.
Captulo3
oxgenodisueltoenlavelocidaddereaccin,determinacindelos
niveles de oxgeno disuelto mnimos para que la reaccin tenga
lugar y desarrollo de mtodos para asegurar niveles de oxgeno
adecuados,deacuerdoaloexpuestoanteriormente.
Disearmtodosparacontrolaradecuadamentelatemperatura.
Desarrollarnormasparaeldesarrollodemtodosenelformatode
microplacasenensayosdeactividadantioxidanteyconseguirque
losfabricantesdiseenlainstrumentacinapropiadaparatalfin.
Ponerapuntomtodosnormalizadosparaelclculodereasbajo
lacurva.
Proporcionardatosconjuntosdecapacidadantioxidanteydeotros
parmetroscomolaconcentracintotaldepolifenolesparaconocer
losperfilesdeactividad.
deducelaimportanciadeproporcionardatoscomplementarios,talescomo
la concentracin total de antioxidantes fenlicos y la de antioxidantes no
fenlicos, ntimamente relacionada con la capacidad antioxidante para
conocer los perfiles de actividad antioxidante de cada alimento. En la
segunda seccin de este captulo se aborda la determinacin de
antioxidantesfenlicosmedianteelusodelaenzimalaccasaenpresencia
denanopartculasdexidodeterbiocomoactivador.
180
Determinacindeantioxidantesenalimentos
Captulo3
BIBLIOGRAFA
(1)
(2)
(3)
(4)
A.J.Khlebnikov.I.A.Schepetkin,N.G.Domina,L.N.Kirpotina,M.T.
Quinn.Improvedquantitativestructureactivityrelationshipmodels
to predict antioxidant activity of flavonoids in chemical, enzymatic,
andcellularsystems.Bioorg.Med.Chem.(2007)15,17491770.
(5)
B.Halliwell.Howtocharacterizeabiologicalantioxidant.FreeRadic.
Res.(1990)9,132.
(6)
M.Carocho,I.C.F.R.Ferreira.Areviewonantioxidants,prooxidants
and relatived controversy: Natural and synthetic compounds,
screening and analysis methodologies and futureperspectives. Food
Chem.Tox.(2013)51,1525.
182
Determinacindeantioxidantesenalimentos
(7)
B.Lorrain,I.Kay,L.Pechamat,P.L.Teissedre.Evolutionofanalysis
ofpolyphenolsfromgrapes,winesandextracts.Molecules.(2013)18,
10761100.
(8)
(9)
(10)
(11)
(12)
B.Ou,M.HampschWoodill,R.L.Prior.Developmentandvalidation
of an improved oxygen radical absorbance capacity assay using
fluorescein as the fluorescent probe. J. Agric. Food Chem. (2001) 49,
46194621.
(13)
(14)
A.SnchezArribas,M.MartnezFernndez,M.Chicharro.Therole
of electroanalytical techniques in analysis of polyphenols in wine.
TrendsAnal.Chem.(2012)34,7896.
183
Chapter3
Thischaptercontainstheinvestigationsperformedtodevelopnew
analytical methodologies for the determination of antioxidants in food
samples. More specifically, these investigations have been focused to the
development of a method to determine the antioxidant capacity in food
samples, and secondly, on the proposal of a method to determine
polyphenols in wines. These studies have given rise to two scientific
articles:
fluorimetric
determination
of
food
184
Determinationoffoodantioxidants
185
Chapter3
o Primary enzymes
o Secondary enzymes
ENZYMATIC
NATURAL
ANTIOXIDANTS
NONENZYMATIC
o Cofactors
(coenzyme Q10)
o Inorganic
species (zinc,selenium)
o Carotenoids (carotene,lycopene,luteine,zeaxanthine)
o Flavonoids (flavonols,flavanols,flavones,flavanones,isoflavonoids,anthocyanins)
o Phenolic acids (hydroxycinnamic acids,hydroxybenzoic
acids)
Figure1.Classificationofnaturalantioxidantsubstances.
186
Determinationoffoodantioxidants
(BHA),
butylated
hydroxytoluene
(BHT),
tertbutylhydroquinone,estersofgallicacid(betterknownasgallates)and
somephenolderivatives,suchase.g.resorcinolderivatives.
OH
OH
OH
butylated hydroxyanisole
(BHA)
tertbutilhydroquinone
(TBHQ)
butylated hydroxytoluene
(BHT)
OH
OH
HO
HO
HO
HO
octyl gallate
octylgallate
GO)
(OG)
propylgallate
propyl
gallate
(GP)
(PG)
OH
HO
4hexylresorcinol
Figure2.Chemicalstructuresofsomesyntheticantioxidants.
187
OH
Chapter3
188
Determinationoffoodantioxidants
aprooxidantwithanantioxidant.Severalassayshavebeendevelopedfor
the assessment of the total antioxidant power of foods, but they do not
provide measurements that correlate mainly owing to the lack of
standardized methods [9]. In general, the chemical assays of antioxidant
activity can be divided into two main groups according to the type of
reactioninvolved:
Assaysbasedonhydrogenatomtransfer(HAT)
Assaysbasedonelectrontransfer(ET)
wherethearyloxyradical(ArO)formedafterthereactionofthephenolic
antioxidant with the peroxyl radical is stabilized by resonance, and AH
and ArOH species are the protected biomolecules and the antioxidant
molecules, respectively. In these assays, a fluorophore competes with the
antioxidantsubstancefortheperoxylradicalsandtheantioxidantactivity
is determined by measuring the difference in the inhibition of the
fluorophore signals obtained in the presence and in the absence of
antioxidant compounds. Some HATbased assays are the oxygen radical
189
Chapter3
developmentofamethodforthedeterminationofantioxidantcapacityare
basedontheapplicationoftheORACapproach.Thismethodinvolvesthe
use of a bisazo initiator, such as 2,2azobis(2methylpropionamidine)
dihydrochloride(AAPH),whichgeneratesperoxylradicalswhenheatedin
thepresenceofdissolvedoxygen[10].
190
Determinationoffoodantioxidants
Toanalyzepotentialinterferencesbytheanalysisofblanksamples
where the contribution of samples and fluorophore can be
analyzed in the absence and in the presence of the radical
generator.
191
Chapter3
Todevelopstandardizedmethodsforthecalculationofareaunder
thecurve.
Toprovidedataofantioxidantcapacityandotherparameters,such
as for instance, the total polyphenol concentration to know the
activityprofile.
significanceofcomplementarydata,suchastotalconcentrationofphenolic
antioxidantsandthatofnonphenolicantioxidants,intimatelyrelatedwith
the antioxidant activity to know the activity profile of each food. The
second section of this chapter tackles the determination of phenolic
192
Determinationoffoodantioxidants
193
Chapter3
basedontheuseofwellmicroplates,wheremeasurementsareperformed
almost simultaneously. This is an aspect that has been investigated and
optimizedinthisDissertation.Morespecifically,thesecondsectionofthis
chapter hasdealt with the development of a highthroughput method for
the determination of polyphenols in wines, where the enzyme
consumption has been lowered by using terbium oxide nanoparticles as
activators.Themethodalsodecreasestheanalysistimesrequiredwhenthe
enzymeisusedintheabsenceofnanoparticles.
LITERATURE
(1)
(2)
(3)
(4)
A.J.Khlebnikov.I.A.Schepetkin,N.G.Domina,L.N.Kirpotina,M.T.
Quinn.Improvedquantitativestructureactivityrelationshipmodels
to predict antioxidant activity of flavonoids in chemical, enzymatic,
andcellularsystems.Bioorg.Med.Chem.(2007)15,17491770.
(5)
B.Halliwell.Howtocharacterizeabiologicalantioxidant.FreeRadic.
Res.(1990)9,132.
(6)
M.Carocho,I.C.F.R.Ferreira.Areviewonantioxidants,prooxidants
and relative controversy: Natural and synthetic compounds,
screening and analysis methodologies and futureperspectives. Food
Chem.Tox.(2013),51,1525.
194
Determinationoffoodantioxidants
(7)
B.Lorrain,I.Kay,L.Pechamat,P.L.Teissedre.Evolutionofanalysis
ofpolyphenolsfromgrapes,winesandextracts.Molecules.(2013)18,
10761100.
(8)
(9)
(10)
(11)
(12)
B.Ou,M.HampschWoodill,R.L.Prior.Developmentandvalidation
of an improved oxygen radical absorbance capacity assay using
fluorescein as the fluorescent probe. J. Agric. Food Chem. (2001) 49,
46194621.
(13)
(14)
A.SnchezArribas,M.MartnezFernndez,M.Chicharro.Therole
of electroanalytical techniques in analysis of polyphenols in wine.
TrendsAnal.Chem.(2012)34,7896.
195
Determinacindeantioxidantesenalimentos
J.Agric.FoodChem.2011,59,22352240
LongWavelengthFluorimetricDeterminationofFood
AntioxidantCapacitybyUsingNileBlueasReagent
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens
DepartmentofAnalyticalChemistry,UniversityofCordoba,CampusofRabanales.
AnnextoMarieCurieBuilding.14071Cordoba.Spain
ABSTRACT
Keywords:nileblue,antioxidantcapacity,longwavelengthfluorimetry,96wellplate
197
Captulo3
1.Introduction
Oxidation processes involving free radicals contribute to the
development of many types of illnesses, such as, e.g., cardiovascular
disease, Alzheimers disorder, and cancer. This redox phenomenon is
produced by reactive oxygen species (ROS), which damage cell
membranes, with this effect being reduced by the ingest of antioxidant
compounds. The antioxidant capacity of foods is mainly given by
polyphenols,suchasflavonoids,andvitaminsCandE,amongothers[1].
From an analytical point of view, there are two types of methods for
assessingthisparameter.Thefirstgroupinvolvesasingleelectrontransfer
reaction, which can be followed by a change in the colour as the oxidant
speciesisreduced.Thesecondinvolvestheuseofhydrogenatomtransfer
reactions,inwhichtheantioxidantandthesubstratecompeteforthefree
radicalsgenerated.Theoxygenradicalabsorbancecapacity(ORAC)assay
isanexampleofthelattermethods.Thisassayinvolvestheuseof2,2azo
bis(2methylpropionamidine)dihydrochloride (AAPH) to give rise to
peroxyl radicals that directly attack absorbing or fluorescent probes,
leadingtothequenchingoftheirabsorbanceorfluorescence,respectively.
Thedyesphycoerythrin[24]andfluorescein(FL)[1,514]havebeenby
far the most used fluorescent probes, although Pyrogallol Red has been
alsodescribedtodevelopphotometricapproachesforthedeterminationof
the antioxidant capacity in berry extracts [15] and human blood plasma
and urine [16]. This assay relies on the performance of photometric
measurementsofPyrogallolRedbleachingbyactionoftheperoxylradical
generator,AAPH.Ithasbeenreportedthatthisdyeallowsfortheseparate
estimationofthecontributionofascorbicacidandsomepolyphenolstothe
198
Determinacindeantioxidantesenalimentos
199
Captulo3
N
Cl-
H2N
N
CH3
CH3
Figure1.ChemicalstructureofNBchloride
200
Determinacindeantioxidantesenalimentos
2.Materialsandmethods
2.1.Instrumentation
A 1420 Multilabel counter Victor 3V microplate reader (Perkin
Elmer and Analytical Sciences, Wallac Oy, Turku, Finland) was used to
perform
fluorescence
measurements.
Different
filters
(nominal
wavelength/passband)wereusedtoselecttheexcitation(485/15nmforFL
and620/8nmfor NB)andemission (535/25nmforFLand680/10nmfor
NB) wavelengths used to monitor ORACFL and ORACNB systems,
respectively.Eachmeasurementwasobtainedin0.5s,andthesubsequent
measurementsforeachwellwithtimeweremadein1minintervals.
2.2.Reagents
All reagents used were of analytical grade. NB chloride was
supplied by Sigma (St Louis, MO), and Trolox and AAPH were obtained
from Aldrich (Milwaukee, WI, and Steinheim, Germany). Dipotassium
hydrogen phosphate was purchased from Merck (Darmstadt, Germany).
Phosphate buffer solutions (0.085 M pH 6.9 and 0.075 M pH 7.5) were
preparedbydissolvingappropriateamountsofdipotassiumhydrogensalt
andadjustingthepHvalueswithhydrochloricacidtodevelopORACNB
and ORACFL methods, respectively. A NB stock solution (29.4 M) was
prepared by dissolving the appropriate amount of the dye in distilled
water by magnetic stirring for 24 h and stored at room temperature.
Workingstandardsolutionsof89nMNBwereprepareddailybydiluting
theappropriatevolumeofthestocksolutioninphosphatebuffersolution
(0.085 M at pH 6.9). A stock 4.5 x 104 M FL solution was prepared by
dissolvingtheappropriateamount
201
Captulo3
3.Procedures
3.1.DeterminationofAntioxidantCapacitybytheORACNBMethod
A volume (20 L) of standard (0.8 8 M Trolox), wine or juice
diluted sample, or blank (0.085 M phosphate buffer at pH 6.9) solutions
was added to each well together with 120 L of 89 nM NB. This mixture
waspreincubatedinsealedplatesat37Cfor15minutes,andthen,60L
of AAPH 0.012 M was added to each well by using an eightchannel
electronicmicropipettoachievethesimultaneousadditionofthisreagent.
Immediately, the plate was inserted into the microplate reader, and the
variationofthefluorescenceintensitywithtimewasmonitoredat37Cfor
60 min, using the instrumental conditions indicated above. Each
measurement was performed in triplicate, and a blank triplicate was
recorded at the beginning of each series to control potential changes that
may occur, owing to the lack of stability of AAPH, which was kept
protected from light at room temperature to ensure identical thermal
conditions at time 0 of the reaction. The decay curves were integrated by
usingOriginsoftware,andthen,thenormalizednetareaunderthecurve
(AUC) was calculated by subtracting the blank signal from the signal
obtainedinthepresenceofthestandardorsampleanddividingtheresult
bytheblanksignal.
202
Determinacindeantioxidantesenalimentos
3.2.DeterminationoftheAntioxidantCapacityusingtheORACFLMethod
instructionsdescribedelsewhere[5],whichinvolvetheuseofamicroplate
reader and FL as reagent. Briefly, 20 L of standard or diluted sample
solutionsweremixedwith120Lof70nMFLinphosphatebuffer(0.075
M, pH 7.4) for 15 min at 37 C, and then, 60 L of 0.012 M AAPH were
added.Thevariationofthefluorescenceintensitywithtimewasmonitored
for 60 min using 485 and 535 nm filters to select excitation and emission
wavelengths,respectively.Thecurvesobtainedwereprocessedinthesame
wayasthatdescribedfortheORACNBmethod.
3.3.DeterminationofAntioxidantCapacityinCommercialWineandFruitJuice
Samples
Several wines (white, semidry and red) and fruit juices (peach,
pineapple and apple) were bought in a local market and analysed
immediatelyaftertheywereopenedaccordingtothefollowingprocedure:
A volume (4.5 mL) of sample was treated with 300 L of 1 M NaOH to
increase the pH to neutral values, and it was raised up to 5 mL with
phosphate buffer. Then, an adequate dilution (1:500 1:10000 dilutions)
with the same phosphate buffer was performed to match the dynamic
rangesofthecalibrationcurvesofeitherORACFLorORACNBmethod.
A volume (20 L) of the diluted sample was treated according to the
proceduresindicatedaboveforbothmethods.
203
Captulo3
4.Resultsanddiscussion
4.1.SelectionoftheLongWavelengthFluorophor
ROS, such as peroxyl radicals (ROO), hydroxyl radicals (OH),
superoxide ion (O2), and singlet oxygen (1O2) are involved in the
physiology of some diseases. Radical chainbreaking antioxidants convert
reactive free radicals into stable and nonaggressive molecules by
mechanisms in which AAPH radicals formed in airsaturated solutions
reactrapidlywithmolecularoxygentogiverisetoperoxylradicals,ROO,
as described elsewhere [6]. The presence of antioxidant compounds gives
rise to the formation of a hydroperoxide and a stable antioxidant radical
that breaks the action of peroxide radicals. Although a wide variety of
antioxidant compounds can be used as reference standards in ORAC
assays [13], such as gallic, caffeic and ascorbic acids, the vitamin E
analogue Trolox has been chosen to develop the ORACNB method
presentedhere.
The reactions involving the formation of free radicals can be
followed by the decrease in the inhibition of the fluorescence from some
organic molecules, such as phycoerythrin and FL, in the presence of
sampleswithantioxidantcapacity[114].However,theshortStokesshiftof
these compounds can give rise to lightscattering phenomena that could
affecttheperformanceoffluorescencemeasurements.
Also, phycoerythrin shows a low photostability, and its price is
relatively high. With the aim of studying the potential use of fluorescent
dyesthatemitintheredregionofthespectrumasalternativereagentsfor
thispurpose,severaloxazineandthiazinedyes,namelyNB,azureAand
azure B, were assessed. Figure 2 shows the curves obtained for each
204
Determinacindeantioxidantesenalimentos
18000
18000
22
16000
a)
16000
14000
12000
10000
11
8000
6000
4000
b)
14000
12000
10000
8000
6000
4000
2000
2000
0
0
20
40
60
80
100
20
40
Time (min)
60
80
100
Time (min)
18000
16000
c)
14000
12000
10000
8000
6000
4000
2000
0
0
20
40
60
Time (min)
80
10
Figure6:CurvesobtainedfortheORACassayusing(a)nileblue,(b)azureA,
and(c)azureBfluorophores:(a)blankand(2)2MTrolox.
blank and standard with all the fluorophors assayed, NB gave the best
results. Also, NB was chosen because it has a wider Stokes shift that the
205
Captulo3
16000
14000
14
12000
10000
8000
6000
4000
2000
0
0
10
20
30
Time (min)
40
50
4.2.OptimisationoftheORACNBMethod
Thevariablesaffectingthesystemwereoptimisedbytheunivariate
method.Eachresultwastheaverageofthreemeasurements.Theanalytical
206
Determinacindeantioxidantesenalimentos
parameter used to optimise the system was the net AUC, calculated as
indicatedaboveintheprocedureoftheORACNBmethod.
The influence of the pH was investigated in the range of 4 11,
using0.075Macetate,phosphate,borateand,carbonatebuffersolutionsto
keepthepHconstantinthebufferingregionofeachsolution.AsFigure4A
shows, there was not appreciable net AUC at pH values below 5.8 and
above8.ThebehaviourofthesystematlowpHvaluescouldbeascribedto
thefactthatthepKavalueofTroloxisabout3.89,showingalowsolubility
at this pH [22]. The solubility increases with the pH, which improves the
valueofthenetAUC,obtainingthebestresultsinthepHrangeof6.07.0.
A pH of 6.9 was chosen, which is slightly lower than that required to
develop the ORACFL method (pH 7.4). The influence of buffer
concentration was studied by assaying phosphate concentrations in the
range of 0.02 0.15 M, finding that a 0.085 M phosphate buffer solution
gavethebestsignal.
NB and AAPH concentrations are two critical variables that are
interrelated.Theinfluenceofthefluorophorconcentrationwasstudiedby
adding a fixed volume (120 L) of solutions with NB concentrations
ranging from 60 to 370 nM (Figure 4B). The system was practically
independentonthisvariableintherange80130nM,choosing89nMNB
for the development of the method. The influence of the peroxyl radical
generator, AAPH, was evaluated in the range of 0.006 0.024 M, finding
thattheAUCsignalsofbothanalyteandblanksolutionsdecrease,withthe
difference also decreasing between both signals, when the AAPH
concentration increases. However, the curves obtained at low AAPH
concentrations were less defined
and
207
showed
low
Captulo3
reproducibility.Thus,a0.012MAAPHconcentrationwaschosen.
0.16
0.10
A)
B)
0.08
0.12
AUC
AUC
0.06
0.08
0.04
0.04
0.02
0.00
0.00
5.5
6.0
6.5
7.0
7.5
8.0
8.5
pH
100
200
300
400
[NB], nM
208
Determinacindeantioxidantesenalimentos
thelimitingstep.ThemethodusingFLasreagent[8]isalsodevelopedat
37C.
4.3.AnalyticalFeatures
Thekineticcurveswereobtainedunderoptimumconditions,using
ex of 620 nm and em of 680 nm to monitor the variation of the
fluorescence intensity with time for 60 min and the net AUC as the
analyticalparameter.Thedynamicrangeofthecalibrationgraphwas0.8
8 M Trolox. The regression equation was AUC = (0.02 0.01) + (0.074
0.006) X, where X was the Trolox concentration expressed in micromolar.
The regression coefficient (R) is 0.993, which is indicative of a good
linearityofthecalibrationcurve.Thedetectionlimit,calculatedfollowing
International Union of Pure and Applied Chemistry (IUPAC)
recommendations[23],was0.45M,whichislower[6,7]orsimilar[8]to
thoseobtainedinotherORACmethodsinvolvingFL.Theprecisionofthe
method was assessed attwo different Trolox concentrations,1 and5 M,
andexpressedasthepercentageofrelativestandarddeviation,giving5.6
and2.9%,respectively.
4.4.Applications
The proposed method was applied to the analysis of wines,
namely, white, semidry and red, and commercial fruit juices, namely,
pineapple,peachandapple.ThesesampleswereanalyzedbybothORAC
FL and ORACNB methods with a simple sample treatment, which
consisted in the adjustment of the pH to neutral values, because the
samples
featured
acidic
pH
209
Captulo3
20000
20000
16000
A)
B)
Relative Fluorescence Intensity
12000
8000
4000
16000
12000
8000
4000
0
0
20
40
60
Time (min)
80
100
20
40
60
80
100
Time (min)
Figure5.Antioxidantcapacitycurvesobtainedinthepresenceof(A)semidry
white wine and (B) pineapple juice samples at different sample dilutions.
Experimentalconditionsareasfollow:[NB],89nM;[AAPH],0.024M,pH,6.9;
[phosphate],0.085M;andtemperature,27C.InpanelA,(1)blankand(2,3
and 4) are 1/4000, 1/2000, and 1/1000 dilutions of the white wine sample,
respectively.InpanelB(1)blank.
210
Determinacindeantioxidantesenalimentos
found in the samples using both ORACNB and ORACFL methods. The
paired t test was applied to the results at a 95% significance level, and it
was found that there were not significant differences in the results
provided by both methods, which confirms the practical utility of the
proposedORACNBmethodtotheanalysisofthesefoodsamples.
Sample
ORACNBa
ORACFLa
whitewine(manzanilla)
1.500.09
1.30.2
semidrywine
7.70.9
4.40.4
redwine
15.30.4
10.40.9
2.2220.001
1.930.09
pineapplejuice
2.70.3
2.3990.006
applejuice
2.70.3
3.50.4
peachjuice
Meanstandarddeviation(SD)(n=3)
Also,thevaluesfoundforbothtypesofsamplesagreewithvalues
reportedintheliterature[9,10].
211
Captulo3
to 113.6%. The mean recovery values obtained were 92.0 and 92.9 % for
wineandjuicesamples,respectively.Thisinternalvalidationalsoconfirms
theusefulnessofthedevelopedORACNBmethodfortheanalysisofreal
samples.
Table2.Recoveryvaluesobtainedforthedifferentsamplesanalyzed
Recoverystudy
Sample
Addeda
Founda,b
Recovery(%)
Whitewine
2.2
2.00.2
92.0
8.9
6.70.9
75.2
11.1
9.20.8
82.9
Semidrywine
4.4
8.8
13.2
3.60.2
8.10.3
12.90.6
81.8
92.1
97.0
Redwine
17.8
35.6
44.4
182
384
444
101.1
106.7
99.1
Peachjuice
2.2
4.4
5.5
2.10.1
3.50.4
4.30.2
95.5
79.5
78.2
Pineapplejuice
2.2
4.4
5.5
2.50.2
4.50.3
5.80.4
113.6
102.3
105.5
Applejuice
4.4
3.20.2
8.8
7.90.5
11.1
111
aUnitsinmillimolarTroloxequivalentsofsample.
bMeanSD(n=3).
72.7
89.6
99.1
212
Determinacindeantioxidantesenalimentos
fluorophor NB for the first time as an analytical reagent. The use of this
reagent instead of other fluorophores previously proposed for this
purpose, such as FL or phycoerythrin, is a useful alternative to avoid
potentialbackgroundsignalsfromthesamplematrix,whichcanappearat
lower wavelengths. Also, the relatively wide Stokes shift of NB allows
analytical measurements to be free of scattering signals, which can be a
limitation when the abovementioned fluorophores are used. Finally, the
probability of photobleaching processes for NB is lower than that for
phycoerythrin,aswellasitscost.
Acknowledgement
The authors gratefully acknowledge financial support from the
MinisteriodeCienciaeInnovacin(MICINN)(GrantCTQ200908621)and
fromtheJuntaofAndalucia(GrantP09FQM4933).
Abbreviationsused
AAPH,2,2azobis(2methylpropionamidine)dihydrochloride;AUC,area
underthecurve;BHA,butylhydroxyanisole;FL,fluorescein;NB,nileblue;
ORAC,oxygenradicalabsorbancecapacity;ROS,reactiveoxygenspecies
213
Captulo3
References
(1)
(2)
(3)
(4)
G.Cao,C.P.Verdon,A.H.B.Wu,H.Wang,R.L.Prior.Automated
assayofoxygenradicalabsorbancecapacitywiththeCOBASFARA
II.Clin.Chem.(1995)41,17381744.
(5)
(6)
(7)
214
Determinacindeantioxidantesenalimentos
(8)
(9)
(10)
(11)
(12)
(13)
(14)
A.TijerinaSenz,I.Elisia,S.M.Innis,J.K.Friel,D.D.Kitts.Useof
ORACtoassessantioxidantcapacityofhumanmilk.J.FoodCompos.
Anal.(2009)22,694698.
(15)
E.Atala,L.Vsquez,H.Speisky,E.Lissi,C.LpezAlarcn.Ascorbic
acidcontributiontoORACvaluesinberryextracts:Anevaluationby
the ORACpyrogallol red methodology. Food Chem. (2009) 113,331
335.
(16)
P. Torres, P. Galleguillos, E.
Lissi,
215
C.
LpezAlarcn.
Captulo3
(18)
M.P.AguilarCaballos,A.GmezHens,D.PrezBendito.Simulta
neous stoppedflow determination of butylated hydroxyanisole and
propyl gallate using a Tformat spectrofluorimeter. J. Agric. Food
Chem.(2000)48,312317.
(19)
H.Tavallali,E.Asrari,M.A.AmeriSiahoe.Sensitivekineticspectro
photometricmethodfortracedeterminationofcerium(IV)basedon
decolarizationofnilebluebypotassiumperiodate.Int.J.Chem.Tech
Res.(2009)1,359362.
(20)
G.M.doNascimento,R.C.deOliveira,N.A.Pradie,P.R.G.Lins,P.
R.Worfel,G.R.Martinez,P.DiMascio,M.S.Dresselhaus,P.Corio.
Redoxmediatorsinglewallcarbonnanotubesmodifiedwithorganic
dyes: Synthesis, characterization and potential cytotoxic effects. J.
Photochem.Photobiol.A(2010)211,99107.
(21)
216
Determinacindeantioxidantesenalimentos
(22)
M.Lcio,C.Nunes,D.Gaspar,H.Ferreira,J.L.F.C.Lima,S.Reis.
Antioxidant activity of vitamin E and Trolox: Understanding of the
factors that govern lipid peroxidation studies in vitro. Food Biophys.
(2009)4,312320.
(23)
G.L.Long,J.D.Winefordner.Limitofdetection:Acloserlookatthe
IUPACsdefinition.Anal.Chem.(1983)55,712A724A.
217
Determinacindeantioxidantesenalimentos
Automaticdeterminationofpolyphenolsinwines
usinglaccaseandterbiumoxidenanoparticles
J.GodoyNavajas,M.P.AguilarCaballos,A.GmezHens
DepartmentofAnalyticalChemistry,UniversityofCordoba,Campus
ofRabanales.AnnextoMarieCurieBuilding.14071Cordoba.Spain
Abstract
The analytical usefulness of the combined use of laccase, terbium
oxide
nanoparticles
(Tb4O7NPs)
and
8hydroxypyrene3sulfonate
trisodium(HPTS)forthedeterminationofpolyphenolcompoundsinwine
samples is described. The system is based on the temporal inhibition by
polyphenols on the decrease of the HPTS fluorescence in the presence of
laccase and on the activating effect of Tb4O7NPs, which increase the
reactionrateofthesystem,shorteninganalysistimes.
219
Captulo3
1.Introduction
220
Determinacindeantioxidantesenalimentos
GmezHens,2011).MostofthesemethodsuseTrolox(6hydroxy2,5,7,8
tetramethylchroman2carboxylicacid),whichisawatersolubleanalogue
of vitamin E, as calibration standard. Although several studies have been
carriedoutusingdifferentwinetypestorelatethephenolcontentwiththe
antioxidant activity, the correlation coefficients obtained are usually low
(FernndezPachn,Villao,GarcaParrilla&Troncoso,2004;DiMajo,La
Guardia, Giammanco, La Neve & Giammanco, 2008; Li, Wang, Li, Li &
Wang,2009),whichistheresultofthedifferentantioxidantpotentialofthe
phenolcompoundspresentinwines.
Laccaseisaphenoloxidaseenzymethathasbeendescribedforthe
determination of both phenolic content and antioxidant activity, using
different experimental conditions. This enzyme catalyzes the oxidation of
phenolic compounds to quinones or radicals by reducing the dissolved
oxygen to water. Several laccasebased amperometric sensors have been
described for polyphenol determination in wines (Gamella, Campuzano,
Reviejo & Pingarrn, 2006; Di Fusco, Tortolini, Deriu & Mazzei, 2010;
Montereali,DellaSeta,Vastarella&Pilloton,2010)usinggallicacidasthe
polyphenol standard. Although the detection limits of these methods are
adequatefortheiruseinroutineanalysis,theyshowalimitedlifetimethat
is ascribed to the adsorption of oxidized products on the surface of the
electrode with the consequent negative effect on the enzyme activity. A
fluorometric method has been reported for the determination of
polyphenolcontentinjuiceandteasamplesusingindocyaninegreenand
positively charged gold nanoparticles (AndreuNavarro, Fernndez
Romero & GmezHens, 2012). The method is based on the temporal
inhibitioncausedbypolyphenolsontheoxidationofthefluorophoreand
221
Captulo3
222
Determinacindeantioxidantesenalimentos
2.Experimental
2.1Instrumentation
Elmer and Analytical Sciences, Wallac Oy, Turku, Finland) was used to
perform
fluorescence
measurements.
Different
filters
(nominal
223
Captulo3
xenon arc source and a R928 photomultiplier tube, was used to perform
fluorescenceexcitationandemissionscansofthedifferentfluorophoresin
solution and photobleaching experiments. A Lambda 35 UV/VIS
spectrometer (PerkinElmer, United Kingdom) was used to perform
photometricmeasurements of the FolinCiocalteumethod. 1/2AreaPlateTM
microplates(PerkinElmer,USA)withatotalvolumeof180 Lwereusedto
recordthekineticcurvesofthesystem.
2.2Reagents
All reagents used were of analytical grade. The fluorophores 8
hydroxypyrene1,3,6trisulfonic acid trisodium salt (HPTS), 2[4
(dimethylamino)styryl]1methylpyridinium iodide (2Di1ASP), 2[4
(dimethylaminophenyl]1,3butadienyl)3ethylbenzothiazolium
toluenesulfonate salt (Styryl 7), nile blue chloride, azure B chloride and
toluidineblueOwereprovidedbySigmaAldrich(St.Louis,Mo,USA),as
wellasTween20,laccaseenzyme(TrametesVersicolor,ECNumber1.10.3.2,
2.05 U/mg), and caffeic acid. The surfactants Triton X100 and hexadecyl
trimethylammoniumbromide(CTAB)werepurchasedfromFluka(Buchs,
Switzerland).Europium(III)oxidenanoparticles(Eu2O3nanopowder,<150
nm), diamond nanoparticles (<10 nm particle size) and gallic acid were
acquired from Sigma. Terbium oxide nanopowder (TEM <150 nm), silver
nanoparticles (10 nm, in aqueous buffer containing citrate as stabilizer),
terbium nitrate pentahydrate and sodium chloride were purchased for
SigmaAldrichandfluoresceinsodiumwaspurchasedforFluka.Ascorbic
acid was acquired from SigmaAldrich while citric acid and sulfuric acid
were purchased from Panreac (Barcelona, Spain). Glucose and sucrose
224
Determinacindeantioxidantesenalimentos
werepurchasedfromSigmaAldrichandFluka,respectively.Dipotassium
hydrogen phosphate, tris(hydroxymethyl)aminomethane (TRIS), sodium
sulfite, malic acid and sodium hydroxide were purchased from Merck
(Darmstadt, Germany). Sodium carbonate, sodium acetate and sodium
dodecyl sulfate (SDS), FolinCiocalteus phenol reagent (2N) and 6
hydroxy2,5,7,8tetramethylchromane2carboxylic acid (Trolox) were
providedfromSigmaAdrich.
Phosphate buffer solution (0.4 M pH 7.0) was prepared by
dissolvinganappropriateamountofdipotassiumhydrogenphosphatesalt
(Merck)indistilledwaterandadjustingthepHwithhydrochloricacid.A
HPTSstocksolution(890M)waspreparedbydissolvingtheappropriate
amount of the fluorophore in distilled water by magnetic stirring for
several minutes and stored at room temperature. Working solutions (10
MHPTS)wereprepareddailybydilutingtheappropriatevolumeofthe
stocksolutioninthephosphatebuffersolution.A10mMstocksolutionof
gallicacidwasprepareddailybydissolvingtheappropriateamountofthe
reagent in distilled water and the different standards solutions were
prepareddilutingtheappropriatevolumeofthestocksolutionindistilled
water. A 4 mg/mL stock Tb4O7 NPs dispersion was prepared daily by
dispersing the appropriate amount of nanopowder in distilled water by
using an ultrasound bath. Working solutions were prepared by diluting
the stock dispersion in the phosphate buffer solution. A laccase stock
solution (4 U/mL) was prepared daily by sonication for 10 s and let
overnighttostandbeforeuse.Workinglaccasesolutionswerepreparedby
diluting the appropriate volume of the stock solution in the phosphate
buffersolution.
225
Captulo3
2.3.Procedures
2.3.1.DeterminationofpolyphenolsusingthelaccaseTb4O7NPmethod
A volume (75 L) of zero standard,gallic acid standards (0.5 12
M) or diluted wine sample was firstly added to each well of the
microplatetogetherwith15Lof10MHPTS.Thismixturewaskeptfor
15minutesat37 oCtoreachconstanttemperatureand,then,avolume(60
L) of a mixture containing 1 mg/mL Tb4O7 NPs and 1 U/mL laccase in
phosphate buffer solution was added to each well by using an electronic
multichannelmicropipette.Immediatelyafter,themicroplatewasinserted
intothemicroplatereader,andthevariationofthefluorescenceintensityof
HPTS with time was monitored at 37 oC for 36 min, using nominal
excitationandemissionwavelengthfiltersof450and535nm,respectively.
Normalized decay curves were obtained by dividing the fluorescence
signals obtained at each time by the fluorescence signal obtained at time
zero for each curve. These curves were later integrated using Origin
software, and then, the net normalized area under the curve (AUC) was
calculatedbysubtractingtheAUCfortheblankfromtheAUCobtainedin
thepresenceofgallicacid.
2.3.2.Analysisofwinesamples
Winesamples(red,whiteandsweetwinesamples)wereboughtat
226
Determinacindeantioxidantesenalimentos
1:8000 for red, 1:100 1:500 for white and 1:500 1:2000 for sweet wine
samples.
2.3.3.FolinCiocalteumethod
Thismethodwasperformedasdescribedelsewhere(Magalheset
al., 2006). Briefly, a volume (2.5 mL) of gallic acid standard (15150 M
preparedindistilledwater)ordilutedwinesamplewasmixedwith500L
ofFolinCiocalteureagent(heteropolyphosphotungstatemolybdate)and5
mL of sodium carbonate (60 g/L). This mixture was kept at room
temperaturefor2hand,afterwards,theabsorbancewasmeasuredat760
nm.
3.ResultsandDiscussion
3.1.Studyofthesystem
This study was carried out to choose the experimental conditions
that allow the development of a fast and automatic fluorometric method
for the determination of polyphenol compounds using laccase. The study
was carried out in an automatic system involving the use of microplates
with a volume of 180 L for each well, so that the sample and reagent
consumptionwaslowerthaninconventionalmicroplates(350400L).
Several potential fluorescent laccase substrates (Styryl 7, 2Di1
ASP, azure B chloride, toluidine blue O, nile blue chloride, sodium
fluorescein and HPTS) (Table 1) were assayed to study their capability to
compete with phenolic antioxidants for the active sites of the enzyme.
Although indocyanine green has been described as a laccase substrate
(AndreuNavarro et al., 2012), it was not assayed because its long
227
Captulo3
excitationandemissionwavelengthswerenotadequateforthemicroplate
readerusedinthisstudy.
Table1.Fluorophoresassayedaspotentialsubstratesoflaccaseenzymein
thisstudy
Fluorophore
O O
HO
S
ONa
NaO
ONa
S
S
O O
O O
8Hydroxypyrene1,3,6trisulfonicacidtrisodiumsalt(HPTS)
I+
N
CH3
CH3
N
CH3
2[4(Dimethylamino)styryl]1methylpyridiniumiodide(2
Di1ASP)
O S O
CH3
H3C
+
N
CH3
S
N
CH3
2[4(Dimethylaminophenyl]1,3butadienyl)3ethyl
benzothiazoliumptoluenesulfonatesalt(Styryl7)
Excitationfilter
Emissionfilter
450/6
535/25
485/15
535/25
531/25
680/10
485/15
535/25
620/8
680/10
620/8
680/10
620/8
680/10
ONa
O
NaO
Sodiumfluorescein
H3C
H2N
Cl+
N CH3
CH3
ToluidineblueO
N
ClH2N
N
CH3
Nilebluechloride
H3C N
S
H
AzureBchloride
CH3
Cl+
N CH3
CH3
228
Determinacindeantioxidantesenalimentos
1.0
1
2
3
0.8
0.6
0.4
0.2
10
20
30
40
Time (min)
Figure1.Behaviorofdifferentfluorophoresinthepresenceoflaccase.1)azure
Bchloride,2)sodiumfluorescein,3)toluidineblueO,4)2Di1ASP,5)HPTS.
Experimentalconditions:In1),3)and4)[fluorophore]=5M,[laccase]=0.2
U/mL.In2)and5)[fluorophore]=1M,[laccase]=0.1U/mL.Alltheassays
werecarriedoutusingphosphatebuffersolution(0.2M,pH7.0).
229
Captulo3
Thepresenceofgallicorcaffeicacidonthesystemcausedadelay
on the fluorescence decrease of HPTS, ascribed to the sequential catalytic
effectoflaccase,whichactsoverthefluorophorewhenthepolyphenolhas
been oxidized. Figure 2 shows the kinetic curves obtained in the absence
andinthepresenceofgallicacid.
1.2
14
1.0
0.8
0.6
0.4
0.2
0.0
10
15
20
Time (min)
Figure 2. Kinetic curves obtained for the HPTSlaccase system in the absence
(curves1and2)andinthepresence(curves3and4)of2Mgallicacid,andin
thepresence(curves1and3)andintheabsence(curves2and4)of1mg/ml
Tb4O7NPs.[HPTS]=10M,[laccase]=1U/mL,pH=7.0,[phosphate]=0.4M,
temperature37C.
230
Determinacindeantioxidantesenalimentos
231
Captulo3
abovetheircriticalmicellarconcentration.ThestronginteractionofHPTS
with CTAB, which has been previously described (Pramanik, Banerjee &
Bhattacharya, 2007), gave rise to a decrease of the reaction rate of the
system, which made difficult to develop an assay with a suitable sample
throughput.ThepresenceofSDSandTritonX100causedadecreaseinthe
fluorescenceintensityofHPTS,decreasingalsotheAUCvaluesobtainedin
the absence and the presence of the analyte. Finally, the fluorescence of
HPTSandthereactionrateofthesystemwerenotaffectedinthepresence
ofTween20.Accordingtotheseresults,surfactantswerenotusedforthe
developmentofthesystem.
3.2.Optimizationofthesystem
Thevariablesaffectingthesystemwereoptimizedbytheunivariate
method using the net AUC as the analytical parameter, which was
calculatedasindicatedintheprocedure.However,itwasnecessarytofind
acompromisedsolutionbetweenthisparameterandthetimerequiredto
obtain each measurement, in order to avoid the excessive duration of the
procedure.Eachresultwastheaverageofthreemeasurements.
TheinfluenceofthepHonthesystemwasstudiedintherangeof
4.09.0 using different buffers solutions, such as acetic acid/acetate,
phosphate and carbonate at a 0.2 M concentration. The fluorescence of
HPTS was very low at pH 4.0 in the presence of acetate ions and it
remained constant without undergoing any oxidation reaction with
laccase.HPTSshowedahighfluorescenceatpHvaluesintherangeof8.0
9.0, but it was not modified in the presence of laccase. However,
appropriate net AUC values were obtained at pH 7.0 7.5. Hexamine,
232
Determinacindeantioxidantesenalimentos
3.3.Analyticalfeatures
The net AUC values were obtained from the normalized kinetic
233
Captulo3
theregressionequationwas:AUC=(0.400.05)+(2.40.6)X,inwhichXis
the gallic acid concentration expressed as micromolar. The regression
coefficient (R) was 0.996, which is indicative of the good linearity of the
calibration curve. The limit of detection (LOD), calculated according to
IUPACrecommendations(Long&Winefordner,1983),was0.14M,while
thevalueobtainedintheabsenceofTb4O7NPswas0.4M,whichshows
thepositiveeffectoftheNPsonthemethod.
1.0
16
0.8
0.6
0.4
0.2
0.0
10
20
30
40
Time (min)
234
Determinacindeantioxidantesenalimentos
The LOD reached in the presence of the NPs is lower than those
describedforgallicacidusingsomeamperometricbiosensors(DiFuscoet
al., 2010; Montereali et al., 2010). Although a LOD of 0.04 M has been
obtained for gallic acid using indocyanine green (AndreuNavarro et al.,
2012), this method involves the sequential analysis of each sample,
requiring about 15 min to carry out each measurement. However, the
automation of the measurements using a microplate reader allows a
samplethroughputof35samplesh1(threemeasurementsforeachsample)
in the new method. The precision of the method, expressed as the
percentage of relative standard deviation, was assessed at 0.7 and 5 M
gallicacidconcentrations,providingvaluesof6.3and2.5%,respectively.
Theselectivityofthemethodwascheckedbyassayingsomenon
phenolic reducing substances, which could be present in wines. A
compoundwasconsiderednottointerfereiftheanalyticalsignalobtained
initspresencewaswithinonestandarddeviationthesignalobtainedinits
absence. Table 2 shows the results obtained after assaying different
potentialinterferingcompounds.Themostseriousinterferencewascaused
byascorbicacid,whichwastoleratedatthesamemolarconcentrationthan
thatoftheanalyte.However,thisinterferencewouldbenegligiblebecause
themaximumlimitallowedforascorbicacidwhenusedasanadditivein
wine is 0.85 mM (Oxford companion to wine, Robinson J. (Ed.), 2006),
which is lower than the usual content of polyphenols in wines. Sulfites
were tolerated in a concentration 20fold of the analyte. Although this
interference could be removed using a pretreatment step (Montereali et
al., 2010), it was not necessary as the presence of sulfites in wines is
controlled according to their total content in reducing substances
235
Captulo3
(OrganisationInternationaledelaVigneetduVin(OIV),2011).Thus,the
maximumlimitofsulfurdioxideis2.5mMforredwine,3.3mMforwhite
wineand6.3mMforsweetwinesamples,whentheycontainupto4g/lof
reducingsubstances.Accordingtotheselimitsandthevaluesforphenolic
antioxidantconcentrationfoundinliterature(Gamellaetal.,2006;DiFusco
et al., 2010), the new method can be directly applied to the analysis of
wines since the concentration of polyphenols is higher than those of
ascorbic acid and sulfites. Trolox, which is usually the standard used for
thedeterminationoftheantioxidantactivity,asindicatedabove,wasalso
assayedintheconcentrationrangeof0.410M,butthecurvesobtained
weresimilartothatobtainedfortheblanksample.Theseresultsshowthat
the new method is suitable for the determination of polyphenols, but it
doesnotallowthedeterminationofantioxidantactivity.
Table2.Influenceofsomereducingagentsontheproposeddeterminationof
phenolicantioxidantsat2Mgallicacid
Compound
Maximumtolerated
interferent/analyteratio*
Malicacid
100
Citricacid
100
Sucrose
100
Glucose
25
Sodiumsulfite
20
Ascorbicacid
*Maximumratioassayedwas100
236
Determinacindeantioxidantesenalimentos
3.4.Applications
Theproposedmethodwasappliedtotheanalysisofred,whiteand
sweet wines. The sample treatment only consisted in the dilution of the
samplestomatchthedynamicrangeofthecalibrationcurve,usinggallic
acidstandardsandthenetAUCasanalyticalparameter.Table3showsthe
content ofantioxidants found in these samples analyzed by the proposed
methodandbytheFolinCiocalteumethod.Thevaluesfoundbyusingthe
new method agree with those reported in the literature (Gamella et al.,
2006; Di Fusco et al., 2010). As it can be seen from the table, the values
providedbyFolinCiocalteumethodwerehigherinallinstancesthanthose
providedbythelaccasemethod,whichisascribedtothecapabilityofthe
later to also detect nonphenolic substances. A recovery study was
performedbyaddingthreedifferentamountsofgallicacidtoeachsample
and subtracting the results obtained from similarly treatment unspiked
samples(Table3).Therecoverypercentagesobtainedrangedfrom81.0to
108.3%andthemeanrecoveriesforred,whiteandsweetwineswere90.9,
92.4and99.5%,respectively.
237
238
1.920.04
2.0280.005
3.640.05
Whitewine(Rueda)
Whitewine(Valdepeas)
Sweetwine(Crdoba)
Units:mMgallicacidequivalentsofsample
MeanSD(n=3)
14.040.18
Redwine(Cuenca)
11.50.1
contenta,b
Redwine(LaRioja)
sample
FolinCiocalteu
method
1.270.09
1.000.03
1.000.06
9.50.5
7.00.4
contenta,b
1
3
5
0.25
0.75
1.25
0.25
0.75
1.25
4
12
20
4
12
20
addeda
1.010.09
3.00.3
4.70.3
0.230.03
0.700.04
1.00.1
0.240.03
0.770.04
1.080.03
3.20.2
11.70.3
18.90.8
4.30.5
10.00.5
161
founda,b
Proposedmethod
Table3.Antioxidantconcentrationfoundandrecoverystudyforthewinesamplesanalyzed
101.5
102.0
95.0
92.0
92.2
83.3
97.2
102.8
86.7
80.0
97.4
94.5
108.3
84.1
81.0
recovery
(%)
Captulo3
Determinacindeantioxidantesenalimentos
4.Conclusions
determinationofpolyphenolsinwinesampleshasbeendemonstratedand
theresultshavebeencomparedwiththoseprovidedbytheFolinCiocalteu
method,whichwasusedasreference.Theresultsobtainedforallsamples
analyzed by the new method were lower owing to its better selectivity
compared to the reference method, which is a common aspect for other
laccasebased enzyme methods previously described for wine analysis
(Gamellaetal.,2006;DiFuscoetal.,2010).
Acknowledgements
AuthorsgratefullyacknowledgefinancialsupportfromtheSpanish
MinisteriodeEconomayCompetitividadMINECO(GrantNo.CTQ2012
32941),theJuntadeAndalucaProgram(GrantNo.P09FQM4933)andthe
FEDERFSEprogram.
239
Captulo3
References
AndreuNavarro, A., FernndezRomero, J. M. & GmezHens, A. (2012).
Determination of polyphenolic content in beverages using laccase,
goldnanoparticlesandlongwavelengthfluorimetry.AnalyticaChimica
Acta,713,16.
Ashrafi,SD.,Rezaei,S.,Forootanfar,H.,Mahvi,A.H.&Faramarzi,M.A.
(2013). The enzymatic decolorization and detoxification of synthetic
dyesbythelaccasefromasoilisolatedascomycete,Paraconiothyrium
variabile.InternationalBiodeteriorationandBiodegradation,85,173181.
Benzina, O., Dassi, D., ZouariMechichi, H., Frikha, F., Woodward, S.,
Belbahri,L.,RodrguezCouto,S.&Mechichi,T.(2013).Decolorization
and detoxification of two textile industry effluents by the laccase/1
hydroxybenzotriazole system. Environmental Science and Pollution
Research,20,51775187.
Campos, A. M., Sotomayor, C. P., Pino, E. & Lissi, E. (2004). A pyranine
based procedure for evaluation of the total antioxidant potential
(TRAP) of polyphenols. A comparison with closely related
methodologiesBiologicalResearch,37,287292.
Dawidowicz, A. L., Wianowska, D. & Olszowy, M. (2012). On practical
problemsinestimationofantioxidantactivityofcompoundsbyDPPH
method (Problems in estimation of antioxidant activity). Food
Chemistry,131,10371043.
Di Fusco, M., Tortolini, C., Deriu, D. & Mazzei, F. (2010). Laccasebased
biosensorforthedeterminationofpolyphenolindexinwine.Talanta,
81,235240.
240
Determinacindeantioxidantesenalimentos
241
Captulo3
Li, H. H., Wang, X., Li, Y., Li, P. & Wang, H. (2009). Polyphenolic
compounds and antioxidant properties of selected China wines. Food
Chemistry,112,454460.
Long,G.L.&Winefordner,J.D.(1983).Limitofdetection.Acloserlookat
theIUPACdefinition.AnalyticalChemistry,55,712A724A.
Lorrain, B., Ky, I., Pechamat, L. & Teissedre, P. L. (2013). Evolution of
analysis of polyphenols from grapes, wines, and extracts. Molecules,
18,10761100.
Magalhes,L.M.,Segundo,M.A.,Reis,S.,Lima,J.L.F.C.&Rangel,A.O.
S. S. (2006). Automatic method for the determination of Folin
Ciocalteu reducing capacity in food products. Journal of Agricultural
andFoodChemistry,54,52415246.
Mediari, M., Rastija, V. & Boji, M. (2011). Recent advances in the
application of high performance liquid chromatography in the
analysis of polyphenols in wine and propolis. Journal of AOAC
International,94,3242.
Mondal, S. K., Ghosh, S., Sahu, K., Sen, P. & Bhattacharyya, K. (2007).
Excited state proton transfer from pyranine to acetate in methanol.
JournalofChemicalSciences,119,7176.
Montereali, M.R., Della Seta, L., Vastarella, W. & Pilloton, R. (2010). A
disposable LaccaseTyrosinase based biosensor for amperometric
detectionofphenoliccompoundsinwine.JournalofMolecularCatalysis
B:Enzymatic,64,189194.
Mukhopadhyay, A., Dasgupta, A. K. & Chakrabarti, K. (2013).
Thermostability,pHstabilityanddyedegradingactivityofabacterial
242
Determinacindeantioxidantesenalimentos
laccaseareenhancedinthepresenceofCu2Onanoparticles.Bioresource
Technology,127,2536.
Nenadis, N., Lazaridou, O. & Tsimidou, M. Z. (2007). Use of reference
compounds in antioxidant activity assessment. Journal of Agricultural
andFoodChemistry,55,54525460.
Nkhili, E. & Brat, P. (2011). Reexamination of the ORAC assay: effect of
metalions.AnalyticalandBioanalyticalChemistry,400,14511458.
NugrohoPrasetyo,E.,Kudanga,T.,Steiner,W.,Murkovic,M.,Nyanhongo,
G. S. & Guebitz, G. M. (2010). Laccasegenerated tetramethoxy
azobismethylenequinone(TMAMQ)asatoolforantioxidantactivity
measurement.FoodChemistry,118,437444.
NugrohoPrasetyo,E.,Kudanga,T.,Steiner,W.,Murkovic,M.,Nyanhongo,
G. S. & Guebitz, G. M. (2009). Antioxidant activity assay based on
laccasegenerated radicals. Analytical and Bioanalytical Chemistry, 393,
679687.
Omata, Y., Saito, Y., Yoshida, Y. & Niki, E. (2008). Simple assessment of
radical scavenging capacity of beverages. Journal of Agricultural and
FoodChemistry,56,33863390.
Organisation Internationale de la Vigne et du Vin (OIV). Maximum
acceptablelimitsofvarioussubstancescontainedinwine.Compendium
ofInternationalMethodsofAnalysis,OIVMAC101:R2011.
Pramanik, S., Banerjee, P. & Bhattacharya, S.C. (2007). Interaction of 8
hydroxypyrene1,3,6trisulphonate
in
alkyltrimethylammonium
243
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244
CAPTULO 4
CHAPTER 4
Upconvertingphosphors
J.GodoyNavajas,T.Riuttamki,T.Soukka.Evaluationofdifferent
donoracceptor pairs for the development of homogeneous
bioaffinity assays using upconversion luminescence resonance
energytransfer.
energaelectrnica(EET)esunprocesoquetienelugarentredosmolculas
omateriales.Unadeestasmolculasactacomodador,emitiendoenerga
aunadeterminadalongituddeonda,mientrasquelaotramolculaacta
como aceptor, absorbiendo la energa emitida. Cuando las dos molculas
son fluorescentes, a este mecanismo se le conoce como transferencia de
energa resonante de fluorescencia (FRET) o de luminiscencia (LRET). No
obstante, la energa no siempre se transmite por fluorescencia y no
necesariamente el aceptor emite fluorescencia ya que pueden utilizarse
tambininhibidoresdefluorescencia.Paraqueesteprocesotengalugarse
tienequecumplirunaseriederequisitos:(1)lamolculadadoradebetener
un elevado rendimiento cuntico; (2) debe existir un solapamiento
247
Captulo4
sustancialentreelespectrodeemisindeldadoryelespectrodeabsorcin
delaceptor;(3)esnecesariounadecuadoalineamientoentrelosmomentos
detransicindelaabsorcinylaemisin;y(4)ladistanciaqueseparaal
dadordelaceptordebesermuycorta,normalmentemenorde10nm[1].
LosUCPshansidousadosparaeldesarrollodediferentesensayos
basadosenlatecnologaLRETutilizandofluorforosconunalongitudde
ondadeabsorcinsimilaralaemisindelUCPencuestin.Porejemplo,
los UCPs se han usado como dadores, mientras que el fluorforo Oyster
556 se utiliz como aceptor para el desarrollo de un inmunoensayo
homogneo para la determinacin de estradiol [2]. Utilizando otros tipos
defluorforos,comosonlasficobiliprotenas(Figura1),sehadescritoun
ensayodetransferenciadeenergaconlosUCPscomodadoresmedianteel
sistemadeafinidadbiotinaestreptavina[3].Enestosestudiosdeafinidad,
losUCPsseunenaestreptavidina(SA)paraformarunconjugadoSAUCP
que acta como dador y un fluorforo orgnico se enlaza a biotina para
que acte como aceptor. Cuando el dador y el aceptor interaccionan, se
produce la transferencia de energa, cuya intensidad se utiliza como
parmetroanaltico.EstametodologaLRETpermiteexcitarenlazonadel
infrarrojocercano(paralosUCPsdopadosconYb(III),laexcitacinocurre
a 980 nm). En esta zona del espectro, la probabilidad de interferencias
debidasalamatrizdelamuestraesrelativamentebaja,loquecontribuyea
disminuir las seales de fondo y, por tanto, a mejorar los lmites de
deteccin. La emisin ocurre en la zona del visible, sobre 600 nm, si se
utiliza ficoeritrina como aceptor. Los ensayos de afinidad desarrollados
por el grupo del Prof. Soukka, basados en metodologas LRET, han
implicadoelusodeUCPsdopadosconEr(III)paraformareldador[2,3]
248
Upconvertingphosphors
Figura1.Fenmenodetransferenciadeenergaresonantedeluminiscencia
(LRET) donde el dador es un UCP, el aceptor es un fluorforo y la
interaccin entre ambos se lleva a cabo mediante el sistema de afinidad
biotinaestreptavidina.
249
Captulo4
BIBLIOGRAFA
(1)
J.Fan,M.Hu,P.Zhan,X.Peng.Energytransfercassettesbasedon
organicfluorophores:constructionandapplicationsinratiometricsensing.
Chem.Soc.Reviews(2013)42,2943.
(2)
energytransferbiosensorwitharomaticpolymernanospheresasthelabel
freeenergyacceptor.Anal.Chem.(2013)85,258264
(5)
250
Upconvertingphosphors
bioaffinity
assays
using
upconversion
luminescenceresonanceenergytransfer.
Chapter4
andacceptorabsorptionspectrahavetooverlapinahighextent;(3)an
adequate alignment of absorption and emission transition moments is
required;and(4)thedistancethatseparatesdonorfromacceptorneeds
tobeveryshort,usuallylowerthan10nm[1].
TheUCPshavebeenusedtodevelopdifferentassaysbasedon
LRETusingfluorophoreswithanabsorptionwavelengthsimilartothe
UCP emission wavelength. For instance, the UCPs have been used as
donors, while the Oyster 556 fluorophore was used as acceptor to
develop a homogeneous immunoassay for the determination of
estradiol [2]. Another energy transfer assay has been reported using
other types of fluorophores, such as phycobiliproteins as acceptors
(Figure 1) and UCPs as donors, using the biotinstreptavidin affinity
system[3].Inthesestudies,theUCPsbindstreptavidin(SA)togiverise
to a SAUCP conjugate, which acts as a donor and an organic
fluorophoreboundtobiotinistheacceptor.Whendonorandacceptor
interact, the energy transfer process occurs, which intensity is used as
analytical parameter. This LRET methodology allows the excitation in
the near IR region of the spectrum (Yb(III)doped UCPs are excited at
980nm).Inthisregionofthespectrum,theprobabilityofinterferences
owingtosamplematrixisrelativelylow,whichcontributestodecrease
background signals, and hence, to improve detection limits. When
phycoerythrin is used as acceptor, the emission happens in the visible
region, at about 600 nm. The affinity assays based on LRET
methodologies developed by the research team of Prof. Soukka have
involvedtheuseofEr(III)dopedUCPstogiverisetothedonor[2,3]
252
Upconvertingphosphors
Morerecently,abiosensorbasedontheLRETphenomenonthat
involves the use of UCPs as donors and nanospheres of polym
phenylenediamine(PMPD)asacceptorsfortheDNAdeterminationin
human serum [4] has been designed. On the other hand, these
nanocrystalscancombineothertypesofnanomaterialstogiverisetoan
efficientenergytransfer.TheenergytransferbetweenUCPsandcarbon
nanoparticleshasbeenthebasisforthedesignofanewhomogeneous
biosensorforthedeterminationof2metalloproteinaseinblood[5].
The study included in this Dissertation is aimed to expand the
applicability of Ho(III) and Tm(III)doped UCPs, which can be used
individually or in multiplexed assays as donors together with the
conventionally used Er(III)doped UCPs. On the other hand, several
253
Chapter4
organicfluorophoresbelongingtoATTOandAlexaFluorfamilieshave
beenassayedinordertoinvestigatetheirpotentialuseasacceptors.
LITERATURE
(1)
J.Fan,M.Hu,P.Zhan,X.Peng.Energytransfercassettesbased
K.Kuningas,T.Ukonaho,H.Pkkil,T.Rantanen,T.Lvgren,
254
Upconvertingphosphors
Evaluationofdifferentdonoracceptorpairsforthe
developmentofhomogeneousbioaffinityassaysusing
upconversionluminescenceresonanceenergytransfer
J.GodoyNavajasa,T.Riuttamkib,T.Soukkab
DepartmentofAnalyticalChemistry.ResearchInstituteonFineChemistry.Campus
Rabanales.AnnextoMarieCurieBuilding.14071Crdoba.Spain
bDepartmentofBiotechnology.UniversityofTurku.Biocity6A.20520Turku.
Finland
Abstract
pairs for the development of affinity assays with biotin as model analyte is
reported. Upconversion luminescence resonance energy transfer (LRET) has
been measured for several streptavidincoated upconverting phosphors
(UCPs)(donors)withdifferentbiotinylatedfluorescentacceptors.Thedonors
assayed were streptavidincoated UCPs of different chemical composition,
such as NaYF4, Yb(III)Er(III); NaYF4, Yb(III)Ho(III) and NaYF4, Yb(III)Tm(III).
Biotinylated derivatives of suitable fluorescent acceptors, namely
phycoerythrin (BPE), Rphycoerythrin (RPE), Alexa Fluor 488 (AF488), Alexa
Fluor546(AF546),AlexaFluor680(AF680)andATTO495,wereassayed.
The results obtained show that the LRET intensity depends on the
UCPconsidered.ThebrightestluminescencewasobtainedwithEr(III)doped
UCP,butthesensitivityoftheassays,intermsofbiotinIC50values,wasnot
influencedbyUCPcomposition.TheuseofTm(III)providesgoodopportunity
to develop LRET assays using biotinylated Rphycoerythrin (bioRPE) as the
acceptor. Thus, the present study opens the possible use of UCPs other than
Er(III)dopedonestodevelopsensitiveLRETaffinityassays.
Keywords:homogeneousaffinityassays,upconversionluminescence,resonance
energytransferassays,nanosphosphors.
255
Captulo4
Introduction
The analytical applications of antiStokes luminescence have
experienced a huge increase in the last decade, with the advent of
lanthanide upconverting phosphors (UCPs). These phosphors are
composed of inorganic host lattices, where NaYF4 is one of the most
commonly used, although the use of Gd and La lattices has also been
reported [17]. These inorganic matrices are usually doped with the
infrared emitting Yb(III) ion together with other lanthanide ions, such as
Er(III),Ho(III)andTm(III),amongothers.
ThephenomenonofantiStokesluminescencehappensbyirradiating
thesenanophosphorswithalasersourceintheinfraredregion,generallyat
980nm,andmeasuringthereaftertheemissionoftheotherlanthanideions
in the green to red region of the spectrum. This effect constitutes an
advantage compared to Stokes luminescence since many undesirable
phenomena that usually affect the performance of photoluminescence
emission techniques, such as the autofluorescence coming from sample
matrix when excited at short wavelengths in the UVvisible region, are
avoided.Samplematricesdonotabsorbatthelongexcitationwavelength
used to excite UCPs, so a high spectral selectivity is attained. Another
advantage of using these nanophosphors is the excellent photostability
owing to their inorganic nature and to the low power required for their
excitation,thusbeingtheselasersourcesmuchcheaperthanthepowerful
lasers required for twophoton excitation. Also, some excellent properties
that are common to conventional Stokes lanthanide sensitized
luminescence, such as narrow emission peaks, long luminescence lifetime
256
Upconvertingphosphors
andtheirlowtoxicitymakethemespeciallysuitableforbioanalyticaland
biomedicalapplications[2,8].Someapplicationsdescribetheiruseaslabels
in heterogeneous assays to determine prostate specific antigen (PSA) [8]
and biotin [9,10]. However, many analytical applications of UCPs have
been focused on the development of luminescence resonance energy
transfer (LRET) assays [1114], which have opened new possibilities for
homogeneous binding assays. LRET phenomenon relies on the
nonradiative energy transfer from one fluorescent molecule (donor) to an
acceptor,whichisalsoafluorophore.
The excellent features of UCPs above mentioned have enabled the
avoidance the influence of background signals from biological samples,
and hence, to obtain relatively low detection limits using homogeneous
assays. The use of UCPs in bioanalysis requires these nanocrystals to be
biocompatibleandreadilydispersedinwater,owingtotheirhydrophobic
shell,e.g.ofoleicacid,aftersomesynthesisprocedures[1,2].Thus,theuse
of functionalization procedures is needed, among which ligand exchange
[1114] and silanization [1,2] have been used for this purpose. Ligand
exchange procedures are simple to perform but it has been reported that
theligandsattachedtothesurfacecanbefinallylost.Thestabilityofsilica
encapsulatedUCPsisbetter,althoughithasbeenobservedinsomecasesa
decrease in the luminescence intensity, which has been ascribed to the
remainingfreeaminogroupsonthesurfaceandtoasubsequentdecrease
in the quantum yield [15]. Furthermore, the performance of LRET assays
relies on the use of streptavidincoated UCPs donors and biotinylated
acceptors, where similar functionalization procedures to those above
mentioned are needed. However, the comparison of the performance of
257
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258
Upconvertingphosphors
2.Experimentalprocedures
2.1.Reagents
Different infrared to visible nanosized UCPs, which were doped
withEr(III),Ho(III)andTm(III)andobtainedbyareprecipitationmethod
[16], were used to perform all the experiments. Fluorescent
phycobiliproteins, such as phycoerythrin and Rphycoerythrin (BPE)
were purchased from Cyanotech Corp (KailuaKona, HI), supplied in 100
mM sodium phosphate buffer, pH 7.0, containing 60% ammonium
sulphate and 0.02% (w/v) sodium azide, with protein concentration of 10
mg/mL.AlexaFluor488,AlexaFluor546andAlexaFluor680succinimidyl
esterswerepurchasedfromMolecularProbesInvitrogenPaysley,UK)and
ATTO 495 succinimidylester was obtained from Sigma. Streptavidin was
purchased from SpaBioSpa (Milan, Italy). Biotinamidohexanoic acid N
hydroxysuccinimide ester (bioLCNHS) was from Pierce (Rockford, IL).
Fluorescentprotein(bioBPE)wasbiotinylatedwitha25foldmolarexcess
of NHSLCbiotin according to a previously described procedure [11]. A
freshly prepared solution of NHSLCbiotin in dimethylformamide was
addedintoproteinsolutioncontaining8.5mg/mLphycobiliproteinsin50
mMcarbonatebuffer,pH9.3.Thereactionmixture,withatotalvolumeof
500Landcontainingabout2%(v/v)DMF,wasincubatedinrotationat17
rpm (Rotamix RK, HetoHolten A/S, Allerod, Denmark) protected from
lightfor3hatroomtemperature.Finally,itwaspurifiedusingNAP5and
NAP10 columns (Amersham Biosciences, Uppsala, Sweden). Polyacrylic
acid ammonium salt, Additol XW330 (MW 30000 50000) (Surface
259
Captulo4
(TMED,
nhexanol,
97%
N(3trimethoxysilyl)propyl)ethylene
purity)
and
3(trihydroxysilyl)propyl
2.2.Instrumentation
A Plate Chameleon (Hidex Oy, Turku) equipped with a 200 mW
infrared laser module (Roithner Lasertechnik) and RG850 nm longpass
excitation filter (Andover Corp., Salem, NH) was used. The
260
Upconvertingphosphors
AnamountofUCP(ca.1mg)isplacedinanEppendorftubeanda
261
Captulo4
luminescencewasmeasuredusingChameleon5instrumentfittedwiththe
appropriatefiltersforeachUCPcomposition.
2.4.MeasurementofUCPconcentration
ThepreparationofUCPstandardsandUCPsampleswasdoneby
diluting thephosphor in the measurement buffer and by sonicating them
for1min.Then,aduplicateanalysiswasdoneinduplicatebyadding150
L to each well. The measurements were obtained for 2 s by using a 550
nm for Er(III) and Ho(III) doped UCPs and 470 nm for Tm(III)doped
UCPs.
2.5.UCPsuspensionandpreprecipitation
The dispersion of the synthesized nanophosphors is made more
reproducibleinsizebyasuspensionandpreprecipitationmethodtogeta
morecolloidalformoftheseUCPsandtoremovethelargestaggregatesin
order to discard them prior to the functionalization step. The suspension
wasperformedbyweighing15mgofUCPinanEppendorftubeand930
L of 50% diethyleneglycol in doubly deionized water were added. The
suspension was vortexed and sonicated using 20 + 20 cycles with 100%
amplitude. The bath was left in swirl for 3 min and the dispersion was
vortexedagainandsonicatedwith20+20cycles.TheEppendorftubewas
incubated again in rotation at 60 C for 1 h and then, sonicated in swirl
until the UCP was totally dispersed, and incubated for 1 h at room
temperature. The mixture was vortexed and sonicated for 20 + 20 cycles
againandincubatedonrotationovernight.
262
Upconvertingphosphors
Thepreprecipitationprocesswasdonejustbeforecoatingandthe
UCPsuspensionwaslettostandfor30minand870Lofthesuspension
wastakenfromthetopandtransferredtoanothertubeandtherestofthe
dispersion,whichcontainedlargeparticles,wasdiscarded.
2.6.Functionalizationofthesurfacewithcarboxylicacidgroups
Coating with poly(acrylic acid): An amount of preprecipitated
UCPs (5 mg, 390 L), was taken and mixed with Additol 0.1% and the
reaction mixture was left to react overnight at 35 C in rotation.
Afterwards, the Additol excess was removed by centrifugating first for 7
min at 8000 rpm, then, the supernatant was replaced with MilliQ water
and the dispersion vortexed and sonicated for 1 min and subjected to
centrifugation again. This process was repeated four times, leaving the
thirdwashaliquotfor45minatroomtemperatureandrotation.Thewash
process was ended by suspending UCPs in MilliQ water, until a final
volumeof300L.
CoatingwithglutaricanhydrideafterUCPsilylation:Anamountof
preprecipitatedUCPs(6mg,470L)wastakenforsilylation.Theprocess
was done by using a reverse micelle microemulsion method, which was
performed first by mixing 11.25 mL of cyclohexane, 2.66 mL of Triton X
100 and 2.7 mL of nhexanol. The mixture was divided in two different
microemulsionfractionsA(7.5mL)andB(therestofthemicroemulsion).
The tube with microemulsion A wasmixed with 435 L UCP suspension
and 90 L of ammonium hydroxide were added and the tube was
sonicatedinabathfor10min.TothetubewithmicroemulsionB,350Lof
water were added to give rise to a waterinoil microemulsion, 9.4 L of
263
Captulo4
TEOS,37.5LofTMEDand37.5LofTHPMPweresubsequentlyadded
and the mixture vortexed after each addition. Both tubes A and B were
combined, vortexed and incubated for 2 h at room temperature with
sonication in the first 10 min and the rest of the time with continuous
vortexing.Afterwards,avolume(2.25mL)ofacetonewasaddedtobreak
the microemulsion and precipitate the UCPs, which were collected after
centrifugation at 2860 rpm for 1 min. The supernatant was transferred to
anothertubeandacetonewasaddedtoreachatotalvolumeof20mLand
centrifuged again (2860 rpm, 3 min), the supernatant was discarded and
the UCPpellet was suspended in a small volume (400 L) of doubly
deionized water. The UCP particles obtained after the two precipitation
processeswerecombinedinasingle2mLEppendorftube.Theprecipitate
waswashedtwicewithethanolandtwicewithdoublydeionizedwater,by
sonicating each washing for 1 min and centrifugating at 8000 rpm for 7
min.Thepelletwasfinallysuspendedindoublydeionizedwatertoobtain
afinalvolumeof500L.ThesefunctionalizedUCPswerestoredatroom
temperature in rotation and the concentration of the silicafunctionalized
NPs was measured following the procedure described in section 2.4. The
next step was to change the aminofunctionalized UCPs into carboxylic
functionalizedUCPs.
silylated amino UCPs with glutaric anhydride. The UCPs obtained in the
above mentioned procedure were centrifuged (12000 rpm, 7 min), the
supernatantliquidwasdiscardedand150Lofdrypyridinewasadded.
Then, glutaric anhydride was added in a 10fold mass ratio compared to
UCP mass, after being dissolved in 120 L of pyridine. The mixture was
264
Upconvertingphosphors
vortexedandsonicatedfor5minandlatertransferredtoabathfor2.5hat
50 C. Afterwards, another batch of glutaric anhydride was diluted and
added and the process was repeated as for the first batch. At the end, a
volume(400L)ofethanolwasaddedandcentrifugedat12000rpmfor7
min. Finally, the pellet was washed twice with doublydeionized water,
suspended in 450 L of doublydeionized water and stored at room
temperatureinslowrotation.
2.7.ConjugationofCOOHfunctionalizedUCPswithstreptavidin
The conjugate of streptavidin (SA) with the functionalized UCPs
wassynthesizedbyusingacarbodiimidereactioninthepresenceofsulfo
NHS and EDC. To start the process, functionalized UCPs were washed
with 1 mL of MES buffer (pH 6.1, 20 mM) and then, they were re
suspended in 500 L. On the other hand, 20 mM EDC and 30 mM sulfo
NHSweredissolvedbyweighingtheappropriateamountbroughttoroom
temperature.Afterwards,avolume(10L)ofsulfoNHSandavolume(10
L) of EDC were added to the UCP dispersion and the resulting mixture
wasincubatedfor45minatroomtemperatureinrotation.Afterthistime,
thedispersionwaswashedwithMESbuffer,centrifugedat12000rpmfor7
min and the dispersion was reconstituted with MES buffer. To the
synthesized sulfosuccinimidyl ester, 33 L of streptavidin (30 mg/mL)
were added to the mixture and incubated for 2.5 h at room temperature
and in rotation. The reaction was stopped by adding 12.8 mL of 2 M
glycine, pH 11, and the mixture was allowed to react for 30 min with
rotation. The conjugate obtained was purified by washing twice with
borate buffer (10 mM, pH 8.5) containing 0.1% Tween 20 and one with
265
Captulo4
2.8.Homogeneousacceptortitration
The amount of biotinacceptor concentration necessary to saturate
SAUCPbindingsiteswasfoundusingahomogeneousacceptortitration,
which involves the use of biotin as the analyte, a donor (a SAconjugate)
and an acceptor (biotinylated fluorophores). The microtiter plates used
wereblackhalfarea96wellplates.
The assay was performed as follows: a volume (32 L) of biotin
12.5 M or same volume of reaction buffer was added to 24 L of a SA
UCP(0.05mg/mL,0.015mg/mlinthefinalreactionmixture).Themixture
266
Upconvertingphosphors
was incubated for 15 min at 23 C and at 900 rpm using a plate tape to
protecttheplatefromlightandtheluminescencewasmeasuredwiththe
Chameleon5instrument,usinganemissionfilterof550nm,for2000ms.
Afterwards, biotinylatedacceptor at different concentrations (0.25, 0.74,
2.2, 7,20 nM in the finalreaction mixture) wasadded and theincubation
was performed at 23 C at 900 rpm. Luminescence measurements were
takenat45minusing600and550nmasemissionfiltersfor2000msinthe
caseofEr(III)andHo(III)dopedUCPs,andof555and470nmforTm(III)
dopedUCPs.Signalsweretreatedasdescribedpreviouslyelsewhere[11].
2.9HomogeneousLRETaffinityassayforDbiotin
Thehomogeneouscompetitiveformatforbiotindeterminationwas
also assayed using Dbiotin concentrations in the range of 0 12.5 M in
theassaytube.TheconcentrationofSAUCPwas0.015mg/mLforEr(III)
and Ho(III) donors and 0.03 mg/mL for Tm(III) donor. The acceptor
concentrationsusedwere0.2and0.4nM,forbioBPE,1.33nMforbioRPE
and4nMforAF680.A12.5Mbiotinconcentrationwasusedtoverifythe
backgroundlevel.
3. ResultsandDiscussion
3.1.
Luminescentfeaturesofdonoracceptorpairs
The LRET study presented has been carried out using the
267
Captulo4
donortotheacceptor,thusresultinginacceptoremission.Thisprocessis
subject to the following two conditions: 1) The excitation spectrum of the
acceptorhastooverlapdonoremission,and2)Thedistancebetweendonor
andacceptorfollowsFrsterequation.
biotin D,
biotinylatedfluorophore
Figure2showstheexcitationandemissionspectraofthedifferent
donoracceptor pairs used in this study. Figure 2.A shows the up
conversion emission spectra of the Er(III)dopedUCP (donor) when
excited at 980 nm and the excitation and emission spectra of
phycoerythrin (BPE) (acceptor). As it can be seen, the overlap of UCP
emissionandBPEexcitationiscomplete.AcriticalaspecttoenhanceFRET
emission is the selection of the appropriate wavelengths of the emission
filter to measure acceptor emission without collecting the signal coming
fromacceptorselfexcitation.Forthisreason,althoughmaximumemission
ofBPEhappensat575nm,a600/40nmfilterwaschosentomeasureLRET
intensity.TheUCPemissionat550nmwasmeasuredbyusing535/50nm
filter. This combination of donoracceptor pairs has been previously used
for the development of several affinity assays [11, 13] and homogeneous
immunoassays [12] to determine estradiol. Other fluorescent acceptors,
268
Upconvertingphosphors
suchasAF546(ex556,em570nm)andAF680(ex680,em702nm),were
alsoassayed.AsitcanbeobservedinFig.2.B)the600/40nmfilterisstill
appropriatefortheEr(III)dopedUCPAF546pair,whilea750/40nmfilter
isusedwhenAF680isusedasacceptor.A665/10nmfilterwasusedinthis
instancetocollectUCPemission.
SimilarluminescencespectrawerealsorecordedforHo(III)doped
(Fig 2.D and 2.E) and Tm(III)doped (Figs 2.F2.H) UCPs as donors using
different fluorescent acceptors. For Ho(III)dopedUCPs, BPE and AF546
wereassayedaspotentialacceptors,usinginbothcases600/40nmfiltersto
measuretheLRETintensity.ThisispossibleowingtothefactthatHo(III)
dopedUCPshavetheirmaximumemissionat535nm.Inasimilarwayas
for Er(III)doped UCPs, the wide excitation spectra of BPE allows its
efficient excitation. The excitation of AF546 is less favoured because only
theemissionofthedonormatchestheexcitationoftheacceptorat50%of
itsmaximumintensity.However,itsemissionwavelengthenablestheuse
of600/40tomeasureLRETwithminimumbackground.
Tm(III)dopedUCPshavealoweremissionwavelengththanEr(III)
andHo(III)centeredat472nm(Figs.2.F2.H).Althoughthebestoverlapis
obtainedfortheTm(III)dopedUCPandAF488pair,comparedtotheuse
ofRPEandATTO495.RegardingthefiltersusedtomeasureLRET,theuse
of600/40nmfilterseemstobethebestoptiontocollecttheemissionfrom
RPE, although some emission from acceptor selfexcitation could still
happen.ForAF488,severalfilterssuchas520/10,555/20and600/40,could
be used to collect emission. As it can be seen, the emission intensity at
600/40isreallypoor.ThebestoptionforATTO495seemedtobethefilter
269
Captulo4
555/20,sincetheintensityobtainedwasaboutthreetimeshigherthanthat
obtainedwiththe600/40nmfilter.
500
550
600
650
200
700
400
450
WAVELENGTH (nm)
600
400
600/40
200
400
450
500
550
600
650
1000
600/40
400
450
500
550
600
WAVELENGTH (nm)
700
800
400
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500
550
600
650
700
650
700
1000
G)
800
600
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400
200
555/20
400
600
WAVELENGTH (nm)
E)
400
200
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WAVELENGTH (nm)
600
700
600
700
F)
800
650
200
800
WAVELENGTH (nm)
1000
600
400
600/40
800
550
600
WAVELENGTH (nm)
D)
1000
500
800
400
450
500
550
600
650
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WAVELENGTH (nm)
1000
H)
800
600
400
600/40
450
400
C)
1000
200
555/20
400
600
600/40
200
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750/40
400
600
B)
1000
600/40
520/10
800
A)
1000
400
450
500
550
600
650
700
WAVELENGTH (nm)
Figure 2. Emission spectra obtained for different UCPs (solid line) and excitation and
emission spectra of different acceptors (dashed lines). In 2.A2.C, Er(III)doped UCP
emissionspectratogetherwithBPE(2.A),AF546(2.B)andAF680(2.C).In2.Dand2.E,
Ho(III)doped UCPs with BPE (2.D) and AF546 (2.E). In 2.F2.H, Tm(III)doped UCPs
withRPE(2.F),AF488,(2.G)andATTO495(2.H).
3.2.
Homogeneousacceptortitrationexperiments
The aim of these titration assays is to find the amount of
270
Upconvertingphosphors
271
Captulo4
800000
600000
400000
200000
4
3
1,2
[Bio-BPE] (nM)
InanattempttoincreasethesignalexhibitedbySAHo(III)doped
UCPs,higherequivalentUCPconcentrations(0.03and0.045mg/mL)were
assayed,usingbioBPEasacceptor(Figure4).However,theLRETsignals
obtainedwere5timeslowerthanthoseobtainedforSAEr(III)dopedUCP
donors.A0.4nMbioBPEand0.03mg/mLSAEr(III)UCPwerechosento
performhomogeneousassaysforbiotindetermination,sincetheyprovide
an adequate LRET signal with a bioBPE concentration relatively low,
reachinganadequatesensitivity.
272
Upconvertingphosphors
160000
120000
80000
40000
0
4
8
12
16
20
-1
[Bio-BPE]
x
10
(nM)
Figure4.InfluenceontheconcentrationofHo(III)dopedUCPontheluminescenceand
on the sensitivity of homogeneous acceptor titration curves. [donor] are: (1) 0.015
mg/mL, in (2) 0.03 mg/mL in (3) 0.045 mg/mL. LRET signals were calculated as
indicatedinFigure3.
was
performed
by
assaying
three
surfactant
273
Captulo4
aminogroupsfromsilicalayercanmodifyUCPintensity[15],whichcould
explain the different LRET intensity obtained for identical donor and
acceptorconcentrations.TheuseofsilicatoencapsulateUCPsseemstobe
a good alternative to the use of Additol since the decrease in the
luminescence intensity is relatively small. Thus, all the homogeneous
assays involving either Er(III), Ho(III) or Tm(III) nanosized UCPs were
performed after functionalization by silylation procedures to obtain the
donors.
1000000
800000
4
600000
3
2
400000
1
200000
0
5
10
15
20
-1
[Bio-BPE] x 10 (nM)
274
Upconvertingphosphors
BPEandRPEareveryfluorescentproteinscommerciallyavailable,
but they are relatively expensive. Thus, the feasibility of other alternative
fluorophores, with suitable wavelengths to produce LRET was checked.
Figure6showsthetitrationcurvesobtainedforSAEr(III)doped(6.A)and
SAHo(III)doped (6.B) UCP donors with bioBPE and bioAF546 as
acceptors. The LRET intensity was much lower for bioAF546 and the
increaseofSAHo(III)dopedUCPconcentrationdidnotimprovetheLRET
signal unlike it happened for the pairs with SAEr(III)doped UCPs and
bioBPE as donor and acceptor, respectively. The same behaviour was
observedforSATm(III)donors,inwhichtheacceptorsusedwerebioRPE,
bioAF488andbioATTO495(Fig.6.C).AllofthemprovidedLRETsignal,
but the highest intensity was obtained using bioRPE, so this pair was
chosen to develop further homogeneous affinity assays for biotin with
Tm(III)dopedUCPsasdonors.
3.3DeterminationofbiotinusinghomogeneousLRETassays
Several donoracceptor pairs were used to perform homogeneous
LRET assays for biotin. Figure 7 shows the curves for biotin obtained for
the donoracceptor pairs studied. These pairs were chosen amongst those
which provided better LRET intensities with minimal acceptor
concentrations.
275
10
15
20
25
276
u p co n versio n L R E T (cts)
15
20
[acceptor] (nM)
10
10
25
12
30
14
[acceptor] (nM)
20000
40000
60000
u p c o nv ersio n LR E T (c ts )
40
2
1
10
80
[bio-RPE] x 10-1, nM
60
[acceptor] (nM)
20
20000
40000
60000
80000
100000
120000
12
100
14
u p co n versio n L R E T (cts )
u p co n vers io n L R E T (c ts )
u p c o n ve rs io n (L R E T ) (c ts )
Figure6.Homogeneoustitrationcurvesobtainedfordifferentdonoracceptorpairs.In6.A)[Er(III)dopedUCP=0.015
mg/mL and acceptors were: (1) bioBPE; (2) AF680 and (3) AF546. In 6.B) [Ho(III)doped UCP] were (1) 0.030 mg/mL
withbioBPEasacceptor,(2)0.015mg/mLwithbioAF546asacceptorandin(3)0.030mg/mLwithAF546asacceptor.In
(C) [Tm(III)doped UCP] was used as donor and the conditions were (1) [donor] = 0.0075 mg/mL and bioAF488 as
acceptor,in(2)[donor]=0.0075mg/mLandbioATTO495asacceptor,in(3)[donor]=0.045mg/mLandbioAF488as
acceptor, in (4) [donor] = 0.045 mg/mL and bioATTO495 as acceptor, in (5) [donor] = 0.015 mg/mL and bioRPE as
acceptor.LRETsignalswerecalculatedasindicatedinFigure3.
100000
80000
100000
C)
500000
100000
200000
300000
120000
200000
300000
400000
500000
1000000
B)
400000
500000
1500000
A)
2000000
2500000
Captulo4
Upconvertingphosphors
4000000
250000
A)
B)
3000000
2000000
1000000
200000
150000
100000
50000
0
0,1
10
100
1000
10000
0,1
[Biotin] (nM)
10
100
1000
10000
[Biotin] (nM)
120000
C)
100000
80000
60000
40000
20000
0,1
10
100
1000
10000
[Biotin] (nM)
Figure7.HomogeneousLRETaffinityassaysforDbiotinwithdonoracceptor
pairsgivingthebestresults.InA)withEr(III)dopedUCP(0.015mg/mL)and
(1)0.4nMBPEand(2)4nMAF680.In(B)Ho(III)dopedUCP(0.030mg/mL)
and0.2nMBPE.In(C)Tm(III)dopedUCP(0.030mg/mL)and1.33nMRPE.
LRETsignalswerecalculatedasindicatedinFigure3.
277
Captulo4
the results obtained for homogeneous acceptor assays and the IC50
obtainedforbiotin.
Table 1. IC50 values for biotin assays using different donor-acceptor pairs
IC50 for biotin (nM)
Acceptor
Donor
Er(III)-doped UCP
(0.015 mg/mL)
Bio-BPE
Bio-BPE
Bio-RPE
Bio-AF680
(0.2 nM)
(0.4 nM)
(1.33 nM)
(4 nM)
---
8.57*
---
14.37
5.7
---
---
---
---
7.20
---
---
---
---
3.35
---
Ho(III)-doped
UCP, 0.015
mg/mL)
Ho(III)-doped
UCP, 0.015
mg/mL)
Tm(III)-doped
UCP
(0.030 mg/mL)
*The concentration of bio-BPE was 0.3 nM
278
Upconvertingphosphors
4.
Conclusions
AsystematicstudyonthesuitabilityofdifferentUCPdonoracceptor
pairshasbeenperformedusingbiotinasmodelanalyte.TheuseofEr(III)
doped UCPs has resulted in higher LRET intensities, the maximum of
which have been obtained using bioBPE as acceptor. Although BPE and
RPE have shown to provide the best LRET intensities, the use of other
fluorophores, such as AF680 allows determinations of biotin with IC50
valuesinthenMrange.
The results obtained show the potential feasibility of multiplexed
assaysusingdifferentLRETdonoracceptorpairs.
References
(1)
(2)
Wang F., Banerjee D., Liu Y., Chen X., Liu X. Analyst (2010) 135,
18391854.
(3)
MisiakM.,ProrokK.,CichyB.,Berdnarkiewicz,StrekW.Thulium
cocentration quenching in the upconverting aTm3+/Yb3+ NaYF4
colloidalnanocrystals.Opt.Mat.(2013)35,11241128.
(4)
Jiang T., Song W., Liu S. Qin W., Synthesis and upconversion
luminescene properties study of NaYbF4:Tm3+ crystals with
differentdopantconcentration.J.FluorineChem.(2012)140,7075.
279
Captulo4
(5)
(6)
(7)
WnukA.,KaczkanM.,FrukaczZ.,PrackaI.,ChadeyronG.,Joubert
M.F., Malinowski M. Infrared to visible upconversion in
holmiumdoped materials. J. Alloys Compounds (2002) 341, 353
357.
(8)
(9)
(10)
(11)
(12)
280
Upconvertingphosphors
transferinahomogeneousimmunoassayforestradiol.Anal.Chem.
(2006)78,46904696.
(13)
Rantanen T., Pkkil H., Jmsen L., Kuningas K., Ukonaho t.,
Lvgren T., Soukka T. Tandem dye acceptor used to enhance
upconversion
fluorescence
resonance
energy
transfer
in
homogeneousassays.Anal.Chem.(2007)29,63126318.
(14)
(15)
Kong Y.L., Wang Z.L., Lin C.K., Quan Z.W., Li Y.Y., Li C.X.
Biofunctionalization of CeF3:Tb3+ nanoparticles. Nanotechnology
(2007)18,075601.
(16)
Hyppnen I., Hls J., Kankare M., Lastusaart M., Pihlgren L.,
Soukka T., Terrae Rarae. Preparation and upconversion
luminescence properties of NaYF4:Yb3+,Er3+ nanomaterials. Terra
Rarae(2009)16,16.
(17)
GodoyNavajasJ.,AguilarCaballosM.P.,GmezHensJ.Synthesis
andcharacterizationofoxazinedopedsilicananoparticlesfortheir
potential use as stable fluorescent reagents. J. Fluoresc. (2010) 20,
171180.
281
CAPTULO 5
CHAPTER 5
Discusin de resultados
Discusinderesultados
Introduccin
En este captulo se discuten las caractersticas de los mtodos
desarrolladosenestaMemoria,realizandounestudiocomparativodesus
ventajas y limitaciones frente a mtodos previamente descritos en la
bibliografa.Tambinsediscutelautilidaddelasmetodologaspropuestas
mediantesuaplicacinalanlisisdealimentos.
El captulo se ha dividido en tres apartados: 1)Nanopartculas de
sliceenanlisisdealimentos,2)Nuevasestrategiasparaladeterminacin
de antioxidantes en alimentos y 3) Nuevas investigaciones en el uso de
upconverting phosphors en sistemas de transferencia de energa de
resonancialuminiscente.
1. Nanopartculasdesliceenanlisisdealimentos.
Como ya se ha indicado, el uso de nanomateriales est teniendo un
importanteaugeendistintasreasdelaQumicaAnaltica[15].Dentro
de la gran diversidad de nanomateriales existentes en la actualidad, la
utilizacin de nanopartculas de slice (SiO2NPs) se ha extendido
rpidamente a lo largo de la ltima dcada gracias a sus excelentes
propiedades que las hacen viables para su uso como marcadores en el
campodelbioanlisis.Enelsiguienteapartadosediscutirnlosresultados
obtenidosconestasNPsentresdelosartculosincluidosenestaMemoria.
285
Captulo5
286
Discusinderesultados
componentessepuedeobtenerunamicroemulsindeaguaenaceite(rica
en disolvente orgnico) o bien una microemulsin de aceiteenagua (rica
en agua). De acuerdo a lo descrito en la bibliografa, se ensay el uso de
ciclohexano, 1hexanol y Tritn X100 para formar la microemulsin, y
mientrasqueelprecursordesliceseleccionadofueelTEOS[2,4,6,810].
Sin embargo, las condiciones experimentales anteriormente descritas no
fueronsatisfactoriasparalasntesisdeNPsdopadasconazulniloovioleta
de cresilo. Los estudios realizados sobre la posible composicin de la
microemulsin pusieron de manifiesto que las NPs sintentizadas en
ausencia de ciclohexano presentaban similares caractersticas a las
obtenidasenpresenciadeestedisolvente,enloreferenteanmerodeNPs
y tamao, pero la intensidad de fluorescencia obtenida fue ligeramente
mayor.ComopuedeobservarseenlasimgenespresentadasenlaFigura
1, obtenidas mediante microscopa electrnica de transmisin (TEM), la
cantidad y tamao de las NPs en ausencia de ciclohexano (Figura 1a) fue
comparable con las NPs obtenidas utilizando 7,5 mL de ciclohexano
(Figura1c).Sinembargo,elusodevolmenesintermediosdeciclohexano
(Figura1b)proporcionaNPsdemayortamaoyunaelevadadistribucin
de tamaos. Segn estos resultados, se prescindi del ciclohexano en el
proceso de sntesis de las NPs dopadas con azul nilo y violeta de cresilo,
utilizndose agua, Tritn X100 y 1hexanol para la formacin de la
microemulsin.
Tras estudiar la influencia de la cantidad de Tritn X100, se
observ que el nmero de NPs dopadas con azul nilo aumentaba con la
cantidad de tensoactivo utilizada, mientras que el tamao de las NPs
287
Captulo5
288
Discusinderesultados
Adems,serequiereunaetapaadicionalenelprocesodesntesis,conun
mayorconsumodereactivosytiempo.
289
Captulo5
Lacantidaddeazulniloutilizadaenelprocesodesntesisesotra
290
Discusinderesultados
deestaintensidaddefluorescenciaconcantidadesdefluorforomayores,
puede atribuirse a la formacin de agregados moleculares de azul nilo,
cuyafluorescenciaesmenorqueladelmonmero[7].
Figura3.Influenciadelacantidaddeazulniloadicionadaenlaintensidadde
fluorescencia de las NPs dopadas con este fluorforo. Condiciones
experimentales:510520mg(0,790,81mmol)deTritnX100,11,3mL(0,63
mol)deagua,100L(0,44mmol)TEOS,3mL(0,024mol)hexanoly70L(0,9
mmol)NH4OH.
Trasestudiarlainfluenciadetodaslasvariablesinvolucradasenel
291
Captulo5
ambosmtodos.Conelfindecompararlascaractersticasluminiscentesde
ambos tipos de NPs se prepararon suspensiones de igual concentracin,
observndosequeeldimetromediodelasNPsobtenidasporelmtodo
de microemulsin es ligeramente mayor que el de las obtenidas por el
segundo mtodo, aunque las diferencias no son excesivamente elevadas.
Encambio,sexistendiferenciasdestacablesenlosvaloresdeintensidades
defluorescencia.Esteestudioserealizpreparandosuspensionesenmedio
acuoso de las NPs obtenidas mediante ambos mtodos, encontrando que
las NPs del mtodo optimizado presentaban una intensidad de
fluorescencia12vecesmayorquelasobtenidasenelmtodoStber,loque
puedeatribuirsealamenordegradacindelvioletadecresiloenelmedio
alcalinoutilizadoduranteelprocesodesntesis,graciasalaproteccinque
leproporcionaelmediomicelar.
Tabla1.ComparacindelaspropiedadesdelasNPsdopadasconvioleta
decresilo.
Mtododemicroemulsin
demicelasinversas
0.1
Stbermethod
Cantidaddepartculas,mg
0.02
0.02
Dimetro,nm
178
133
2.94x109
7.06x109
125563
10138
RelacinIntensidad/Npartculas
4.27x105
1.44x106
Intensidadespecfica
1.45x107
1.17x106
Concentracin,mg/mL
NmerodePartculas
Intensidada
0.1
292
Discusinderesultados
293
Captulo5
294
Discusinderesultados
295
Captulo5
296
Discusinderesultados
protenasdesojasehaincrementadosustancialmentedebidoalosaspectos
beneficiosos para la salud que se han descrito para estas protenas, tales
como un descenso del colesterol en sangre, prevencin de enfermedades
como el cncer, la diabetes o la obesidad, as como proteccin frente a
enfermedades que daan el intestino o el rin. Sin embargo, tambin se
ha descrito que el consumo de estas protenas puede originar reacciones
alrgicas.
En esta Memoria se describe el desarrollo de un inmunoensayo
heterogneo competitivo para la determinacin de protenas de soja en
muestras de alimentos utilizando SiO2NPs dopadas con azul nilo para
formarelmarcador,estudindosedosmodalidadesdeinmunoensayo,con
capturadeanalitoyconcapturadeanticuerpo,comoseesquematizaenlas
Figuras 4 y 5. Las NPs fueron unidas a protenas de soja y a anticuerpos
antiprotena de soja para estudiar su viabilidad como trazadores en los
dosformatosdeinmunoensayo.Elprimerodeellos(Figura4),concaptura
deanalito,implicalainmovilizacindeanticuerposantiIgGdeconejoen
297
Captulo5
lasuperficiedelospocillosseguidadelainmovilizacindelosanticuerpos
antiprotenadesojayelposteriorusodelasNPsunidasalaprotenade
soja como marcador. Se utilizaron anticuerpos antiIgG debido al menor
costequesuponenfrentealusodeanticuerposantiprotenadesoja,para
recubrirlatotalidaddelospocillos.Elusodedichosanticuerpossobrelos
que se unen los anticuerpos frente al analito disminuye la concentracin
necesariadeestosltimosy,porlotanto,elcostetotaldelinmunoensayo.
La superficie de los pocillos que forman la microplaca fue tratada con
albmina de suero bovino (BSA) a diferentes concentraciones para evitar
uniones no especficas entre el marcador y los anticuerpos antiIgG
inmovilizados previamente. No obstante, los ensayos realizados
demostraron que el procedimiento no fue suficientemente adecuado ya
que,peseadichorecubrimientoconBSA,anseproducandichasuniones
noespecficas.
298
Discusinderesultados
Para solucionar este problema, se opt por hacer uso del formato
de inmunoensayo con captura de anticuerpo (Figura 5). Para ello, las
protenas de soja se inmovilizaron sobre la superficie de la microplaca,
adicionando posteriormente BSA para evitar posibles uniones no
especficasconelmarcador,formadoporanticuerposantiprotenasdesoja
marcados con las NPs. A continuacin, tras adicionar la muestra y el
marcador a los pocillos, se produjo el inmunoensayo basado en la
competencia entre la protena de soja presente en la muestra y la
inmovilizadaenlaplacaporenlazarsealmarcador.Despusdeincubar,se
elimina la mezcla de reaccin, se lavan los pocillos para eliminar las
especies no unidas a la superficie y se llevan a cabo las medidas de
fluorescenciaenlasuperficiedelospocillos.
Lasmedidasserealizaronaex620yem680nm,obtenindoseun
intervalolinealde0,110mg/Lyunlmitededeteccinde0,05mg/L,el
299
Captulo5
cual es 10 veces menor que el obtenido con el kit comercial ELISA [41].
Adems, a diferencia del mtodo ELISA, en este caso no es necesaria la
adicin de sustrato enzimtico, reducindose as el nmero de etapas del
ensayoyladuracindelanlisis.
Laprecisin,expresadacomodesviacinestndarrelativa,ensayada
a dos concentraciones diferentes, 0,5 y 5 mg/L, fue de 9,6 % y 6,1%
respectivamente.Laselectividaddelmtododependeprincipalmentedela
selectividad de los anticuerpos antiprotena de soja. De acuerdo con las
especificaciones indicadas por el proveedor de estos anticuerpos, estos
presentan cierta reactividad cruzada con extractos de harina de arroz y
ovoalbmina tras su tratamiento con urea caliente. En cambio, no
interaccionan con extractos de carne, maz o casenas, as como arroz o
patata. Adems, en un estudio previo en el que se utiliza el mismo
anticuerpo antiprotena de soja, se describe que las concentraciones de
BSA, globulinas, mioglobina y hemoglobina toleradas son 80 veces
mayoresquelaconcentracindeanalito[42].
El mtodo fue aplicado al anlisis de muestras de leche, yogur y
zumo de frutas que contenan soja. Estas muestras tambin fueron
analizadasconunkitcomercialELISA[41]y,enamboscasos,lasmuestras
fueron sometidas a un tratamiento de desnaturalizacinrenaturalizacin
de acuerdo con las recomendaciones procedentes del distribuidor de los
anticuerposantiprotenadesoja.Losresultadosobtenidossemuestranen
la Tabla 2, los cuales fueron sometidos al test de la t por parejas con un
nivel de confianza del 95 %, sin encontrarse diferencias estadsticamente
significativasentreelmtodopropuestoyelkitcomercial.
300
Discusinderesultados
Tabla2.Determinacindeprotenasdesojaenmuestrasdealimentos
Muestra
MtodoPropuestoa,b
Lechedesoja
Zumodefrutaysoja
KitcomercialELISAa,b
746
843
8,40,7
10,50,3
644
603
Yogurdesoja
MediaDE(n=3)
Unidades:lechedesojaeng/L;zumodefrutasysojayyogurdesojaeng/Kg
Tabla3.Recuperacionesdeprotenasdesojademuestrasdealimentos
Aadidoa,b
Encontradoa,b
Recuperacin(%)
Lechedesoja
24
272
111,0
72
694
95,7
123
11010
89,4
Zumodefrutasysoja
2,7
2,20,2
81,5
8,1
7,70,6
95,1
13,5
121
88,9
Yogurdesoja
23
212
91,3
65
614
93,2
113
957
84,1
Muestra
MediaDE(n=3)
Unidades:lechedesojaeng/L;zumodefrutasysojayyogurdesojaeng/Kg
301
Captulo5
302
Discusinderesultados
303
Captulo5
(clorhidrato
de
N(3dimetilaminopropil)Netilcarbodiimida),
la
304
Discusinderesultados
Aadidoa,b
(g/Kg)
1,5
Encontradoa,b
(g/Kg)
1,60,1
Recuperacin
(%)
106,7
1,960,09
98,0
4,10,2
102,5
1,5
1,50,1
100,0
2,00,1
100,0
3,40,3
85,0
1,5
1,250,07
83,3
1,70,1
85,0
4,30,3
107,5
Lechedesnatada
Lechesemidesnatada
Lecheentera
MediaDE(n=3)
305
Captulo5
Desdeelpuntodevistaanaltico,existendostiposdemtodospara
ladeterminacindelacapacidadantioxidantedelosalimentos.Elprimer
grupoimplicareaccionesdetransferenciadeelectrones,lascualespueden
306
Discusinderesultados
seguirsemediantealgncambiodecolorodefluorescenciadetectableen
el medio de reaccin. El segundo grupo se basa en reacciones de
transferencia de protones, en las que el antioxidante y un sustrato
compitenporlosradicaleslibresgenerados.ElmtodoORAC(capacidad
deabsorcinderadicalesdeoxgeno),pertenecienteaestesegundogrupo,
utiliza un generador de radicales libres que ataca directamente a un
cromforooaunfluorforo,experimentandostosunadisminucindesu
absorbancia o de su fluorescencia, respectivamente. Para este ensayo se
hanutilizadocromforos,talescomoelrojodepirogalol[50],yenmucha
mayorextensindiferentestiposdefluorforostalescomolaficoeritrina
y la fluorescena [51 57]. A pesar de la buena sensibilidad que ofrecen
estos compuestos, el desplazamiento Stokes que muestran es bastante
estrecho (alrededor de 27 nm), lo cual favorece los fenmenos de
dispersin de la radiacin. Adems, el coste de la ficoeritrina es
relativamente elevado y suele sufrir fenmenos de fotodescomposicin,
mientras que la emisin de la fluorescena normalmente solapa con las
sealesdefondoprocedentesdelamatrizdelamuestra.ElusodeLWFs
puede evitar estos inconvenientes, aunque su potencial en ensayos de
actividadantioxidantenohasidoinvestigadohastaahora.
En esta Memoria se describe un mtodo analtico para la
determinacin de la capacidad antioxidante de algunos alimentos
medianteelensayoORACutilizandoazulnilocomoreactivofluorescente,
diclorhidrato de 2,2azobis(2amidinopropano) (AAPH) como generador
deradicaleslibres,ycido6hidroxi2,5,7,8tetrametilcroman2carboxlico
(Trolox) como analito de referencia. Las medidas de fluorescencia se
307
Captulo5
llevaronacaboenmodocinticoregistrandosuvariacinconeltiempoy
utilizandocomoparmetroanalticoelreanetabajolacurvanormalizada,
AUCneta = (AUCmuestra AUCblanco)/AUCblanco), obtenida para diferentes
concentracionesdeanalito.
Como
se
ha
comentado
anteriormente,
los
fluorforos
308
Discusinderesultados
18000
18000
16000
a)
Intensidadfluorescenciarelativa
Relative fluorescence intensity
Intensidadfluorescenciarelativa
Relative fluorescence intensity
16000
14000
12000
10000
8000
6000
4000
2000
b)
14000
12000
10000
8000
6000
4000
2000
0
0
20
40
60
80
100
20
40
60
80
100
Tiempo
(min)
Time (min)
Time(min)
(min)
Tiempo
18000
Relative
Intensidadfluorescenciarelativa
fluorescence intensity
16000
c)
14000
12000
10000
8000
6000
4000
1
2000
0
20
40
60
Tiempo
(min)
Time (min)
80
10
Figura6:CurvasobtenidasparaelensayoORACconlosfluorforos(a)azul
nilo,(b)azureA,y(c)azureB:(1)blancoy(2)enpresenciadeTrolox(2M).
309
Captulo5
0.10
0,10
reabajo lacurvanormalizada
AUC
A)
0.08
0,08
0.06
0,06
0.04
0,04
0.02
0,02
0,00
0.00
5.5
5,5
6.0
6,0
6.5
6,5
7.0
7,0
pH
7.5
7,5
8.0
8,0
8.5
8,5
310
Discusinderesultados
B)
0.12
0,12
0.08
0,08
AUC
reabajo lacurvanormalizada
0.16
0,16
0,04
0.04
0,00
0.00
0
0
100
100
200
200
[NB], nM
[azulnilo](nM)
300
300
400
400
Elintervalolinealdinmicoobtenidofuede0,88MdeTroloxy
el lmite de deteccin fue de 0,45 M, el cual es menor o similar a los
obtenidos en otros mtodos ORAC donde se utiliza la fluorescena como
fluorforo. La precisin del mtodo se ensay a dos concentraciones
311
Captulo5
Vinoblanco(Manzanilla)
1,500,09
1,30,2
Vinosemiseco
7,70,9
4,40,4
Vinotinto
15,30,4
10,40,9
2,2220,001
1,930,09
Zumodepia
2,70,3
2,3990,006
Zumodemanzana
2,70,3
3,50,4
Zumodemelocotn
MediaDE(n=3)
ExpresadacomomiliequivalentesdeTroloxenlamuestra
312
Discusinderesultados
Sellevacabounestudioderecuperacionesparavalidarelmtodo
Estudioderecuperaciones
Muestra
Vinoblanco
Aadidoa
2,2
8,9
11,1
Encontradoa,b
2,00,2
6,70,9
9,20,8
Recuperacin(%)
92,0
75,2
82,9
Vinosemiseco
4,4
8,8
13,2
3,60,2
8,10,3
12,90,6
81,8
92,1
97,0
Vinotinto
17,8
35,6
44,4
182
384
444
101,1
106,7
99,1
Zumodemelocotn
2,2
4,4
5,5
2,10,1
3,50,4
4,30,2
95,5
79,5
78,2
Zumodepia
2,2
4,4
5,5
2,50,2
4,50,3
5,80,4
113,6
102,3
105,5
Zumodemanzana
4,4
3,20,2
8,8
7,90,5
11,1
111
aUnidadesenmiliequivalentesdeTroloxenlamuestra
bMediaDE(n=3)
313
72,7
89,6
99,1
Captulo5
Losresultadosobtenidosmuestranlautilidaddelafluorescenciaa
largalongituddeondaparaladeterminacindelacapacidadantioxidante
de alimentos utilizando el fluorforo azul nilo. El uso de este reactivo en
lugardeotrosfluorforospreviamenteutilizadosconestepropsito,como
sonlafluorescenaylaficoeritrina,suponeunaalternativaparareducir
posiblessealesdefondoprocedentesdelamatrizdelamuestra,lascuales
pueden aparecer al excitar a longitudes de onda ms cortas. Adems, el
relativamente ancho desplazamiento Stokes que presenta el azul nilo
minimiza los efectos de dispersin de la radiacin, muy comunes al usar
fluorforos con desplazamientos Stokes relativamente estrechos. Por
ltimo,laprdidadefluorescenciadebidaalafotodescomposicindelazul
nilo es menor que en el caso de la ficoeritrina, siendo tambin su coste
inferior.
314
Discusinderesultados
315
Captulo5
HO
O
ONa
NaO
O
ONa
O
Figura9.Estructuradelfluorforo8hidroxipireno1,3,6trisulfonato
trisdico(HPTS).
316
Discusinderesultados
1,2
1,0
14
0,8
0,6
0,4
0,2
0,0
10
15
20
Tiempo (min)
Figura10.CurvascinticasobtenidasparaelsistemaHPTSlaccasaenausencia
(curvas 1 y 2) y en presencia (curvas 3 y 4) de 2 M de cido glico, y en
presencia(curvas1y3)yenausencia(curvas2y4)de1mg/mLdeTb4O7NPs.
[HPTS]=10M,[laccasa]=1U/mL,pH=7,0,[fosfato]=0,4M,temperatura=
370C.
TambinseestudilaposibilidaddeutilizarionesTb(III)enlugar
delasTb4O7NPsparaaumentarlavelocidaddereaccindelsistema.Con
317
Captulo5
estefinseutilizaronconcentracionesequivalentesdeTb(III)alasutilizadas
enformadeNPs,obtenindosereasbajolacurvanetasmenores,locual
ratifica la utilidad de estas Tb4O7NPs como activadoras en el sistema
enzimtico.
medianteelmtodounivarianteutilizandoelreabajolacurvaneta(AUC)
como parmetro analtico. Sin embargo, fue necesario alcanzar una
solucin de compromiso entre este parmetro y el tiempo necesario para
llevar a cabo la medida, con el fin de obtener una sensibilidad y una
duracindelensayoadecuadas.
LaactividaddelalaccasaylaconcentracindeTb4O7NPssondos
318
Discusinderesultados
319
Captulo5
1,0
0,8
16
0,6
0,4
0,2
0,0
0
10
20
30
40
Tiempo (min)
320
Discusinderesultados
Tabla7.Influenciadealgunosagentesreductoresenelmtodopropuesto
para la determinacin de antioxidantes fenlicos a una concentracin de
cidoglicode2M
Compuestos
Relacinmaximatolerada
interferente/analito*
cidomlico
100
cidoctrico
100
Sacarosa
100
Glucosa
25
Sulfitosdico
20
cidoascrbico
*Laconcentracinmximaensayadafue100vecesmayorqueladelanalito
El mtodo propuesto fue aplicado al anlisis de muestras de vino
tinto,blancoydulce,lascualessolorequirieroncomotratamientopreviola
dilucinadecuadaparaquelaconcentracindepolifenolespresentealser
321
Captulo5
Tabla8.Concentracindeantioxidante(expresadocomoequivalente
milimolardecidoglico)
Muestra
Mtodopropuestoa,b MtodoFolinCiocalteua,b
Vinotinto(Rioja)
7,00,4
11,50,1
Vinotinto(Cuenca)
9,50,5
14,040,18
Vinoblanco(Rueda)
1,000,06
1,920,04
1,000,03
2,0280,005
1,270,09
3,640,05
Vinoblanco
(Valdepeas)
Vinodulce(Montilla)
a
MediaDE(n=3)
Unidades:equivalentemMdecidoglico
322
Discusinderesultados
Tabla9.Valoresderecuperacinparalasmuestrasdevinoanalizadas.
Muestra
Aadidoa
Encontradoa,b Recuperacin(%)
Vinotinto(LaRioja)
4,30,5
108,3
12
10,00,5
84,1
20
161
81,0
3,20,2
80,0
12
11,70,3
97,4
20
18,90,8
94,5
0,25
0,240,03
97,2
0,75
0,770,04
102,8
1,25
1,080,03
86,7
Vinoblanco
0,25
0,230,03
92,0
(Valdepeas)
0,75
0,700,04
92,2
1,25
1,00,1
83,3
1,010,09
101,5
3,00,3
102,0
4,70,3
95,0
Vinotinto(Cuenca)
Vinoblanco(Rueda)
Vinodulce(Crdoba)
MediaDE(n=3)
Unidades:equivalentesmMdecidoglico
323
Captulo5
324
Discusinderesultados
la
gran
variedad
de
fluorforos
existentes,
las
ficobiliprotenas,talescomolaficoeritrina(BPE)ylaRficoeritrina(RPE),
han sido utilizadas anteriormente como aceptores en diferentes ensayos
LRET [77 79]. Su uso se debe principalmente a su elevada emisin de
fluorescencia, aunque tienen el inconveniente de un coste relativamente
elevado. Por este motivo se ha estudiado la posibilidad de utilizar otros
tipos de fluorforos de menor coste para el desarrollo de ensayos de
afinidad LRET, como los Alexa Fluor o los ATTO. Para llevar a cabo este
estudio sistemtico entre dadores y aceptores, los UCPs fueron marcados
conestreptavidina(SA)mientrasquelosfluorforosfueronmarcadoscon
biotina(Bio).
EnlaFigura12semuestranlosespectrosdeemisindelosdadores
y los espectros de excitacin de los aceptores. Las Figuras 12.A12.C
correspondenalacombinacindelUCPdopadoconEr(III)conlaprotena
fluorescenteBPEylosfluorforosAlexaFluor546(AF546)y680(AF680),
observndose un buen solapamiento entre los espectros de emisin y
excitacindedadoryaceptor,respectivamente.
325
Captulo5
326
400
200
550
600
650
400
700
450
600
400
200
400
450
500
550
600
650
500
550
600
650
700
800
400
200
450
500
550
600
650
700
700
G)
1000
800
600
600/40
400
200
555/20
200
600
600
400
400
600/40
600
450
500
800
400
700
800
700
F)
650
E)
1000
1000
600
200
600/40
800
600/40
D)
550
400
1000
500
600
400
450
500
550
600
650
700
H)
1000
800
600
400
600/40
500
200
C)
800
200
555/20
450
400
400
600
520/10
800
1000
750/40
600
800
B)
1000
600/40
A)
1000
600/40
Discusinderesultados
400
450
500
550
600
650
700
327
Captulo5
4000000
600000
A)
B)
500000
3000000
2000000
1000000
400000
300000
200000
100000
0
0,1
250000
10
100
[Biotina] (nM)
1000
0,1
10000
100
1000
10000
1000
10000
120000
D)
100000
10
[Biotina] (nM)
C)
200000
150000
100000
50000
80000
60000
40000
20000
0
0,1
10
100
1000
10000
0,1
10
100
[Biotina] (nM)
[Biotina] (nM)
Figura 13. Ensayos homogneos LRET de afinidad para biotina con los pares
dadoraceptorqueproporcionanlosmejoresresultados.A)UCPEr(III)(0,015
mg/mL)y0,4nMBPE.B)UCPEr(III)(0,015mg/mL)y4nMAF680.C)UCP
Ho (III) (0,030 mg/mL) y 0,2 nM BPE. D) UCPTm (III) (0,030 mg/mL) y 1,33
nMRPE.Lasintensidadesdeluminiscenciafueroncalculadasrestandolaseal
de fondo (exceso de biotina) a la seal mxima (ausencia de biotina), cts:
cuentas.
328
Discusinderesultados
Aceptor
Dador
BioBPE
BioBPE
BioRPE
BioAF680
(0,2nM)
(0,4nM)
(1,33nM)
(4nM)
8,57*
14,37
5,7
7,20
3,35
UCPdopadocon
Er(III)
(0,015mg/mL)
UCPdopadocon
Ho(III)(0,015mg/mL)
UCPdopadocon
Ho(III)(0,015mg/mL)
UCPdopadocon
Tm(III)
(0,030mg/mL)
*LaconcentracindeBioBPEfuede0,3nM
329
Captulo5
posibleutilidaddediferentesUCPscomodadoresdeenergaydiferentes
fluorforoscomoaceptoresdeenergaensistemasLRET,cabedestacarlo
siguiente: 1) El dador UCPEr(III) proporciona los mejores resultados al
utilizarBioBPEcomoaceptor;2)LautilizacindelfluorforoAF680como
aceptor alternativo a las ficobiliprotenas permite tambin obtener una
elevadasealfluorescente,aunqueelvalordeIC50alcanzadoparabiotina
es mayor que los obtenidos en presencia de las ficobiliprotenas. No
obstante, la utilizacin de dicho fluorforo tambin permite la
determinacin de biotina a nivel nanomolar, evitando el mayor coste que
suponeelusodelasficobiliprotenas.
ComoresumendelasinvestigacionesdescritasenestaMemoria,se
presentan en el siguiente esquema las principales ventajas y limitaciones
delasmetodologasanalticasdesarrolladasenestaMemoria:
Ventajas
Limitaciones
SiO2NPsdopadasconLWFs
Sntesisreproduciblesmedianteelmtodode No
existen
comercialmente
inversas
disponibles
NPs
opciones
sntesisdependende
de
funcionalizacin
caractersticas
fsicoqumicas del
organosilano
fluorforo
330
las
Discusinderesultados
Se
obtienen
menores
lmites
de
menos
etapas,
se
deteccin.
Se
requieren
simplificanlosensayos.
Semejoralaselectividadespectral
Ensayosparadeterminacindesustanciasantioxidantes
EnsayoORACalargalongituddeonda
de
dispersindelaradiacin
mtodos
Mejoraselectividadespectral
normalizados
ElusodeTb4O7NPspermite:
La enzima no se
puedereutilizar
requerida
o Acortarlostiemposdeensayo
Tb4O7NPsdisponiblescomercialmente
EstudiosistemticodeUCPsenfenmenosLRET
La
luminiscencia
antiStokes
utiliza No
pueden
utilizar (espectro)
interferenciadelamatrizdelamuestra
fluormetros
331
se
convencionales
Captulo5
Referencias
(1)
(2)
J.Yan,M.C.Estvez,J.E.Smith,K.Wang,X.Ho,L.Wang,W.Tan.
Dyedoped nanoparticles for bioanalysis. Nanotoday (2007) 2, 44
50.
(3)
(4)
(5)
(6)
(7)
(8)
332
Discusinderesultados
(9)
XL. Chen, JL. Zou, TT. Zhao, ZB. Li. Preparation and
fluoroimmunoassay application of new redregion fluorescent
silicananoparticles.J.Fluoresc.(2007)17,235241.
(10)
W. Yang, CG. Zhang, HY. Qu, HH. Yang, JG. Xu. Novel
fluorescent
silica
nanoparticle
probe
for
ultrasensitive
immunoassays.Anal.Chim.Acta(2004)503,163169.
(11)
(12)
G.A.Saleh,H.F.Askal,I.H.Refaat,F.A.M.AbdelAal.Reviewon
recent
separation
methods
for
determination
of
some
fluoroquinolones.J.Liq.Chromatogr.(2013)36,14011420.
(13)
of
flavonoids
and
their
metabolites
by
chromatographictechniques.TrendsAnal.Chem.(2013)47,4767.
(14)
B.Tang,K.H.Row.Developmentofgaschromatographyanalysis
offattyacidsinmarineorganisms.J.Chromatogr.Sci.(2013)51,599
607.
(15)
(16)
(17)
by
highperformance
333
liquid
chromatography,
Captulo5
(18)
S.Magiera.Fast,simultaneousquantificationofthreenovelcardiac
drugs in human urine by MEPSUHPLCMS/MS for therapeutic
drugmonitoring.J.Chromatogr.B(2013)938,8695.
(19)
R.Ciayadi,G.F.Kelso,M.K.Potdar,S.J.Harris,K.L.Walton,C.
A. Harrison, M. T. W. Hearn. Identification of protein binding
partnersofALK5Kinaseinhibitors.Bioorgan.Med.Chem.(2013)21,
64966500.
(20)
G.Pierri,D.Kotoni,P.Simone,C.Villani,G.Pepe,P.Campiglia,P.
Dugo, F. Gasparrini. Analysis of bovine milk caseins on organic
monolithic
columns:An
integrated
capillary
liquid
(21)
T.F.McGrath,C.T.Elliot,T.L.Fodey.Biosensorsfortheanalysis
of microbiological and chemical contaminants in foods. Anal.
Bioanal.Chem.(2012)403,7592.
(22)
(23)
S.Girotti,S.Ghini,E.Maiolini,L.Bolelli,E.N.Feri.Traceanalysis
of pollutants by use of honeybees, immunoassays, and
chemiluminescencedetection.Anal.Bioanal.Chem.(2013)405,555
571.
334
Discusinderesultados
(24)
(25)
D.L.Brandon,M.Friedman.ImmunoassaysofSoyProteins.J.Agr.
FoodChem.(2002)50,66356642.
(26)
(27)
(28)
(29)
nanoparticles
used
as
labels
in
solidphase
immunoassays.TrendsAnal.Chem.(2012)31,144156.
(30)
A.GmezHens,J.M.FernndezRomero,M.P.AguilarCaballos.
Nanostructuresasanalyticaltolosinbioassays.TrendsAnal.Chem.
(2008)27,394406.
(31)
D.Knopp,D.Tang,R.Niessner.Review:Bioanalyticalapplications
of biomoleculefunctionalized nanometersized doped silica
nanoparticles.Anal.Chim.Acta(2009)647,1430.
335
Captulo5
(32)
(33)
(34)
X.Zhao,L.R.Hilliard,S.J.Mechery,Y.Wang,R.P.Bagwe,S.Jin,
W.Tan.Arapidbioassayforsinglebacterialcellquantitationusing
bioconjugatednanoparticles.P.Ntl.Aca.Sci.USA(2004)101,15027
15032.
(35)
(36)
HH. Yang, HY. Qu, P. Lin, SH. Li, MT. Ding, JG. Xu.
Nanometer fluorescent hybrid silica particle as ultrasensitive and
photostablebiologicallabels.Analyst(2003)128,462466.
(37)
silica
nanoparticles
probe
for
ultrasensitive
immunoassays.Anal.Chim.Acta(2004)503,163169.
(38)
(39)
336
Discusinderesultados
(40)
Z.Ye,M.Tan,G.Wang,J.Yuan.Preparation,Characterization,and
timeresolvedfluorometricapplicationofailicacoatedterbium(III)
fluorescentnanoparticles.Anal.Chem.(2004)76,513518.
(41)
L.LHocine,J.I.Boye,C.Munyana.Detectionandquantificationof
soy allergens in food: study of two commercial enzymelinked
immunosorbentassays.J.FoodSci.(2007)72,C145C153.
(42)
(43)
(44)
(45)
H.Watanabe,A.Satake,M.Matsumoto,Y.Kido,A.Tsuji,K.Ito,M.
Maeda. Monoclonalbased enzymelinked immunosorbent assay
and immunochromatographic rapid assay for monensin. Analyst
(1998)123,25732578.
(46)
Monensin
ELISA
kit
(Cat
KA1422
V.01),
http://www.abnova.com/.
(47)
337
Captulo5
(48)
(49)
M.Ciz,H.Cizova,P.Deneb,M.Kratchanova,A.Slavov,A.Lojek.
Different methods for control and comparison of the antioxidant
propertiesofvegetables.FoodControl(2010)21,518523.
(50)
(51)
(52)
(53)
(54)
(55)
338
Discusinderesultados
(56)
L.Mller,S.Groyke,A.M.Popken,V.Bhm.Antioxidantcapacity
and related parameters of different fruit formulations. Food Sci.
Technol.(2010)43,992999.
(57)
(58)
B.Lorrain,I.Ky,L.Pechamat,P.L.Teissedre,Evolutionofanalysis
of polyphenols from grapes, wines, and extracts. Molecules (2013)
18,10761100.
(59)
(60)
(61)
(62)
L.M.Magalhes,M.A.Segundo,S.Reis,J.L.C.Lima,A.O.S.S.
Rangel.AutomaticmethodforthedeterminationofFolinCiocalteu
reducing capacity in food products. J. Agr. Food Chem. (2006) 54,
52415246.
339
Captulo5
(63)
(64)
Kizek.
Fully
determination
of
automated
antioxidant
spectrometric
activity:
protocols
advantages
for
and
disadvantages.Molecules(2010)15,86188640.
(65)
(66)
E.Nkhili,P.Brat.ReexaminationoftheORACassay:effectofmetal
ions.Anal.Bioanal.Chem.(2011)400,14511458.
(67)
(68)
(69)
(70)
340
Discusinderesultados
detectionofphenoliccompoundsinmustandwine.J.Mol.Catal.B
Enzym.(2010)64,189194.
(71)
(72)
(73)
(74)
(75)
J.Robinson(Ed.)Oxfordcompaniontowine3rdedition.(2006)35
36.
(76)
(77)
(78)
K.Kuningas,T.Ukonaho,H.Pakkil,T.Rantanen,J.Rosenberg,T.
Lvgren, T. Soukka. Upconversion fluorescence resonance energy
341
Captulo5
transferinahomogeneousimmunoassayforestradiol.Anal.Chem.
(2006)78,46904696.
(79)
fluorescence
resonance
energy
transfer
homogeneousassays.Anal.Chem.(2007)79,63126318.
342
in
Discussionoftheresults
Introduction
The features of the methods developed in this Dissertation are
Synthesisandcharacterizationoflongwavelengthfluorophore
dopedsilicananoparticles
343
Chapter5
344
Discussionoftheresults
andTEOSwasselectedasthesilicaprecursor[2,4,6,810].However,the
useoftheexperimentalconditionspreviouslydescribeddidnotgiveriseto
satisfactoryresultsforthesynthesisofthecresylvioletandnilebluedoped
silicaNPs.Severalassayswerecarriedouttooptimizethemicroemulsion
composition.ThisstudyshowedthattheNPssynthesizedintheabsenceof
cyclohexane had similar features (number and size) as those obtained
using cyclohexane, but their fluorescence intensity was slightly higher.
Figure 1 shows transmission electronic microscopy (TEM) images of the
NPs obtained in the presence of different volumes of ciclohexane. As can
be seen, the amount and sizes of the NPs synthesized in the absence of
cyclohexane(Figure1a)werecomparabletothoseachievedusing7.5mLof
cyclohexane (Figure 1c). However, the use of intermediate volumes of
cyclohexane(Figure1b)providedlargerNPswithwidersizedistributions.
Accordingtotheseresults,theuseofcyclohexanewasprecludedandonly
water,TritonX100and1hexanolwereselectedasthecomponentsofthe
microemulsion.
ThestudyoftheinfluenceofTritonX100amountshowedthatthe
numberofnilebluedopedNPsincreasesandtheirsizedecreasesasTriton
X100amountincreases.Thebestresultswereobtainedusing510520mg
ofTritonX100(Figure1f)becauseloweramountsgaveNPsofsizeshigher
than200nm(Figure1dand1e).VerysmallandpartiallyaggregatedNPs
wereobtainedwhentheTritonX100amountwascloseto1.0g.
The feasibility of a slight modification of the reversemicelle
microemulsion procedure for fluorophoredoped SiO2NPs by introducing
theuseofsilicaprecursorswasalsostudied.Thismodificationincludesa
345
Chapter5
previousreactionstepinwhichthecovalentlinkingbetweentheprimary
aminogroupsofthefluorophoreand3aminopropyltriethoxysilane(APS)
wasassayedinordertopreventthedyeleakagefromthesilicamatrix.
346
Discussionoftheresults
Nevertheless, as can be seen in Figure 1g, the NPs amount collected was
substantially lower than that obtained in the absence of APS. Also, these
NPs showed a wide size distribution. Thus, the use of these NPs as
potential labels for further assays was discarded as they do not improve
the features of the NPs obtained without this modification. Also, this
synthesis procedure requires an additional step and an increased reagent
andtimeconsumption.
It was checked that the Triton X100/hexanol ratio is a critical
variable,asFigure2shows.Asitcanbeseen,aratioof170mgTritonX
100/mL hexanol provided the maximum fluorescence intensity of the
synthesizedmaterial.
Figure2.InfluenceofTritonX100/hexanolratioonthefluorescenceintensity
ofnilebluedopedNPs.Experimentalconditions:11.3mL(0.63mol)ofwater,
100L(0.44mmol)TEOS,0.2mLof1x103M(0.2mol)nileblue,3mL(0.024
mol)hexanol,70L(0.9mmol)NH4OH.
347
Chapter5
348
Discussionoftheresults
Afterstudyingofallthevariablesinvolvedinthesynthesisprocess,
Reversemicelle
Stbermethod
microemulsionmethod
Concentration,mg/mL
0.1
0.1
Particleamount,mg
0.02
0.02
Diameter,nm
178
133
2.94x109
7.06x109
125563
10138
Intensity/particleratio
4.27x105
1.44x106
Specificintensity
1.45x107
1.17x106
Particlenumber
Intensitya
349
Chapter5
correspondingmaximumexcitationwavelengthforonehourtoinvestigate
their potential photobleaching and the results were compared to those
obtainedforthedyesinsolution.Thefluorescenceintensityofcresylviolet
andnileblueinsolutionshowedafluorescencedecreaseofabout72%and
65%,respectively,butthefluorescenceintensityofbothcresylvioletand
nile bluedoped NPs remained almost constant. The same study was
carriedoutfortheSiO2NPspreparedbyusingthenileblueAPSmixture,
obtaining a decrease of about a 10 % of the initial fluorescence intensity.
Also,thestabilityofthefluorescentSiO2NPswascheckedovertimeandit
was found that the luminescence intensity was constant for at least one
month.
Insummary,itcanbeconfirmedthatthemodifiedreversemicelle
microemulsionmethodtosynthesizetwotypesofLWFsdopedsilicaNPs,
usingnileblueandcresylviolet,providesNPswithsimilarcharacteristics
(size, amount and average diameter) as those obtained by means of the
Stber method, which is considered as a reference method. However, the
fluorescenceintensityoftheNPssynthetizedbythefirstmethodishigher
than that of NPs obtained by the Stber method. This behavior can be
350
Discussionoftheresults
Utilization
of
silica
nanoparticles
in
heterogeneous
351
Chapter5
352
Discussionoftheresults
Determinationofsoyproteinsbyheterogeneousimmunoassay
353
Chapter5
diabetes and obesity, and protection against bowel and kidney diseases.
However,ithasalsobeendescribedthatsoyproteinscanproduceallergic
responsesinsomepeople.
A competitive heterogeneous immunoassay for the determination
of soy proteins in food samples, using nile bluedoped SiO2NPs as labels,
has been developed in this Dissertation. For this purpose, two different
formatseitherinvolvinganalytecaptureorantibodycapturewereassayed,
respectively (Figures 4 and 5). These NPs were coupled to either soy
proteinsortoantisoyproteinantibodiestostudythepotentialuseofthe
corresponding tracers for the development of the two immunoassay
formats.Theanalytecaptureformat(Figure4)involvestheimmobilization
of antisoy protein onto the wells, after the previous immobilization of
antirabbit IgG antibodies, and the use of the tracer composed of soy
protein linked to nile bluedoped SiO2NPs. The use of antirabbit IgG
antibodies to immobilize the antisoy protein antibodies reduces the
consumption of these antibodies and, therefore, the total cost of the
immunoassay.However,althoughthewellsurfacewascoatedwithBSAat
different concentrations, nonspecific interactions of the tracer with the
antirabbit IgG antibodies were still observed, so that this approach was
unsuitable.
354
Discussionoftheresults
Figure4.Competitiveheterogeneousimmunoassaywithantigencaptureusing
nilebluedopedSiO2NPsaslabellinkedtosoyprotein.
355
Chapter5
antibodiesselectivity.Accordingtotheantibodyspecificationsheet,these
antibodiespresentsomecrossreactivitytowardshotureaextractsofwheat
and ovalbumin, but no crossreactivity is observed in extracts form meat,
356
Discussionoftheresults
Themethodwasappliedtotheanalysisofmilk,yoghourtandfruit
juice samples containing soy proteins. These samples were also analyzed
using a commercial ELISA kit [41]. Samples were subjected to a
denaturationrenaturation treatment according to the procedure indicated
by the antisoy protein antibody manufacturer. Table 2 shows the soy
protein content found by the reported immunoassay and by the ELISA
method, which were compared by a paired ttest carried out at a 95 %
significance level, finding that there were not statistically significant
differencesamongthem.
Table2.Determinationofsoyproteincontentinfoodsamples
Sample
Soymilk
Fruitandsoyjuice
Soyyoghourt
Reportedmethoda,b
CommercialELISAa,b
746
843
8.40.7
10.50.3
644
603
MeanSD(n=3)
Units:fruitandsoyjuice,soyyoghourt,g/Kg;soymilk,g/L
357
Chapter5
Table3.Recoveryofsoyproteinaddedtofoodsamples
Addeda,b
Founda,b
Recovery(%)
Soymilk
24
72
123
272
694
11010
111.0
95.7
89.4
Fruitandsoyjuice
2.7
8.1
13.5
2.20.2
7.70.6
121
81.5
95.1
88.9
Sample
Soyyoghourt
23
212
65
614
113
957
aMeanSD(n=3)
bUnits:fruitandsoyjuice,soyyoghourt,g/kg;soymilk,g/L
91.3
93.2
84.1
Determinationofmonensinbyheterogeneousimmunoassayin
milksamples
358
Discussionoftheresults
359
Chapter5
surface measurements as the distance between the well bottom and the
detector is shorter than that for conventional plates, which minimizes
fluorescencesignallossesandimprovesthesensitivity.
As in the previous section, two heterogeneous immunoassays,
involvingantibodyorantigencaptureformat,wereassayedtochoosethe
bestoptionformonensindeterminationusingnilebluedopedSiO2NPsas
labels. The first format was based on the immobilization of a BSA
monensin conjugate and the use of an antimonensin antibodyNP tracer.
The second assay needed a monensinNP tracer, synthesized via a
carbodiimide reaction using EDAC (N(3methylaminopropyl)N
ethylcarbodiimide hydrochloride), and the immobilization of anti
monensin antibodies by coating the wells with antisheep IgG. A
significantdifferencebetweenthefluorescenceintensityvaluesobtainedin
theabsenceandinthepresence(0.2g/L)ofmonensinwasreachedinboth
assays,butthisdifferencewasabout3foldhigherfortheantibodycapture
format.Thus,thisformatwaschosentodevelopthenewimmunoassayfor
monensindetermination.
The method presents a dynamic range of 0.05 5 g/L and the
detection and quantification limits are 0.015 and 0.05 g/L, respectively,
whichwouldcorrespondto0.12and0.40g/Kginthemilksamples.The
lattervalueisabout5timeslowerthanthemaximumresiduelimit(MRL)
set by the 37/2010/EC Commission Regulation for monensin in milk
samples. Also, the detection limit is four times lower than that reported
using ELISA with chemiluminescence detection [32]. The precision,
expressedasthepercentageofrelativestandarddeviationandassayedat
360
Discussionoftheresults
twodifferentmonensinconcentrations,0.2and1g/L,gavevaluesof5.8
and4.0%,respectively.
The method was applied to the analysis of skimmed, semi
skimmedandwholemilksamples.Thesampletreatmentwasquitesimple
as it only required the deproteinization of samples and a further solvent
evaporation step. A recovery study was also carried out to validate the
method(Table4),obtainingvaluesintherangeof83.3107.5%.
Table4.Recoveriesobtainedformonensinaddedtomilksamples
Sample
Monensn
a,b
a,b
Found
Recovery
Added
(g/Kg)
(g/Kg)
(%)
Skimmedmilk
1.5
1.60.1
106.7
2
1.960.09
98.0
4
4.10.2
102.5
Semiskimmedmilk
1.5
2
4
1.50.1
2.00.1
3.40.3
100.0
100.0
85.0
Wholemilk
1.5
2
4
1.250.07
1.70.1
4.30.3
83.3
85.0
107.5
MeanSD(n=3)
dopedSiO2NPsaslabelstodeterminemacromolecules(proteins)andsmall
molecules (antibiotics) in food analysis. This approach provides lower
detectionlimitsthanELISAandavoidsthelaststepfortheenzymeactivity
measurement. Likewise, the proposed methods have contributed to
expandtheuseofnanotechnologyinthisanalyticalarea.
361
Chapter5
2. Newstrategiesforthedeterminationofantioxidantsinfoods
Oxidation processes involving free radicals contribute to the
development of many illnesses, such as cardiovascular disease,
Alzheimersdisorderandcancer.Thisredoxphenomenonisproducedby
reactive oxygen species (ROS), which damage cell membranes, but this
effect can be minimized by the intake of antioxidant compounds. The
antioxidant capacity of foods is mainly ascribed to polyphenols, such as
flavonoids,andvitaminsCandE,amongothers[49].
Amethodforthedeterminationoftheantioxidantcapacityofsome
food samples has been described in this Dissertation, which shows the
usefulness of longwavelength measurements. Another method for the
determination of polyphenols using nanomaterials as laccase enzyme
activators is also included. The features of both methods and their
applicabilityarediscussedbelow.
Longwavelength
fluorimetric
determination
of
food
antioxidantcapacityusingnileblueasreagent
From an analytical point of view, there are two types of methods
for assessing food antioxidant capacity. The first group involves a single
electrontransfer reaction, which can be followed by a change in the
solution color or fluorescence. The second group is based on the use of a
hydrogen atom transfer reaction, where the antioxidant and a substrate
compete for the free radicals generated. The oxygen radical absorbance
capacity (ORAC) assay, which belongs to the second group, involves the
useofafreeradicalgeneratorthatdirectlyattackseitherachromophoreor
362
Discussionoftheresults
afluorophore,leadingtothedecreaseoftheirabsorbanceorfluorescence,
respectively.Thechromophorepyrogallolred[50]andthefluorophores
phycoerythrin and fluorescein [51 57], have been used to develop this
assay.Despitethehighsensitivitythattheabovementionedfluorophores
offer, these compounds feature a relatively short Stokes shift (about 27
nm), which favors scattering phenomena. Also, phycoerythrin is
relatively expensive and suffers from photobleaching, while fluorescein
emission is usually overlapped by the static background signals from the
sample matrix. The use of longwavelength fluorophores can avoid these
problems, but their potential application to the determination of the
antioxidantcapacityhasnotbeenstudieduptonow.
An analytical method has been described in this Dissertation for
food antioxidant capacity determination by the ORAC assay using nile
blue
as
fluorescent
probe,
2,2azobis(2methylpropionamidine)
363
Chapter5
18000
18000
16000
a)
16000
14000
12000
10000
8000
6000
4000
2000
b)
14000
12000
10000
8000
6000
4000
2000
0
0
20
40
60
80
100
20
40
Time (min)
60
80
100
Time (min)
18000
16000
c)
14000
12000
10000
8000
6000
4000
2000
0
0
20
40
60
Time (min)
80
10
Figure6:CurvesobtainedfortheORACassayusing(a)nileblue,(b)azureA,
and(c)azureBfluorophores:(1)blankand(2)2MTrolox.
364
Discussionoftheresults
using0.075Macetate,phosphate,borate,andcarbonatebuffersolutionsto
keepthepHconstantinthebufferingregionofeachsolution.AsFigure7
shows, the net AUC values were very low at pH below 5.8 and above 8,
obtainingthebestresultsinthepHrangeof6.07.0.Thebehaviorofthe
systematlowpHvaluescouldbeascribedtothefactthatthepKaofTrolox
isabout3.89,showingalowsolubilityatthispH.
0.10
A)
0.08
AUC
0.06
0.04
0.02
0.00
5.5
6.0
6.5
7.0
pH
7.5
8.0
8.5
rangeof60370nM(Figure8),obtainingthatthesystemwaspractically
independentofthisvariableintherangeof80130nM,choosing89nM
365
Chapter5
0.16
B)
AUC
0.12
0.08
0.04
0.00
100
200
[NB], nM
300
400
Figure8:InfluenceofnileblueconcentrationontheORACnilebluemethod.
Experimental conditions: [AAPH] = 0.012M; [Trolox] = 2 M; pH = 6.9;
[phosphate]=0.085M;temperature=37C.
366
Discussionoftheresults
Thedynamicrangeofthecalibrationgraphwas0.88MTrolox
and the detection limit was 0.45 M, which is lower or similar to those
obtained in other ORAC methods involving fluorescein. The precision of
the method was assessed at two different Trolox concentrations, 1 and 5
M,obtainingrelativestandarddeviationsof5.6and2.9%,respectively.
commercial fruit juices and the results were compared to those obtained
withthetraditionalORACmethod.Thetreatmentofthesampleswasthe
sameforbothmethods,whichjustrequiredtoadjustthepHuntilneutral
valuesandthefurthersampledilutiontomatchthedynamicrangesofthe
corresponding calibration curves. Table 5 lists the content found in the
samplesusingbothmethods.Thepairedttestwasappliedtotheresultsat
the95%significancelevel,anditwasfoundthattherewerenotsignificant
differences in the results provided by both methods, which confirms the
practicalusefulnessofthenewmethoddescribedinthisDissertation.
Table5.AntioxidantcapacityoffoodsamplesanalyzedbyORACnileblue
andORACfluoresceinmethods
Sample
ORACnilebluea,b ORACfluoresceina,b
Whitewine(Manzanilla)
1.500.09
1.030.2
Semidrywine
7.70.9
4.40.4
Redwine
15.30.4
10.40.9
2.2220.001
1.930.09
Pineapplejuice
2.70.3
2.3990.006
Applejuice
2.70.3
3.50.4
Peachjuice
Meanstandarddeviation(SD)(n=3)
ExpressedasmillimolarTroloxequivalentsofsample
a
b
367
Chapter5
wasperformedbyaddingthreedifferentamountsofTroloxtoeachsample
and subtracting the results obtained from similarly treated unspiked
samples.Table6showstherecoverypercentages,whichrangedfrom72.7
%to113.6%.Themeanrecoveryvaluesobtainedwere92.0and92.9%for
wineandjuicesamples,respectively.Thisinternalvalidationalsoconfirms
theusefulnessofthedevelopedmethodfortheanalysisofrealsamples.
Tabla6.Recoveryvaluesobtainedforthedifferentsamplesanalyzed
Recoverystudy
Addeda
Founda,b
Recovery(%)
Whitewine
2.2
8.9
11.1
2.00.2
6.70.9
9.20.8
92.0
75.2
82.9
Semidrywine
4.4
8.8
13.2
3.60.2
8.10.3
12.90.6
81.8
92.1
97.0
Redwine
17.8
35.6
44.4
182
384
444
101.1
106.7
99.1
Peachjuice
2.2
4.4
5.5
2.10.1
3.50.4
4.30.2
95.5
79.5
78.2
Pineapplejuice
2.2
4.4
5.5
2.50.2
4.50.3
5.80.4
113.6
102.3
105.5
4.4
3.20.2
8.8
7.90.5
11.1
111
aUnitsinmillimolarTroloxequivalentsofsample
bMeanSD(n=3)
72.7
89.6
99.1
Sample
Applejuice
368
Discussionoftheresults
fluorimetryforthedeterminationoftheantioxidantcapacityinfoodusing
the fluorophore nile blue. The use of this reagent instead of other
fluorophorespreviouslyreportedforthispurpose,suchasfluoresceinor
phycoerythrin,isausefulalternativetoavoidpotentialbackgroundsignals
fromthesamplematrix,whichcanappearatlowerwavelengths.Also,the
relatively wide Stokes shift of nile blue reduces the scattering signals.
Finally, the probability of photobleaching for nile blue is lower than that
forphycoerythrin,aswellasitscost.
Automaticdeterminationofpolyphenolsinwineusinglaccase
andterbiumoxidenanoparticles
369
Chapter5
Laccaseisaphenoloxidaseenzymethathasbeendescribedforthe
determination of both phenolic content and antioxidant activity, using
different experimental conditions. As it has been already mentioned, this
enzyme catalyzes the oxidation of phenolic compounds to quinones or
radicals by reducing thedissolvedoxygen to water, which has beenused
todevelopamperometricsensors[6870]aswellasphotometric[7173]
andfluorimetric[74]methods.
Theusefulnessofterbiumoxidenanoparticles(Tb4O7NPs),laccase
and the fluorophore 8hydroxypyrene1,3,6trisulfonic acid trisodium salt
(HPTS)forthedeterminationofpolyphenolsinwinesamplesisdescribed
in this Dissertation. As for the method reported in the previous section,
fluorescence measurements were carried out in the kinetic mode, by
measuring the change of fluorescence over time and using as analytical
parameter the net AUC values obtained for different concentrations of
gallicacid,whichwasusedasthecalibrationstandard.
Firstly, several potential fluorescent laccase substrates (Styryl 7, 2
Di1ASP, azure B chloride, toluidine blue O, nile blue chloride, sodium
fluorescein and HPTS) were assayed to study their capability to compete
with phenolic antioxidants for the active sites of the enzyme. Although
indocyaninegreenhasbeendescribedasalaccasesubstrate[74],itwasnot
assayed because its long excitation and emission wavelengths were not
adequateforthemicroplatereaderusedinthisstudy.Theresultsobtained
from the study of the behavior of the different fluorophores assayed
proved that only HPTS showed a relatively fast and remarkable decrease
initsfluorescenceintensityinthepresenceoflaccase.
370
Discussionoftheresults
HO
O
ONa
NaO
ONa
O
system was the study of the potential activator effect of different NPs on
the catalytic behavior of laccase. Several NPs, such as diamond, Eu2O3,
Tb4O7,AgandmagneticgoldNPswereassayedintheconcentrationrange
of 0.1 2 mg/mL, finding that only Tb4O7NPs notably increased the
reaction rate of the system. As can be seen in Figure 10, both blank and
analyte AUC decreased, which allows an increase in the sample
throughput.Also,theeffectofTb4O7NPsonthesystemwascheckedinthe
absenceoflaccase,findingthattheNPsdidnotmodifythefluorescenceof
HPTS.However,diamondandmagneticgoldNPscausedthefluorescence
quenching of HPTS, AgNPs inhibited the catalytic effect of laccase, while
371
Chapter5
nochangeinthekineticbehaviorofthesystemwasfoundinthepresence
ofEu2O3NPs.
Figure10.KineticcurvesobtainedfortheHPTSlaccasesystemintheabsence
(curves1and2)andinthepresence(curves3and4)of2Mgallicacid,andin
thepresence(curves1and3)andintheabsence(curves2and4)of1mg/mL
Tb4O7NPs.[HPTS]=10M,[laccase]=1U/mL,pH=7.0,[phosphate]=0.4M,
temperature=37C.
Tb4O7NPs,wasalsostudiedusingaconcentrationequivalenttothatofthe
NPs, but lower net AUC values were obtained. These results show the
usefulnessoftheseNPsasactivatorsoftheenzymaticsystem.
Thevariablesaffectingthesystemwereoptimizedbytheunivariate
method using the net AUC as the analytical parameter. However, it was
372
Discussionoftheresults
necessarytofindacompromisedsolutionbetweenthisparameterandthe
time required for each measurement, in order to obtain an adequate
sensitivitybutavoidingtheexcessivedurationoftheassay.
TheinfluenceofthepHonthesystemwasstudiedintherangeof
4.0 9.0 using different buffer solutions. The fluorescence of HPTS was
very low at acid pH values and very high in the range of 8.0 9.0, but
laccase did not show any enzymatic activity at the higher pH values.
However,theHPTSfluorescenceintensitywashighenoughatpH7.07.5
anddecreasedinthepresenceoflaccase.Thus,apHof7.0wasselectedfor
themethoddevelopment,usinga0.4Mphosphatebuffersolutiontoadjust
this pH value. The study of the influence of the HPTS concentration
demonstratedthatthesystemwasindependentofthisvariableintherange
of 8 12 M, and the intermediate value (10 M) was chosen as the
optimumconcentration.
ThelaccaseactivityandtheTb4O7NPsconcentrationaretwocritical
variables that have a remarkable effect on both the net AUC and the
reactionrate.Bothvariableswerestudiedintherangesof0.52U/mLand
0.2 2 mg/mL for laccase activity and Tb4O7NPs concentration,
respectively.Highvaluesofbothvariablesgaverisetoveryhighreaction
rates and low net AUC values, which negatively affected the method
sensitivity. On the contrary, very low values of these variables caused a
notable increase of the net AUC and, therefore, the improvement of the
method sensitivity, but the duration of the assay was excessively long.
Thus, the values selected to obtain appropriate net AUC and duration of
theprocesswere1U/mLlaccaseactivityand1mg/mLNPsconcentration.
373
Chapter5
thismethodwasthenetAUC,obtainedfromthenormalizedkineticcurves
for different gallic acid concentrations. Figure 11 show the kinetic curves
obtained to ex 450 nm y em 535 nm under the optimum experimental
conditions.
1.0
16
0.8
0.6
0.4
0.2
0.0
10
20
30
40
Time (min)
Figure 11. Kinetic curves obtained for different gallic acid concentrations: (1)
(1) 0 M, (2) 0.5 M, (3) 1 M (4) 2 M (5) 5 M y (6) 10 M. Experimental
conditions: [HPTS] = 10 M, [laccasa] = 1 U/mL, [NPs] = 1 mg/mL, pH = 7.0,
[phosphate]=0.4M,temperature=37oC.
Thedynamicrangeofthecalibrationgraphwas0.512Mgallic
acidandthedetectionlimitwas0.14M,whichisaboutthreetimeslower
than that obtained in the absence of Tb4O7 NPs (0.4 M). This difference
shows the positive effect of the NPs on the method, improving both the
reaction rate and the detection limit. The detection limit reached in the
presence of the NPs is lower than those described for gallic acid using
374
Discussionoftheresults
Theselectivityofthemethodwascheckedbyassayingsomenon
phenolicreducingsubstances,whichcouldbepresentinthewinesamples
used to demonstrate the applicability of the method. Table 7 shows the
results obtained after assaying different potential interfering compounds.
The most serious interference was caused by ascorbic acid, which was
tolerated at the same molar concentration than that of the analyte.
However,thisinterferencewouldbenegligiblebecausethemaximumlimit
allowedforascorbicacidwhenusedasanadditiveinwineis0.85mM[75],
which is lower than the usual content of polyphenols in wines. Sulfites
weretoleratedinaconcentration20foldthatoftheanalyte.Althoughthis
interference could be removed using a pretreatment step [70], it was not
necessary as the presence of sulfites is controlled according to their total
contentinreducingsubstances[76].
375
Chapter5
Table7.Influenceofsomereducingagentsontheproposeddetermination
ofphenolicantioxidantsat2Mgallicacid
Compound
Maximumtolerated
interferent/analyteratio*
100
Malicacid
Citricacid
100
Sucrose
100
Glucose
25
Sodiumsulfite
20
Ascorbicacid
*Maximumratioassayedwas100
The method was applied to the analysis of red, white and sweet
wines. The sample treatment only required the dilution of the sample to
match the dynamic range of the calibration curve, using gallic acid
standards and the net AUC as analytical parameter. Table 8 shows the
content of polyphenols found in these samples analyzed by the new
methodandbytheFolinCiocalteumethod,whichisusuallyconsideredas
areferencemethod.
Table8.Antioxidantconcentration(expressedasmillimolarequivalentsof
gallicacid)foreachsample
Sample
Proposedmethod FolinCiocalteumethoda,b
Redwine(Rioja)
7.00.4
11.50.1
Redwine(Cuenca)
9.50.5
14.040.18
Whitewine(Rueda)
1.000.06
1.920.04
Whitewine
(Valdepeas)
1.000.03
2.0280.005
Sweetwine(Montilla)
1.270.09
3.640.05
MeanSD(n=3)
Units:mMgallicacidequivalentsofsample
376
Discussionoftheresults
FolinCiocalteumethodwerehigherinallinstancesthanthoseprovidedby
the laccasemethod, which isascribed to the capability of the later toalso
detect nonphenolic substances. The values obtained by the new method
are similar to those obtained using other methods for the analysis of this
typeofsamples[68,69].
A recovery study was performed by adding three different
amountsofgallicacidtoeachsampleandsubtractingtheresultsobtained
fromsimilarlytreatedunspikedsamples.AsitcanbeseenintheTable9,
the recovery percentages obtained ranged from 81.0 to 108.3 % and the
meanrecoveriesforred,whiteandsweetwineswere90.9,92.4and99.5%,
respectively.
Table4.Recoverystudyforthewinesamplesanalyzed
Founda,b
Sample
Addeda
Redwine(LaRioja)
4
4.30.5
12
10.00.5
20
161
Redwine(Cuenca)
4
3.20.2
12
11.70.3
20
18.90.8
Whitewine(Rueda)
0.25
0.240.03
0.75
0.770.04
1.25
1.080.03
Whitewine(Valdepeas)
0.25
0.230.03
0.75
0.700.04
1.25
1.00.1
Sweetwine(Montilla)
1
1.010.09
3
3.00.3
5
4.70.3
bMeanSD(n=3)
aUnits:mMgallicacidequivalentsofsample
377
Recovery(%)
108.3
84.1
81.0
80.0
97.4
94.5
97.2
102.8
86.7
92.0
92.2
83.3
101.5
102.0
95.0
Chapter5
DepartmentofBiotechnologyoftheUniversityofTurku(Finland).Dueto
the short duration of this period, the research was focused to the
systematic evaluation of several donors and acceptors to design a
luminescence resonance energy transfer system (LRET) using the anti
Stokes phenomenon in biotinstreptavidin affinity assays. This study
aimed the selection of the most appropriate system for its subsequent
analyticalapplication.
378
Discussionoftheresults
the
wide
variety
of
existing
fluorophores,
Figure12showstheexcitationandemissionspectraofthedifferent
379
Chapter5
Similar spectra were obtained when the UCPs doped with Ho(III)
(Figure12.D12.E)wereassayedwithBPEandAF546.Inasimilarwayto
the Er(III)doped UCP behavior, the wide excitation spectrum of BPE
allowsitsefficientexcitationwhiletheenergytransferusingAF546shows
200
700
400
450
400
200
600
650
400
200
550
600
WAVELENGTH (nm)
700
800
600
400
200
400
450
500
550
600
650
700
650
700
G)
1000
800
600
400
600/40
600
500
600
WAVELENGTH (nm)
WAVELENGTH (nm)
800
450
500
800
700
F)
700
E)
1000
WAVELENGTH (nm)
1000
200
200
400
450
500
550
600
WAVELENGTH (nm)
650
700
H)
1000
800
600
400
600/40
550
650
400
200
555/20
500
600
450
550
600
600/40
600
500
800
WAVELENGTH (nm)
800
400
650
600/40
600
D)
1000
550
200
WAVELENGTH (nm)
400
500
400
555/20
450
400
600
520/10
800
C)
1000
750/40
400
B)
1000
600/40
600
800
600/40
A)
1000
600/40
aloweryield.
400
450
500
550
600
650
700
WAVELENGTH (nm)
Figure 12. Emission spectra from UCPs and acceptors used in this study. In
12.A12.C, Er(III)doped UCP emission spectra together with BPE (12.A),
AF546 (12.B) and AF680 (12.C). In 12.D and 12.E, Ho(III)doped UCPs with
BPE (12.D) and AF546 (12.E). In 12.F12.H, Tm(III)doped UCPs with RPE
(12.F),AF488(12.G)andATTO495(12.H).
380
Discussionoftheresults
TheTm(III)dopedUCPswerecombinedwithRPE,AlexaFluor488
fluorophorestothephycobiliproteinsBPEandRPE,inordertoavoidtheir
relatively high cost, was studied. Figure 13 shows the titration curves
obtained with Er(III)SA (13.A), Ho(III)SA (13.B) and Tm(III)SAdoped
UCPs as donors, combined with different fluorophores. Luminescence
emissionwasobtainedinallinstances,butthebestresultswereachieved
with the Er(III)BPE, Er(III)AF680, Ho(III)BPE and Tm(III)RPE pairs,
whichwereusedtoobtainthetitrationcurvesforbiotin.Asitcanbeseen
from Figure 13, the highest luminescence intensities were obtained for
Er(III)dopedUCPswithBPEandAF680.
381
Chapter5
600000
4000000
500000
3000000
2000000
1000000
0
1
10
100
[Biotin] (nM)
1000
300000
200000
100000
0,1
10000
10
100
1000
10000
120000
250000
D)
C)
100000
200000
150000
100000
50000
80000
60000
40000
20000
[Biotin] (nM)
400000
0,1
B)
A)
0,1
10
100
1000
10000
0,1
10
100
1000
10000
[Biotin] (nM)
[Biotin] (nM)
382
Discussionoftheresults
summarizesthedifferentassayscarriedoutandtheIC50valuesobtained
forbiotin,thelowestvalueforthisparameterwasachievedwiththepair
SATm(III)UCPwithRPE,inspite ofitslowLRETintensity.Thehighest
IC50 was obtained for the SAEr(III)doped UCP with AF680, which was
about14.4nM.Thevaluesobtainedarecomparableand,insomeinstances,
better than those obtained by other previously reported methods [77, 78]
usingEr(III)dopedUCPsasdonorsandBPEasacceptor.
Table10.IC50valuesforbiotinassaysusingdifferentdonoracceptorpairs
Acceptor
Donor
IC50forbiotin(nM)
BioBPE
(0.2nM)
BioBPE
(0.4nM)
BioRPE
(1.33nM)
BioAF680
(4nM)
8.57*
14.37
5.7
7.20
3.35
Er(III)doped
UCP
(0.015mg/mL)
Ho(III)doped
UCP,0.015
mg/mL)
Ho(III)doped
UCP,0.015
mg/mL)
Tm(III)doped
UCP
(0.030mg/mL)
*TheconcentrationofBioBPEwas0.3nM
systematicstudycarriedouttoevaluatethepotentialusefulnessofseveral
383
Chapter5
UCPsasdonorsandseveralfluorophoresasacceptorsinLRETsystems:1)
The donor UCPEr(III) provides the best results when it is used together
with BioBPE as acceptor; 2) The use of the fluorophore AF680 as
alternativeacceptortophycobiliproteinsprovidesahighfluorescentsignal,
althoughtheIC50valuereachedforbiotinishigherthanthatobtainedin
presenceofthephycobiliproteins.Nevertheless,theuseofthisfluorophore
also allows the determination of biotin at nanomolar concentration level,
avoidingtherelativelyhighcostofthesephycobiliproteins.
As a final summary of this chapter, the main advantages and
limitations of the new analytical methodologies developed in this
Dissertationareincludedinthefollowingtable:
Advantages
Limitations
LWFdopedSiO2NPs
Synthesis procedures using reverse
micelle
microemulsion
method
are
commerciallyavailable
A previous study of the
reproducible
Increased stability compared to the
fluorophoresinsolution
synthesis
conditions
needs
be
to
done
according to physical
chemical properties of
organosilanereagents
theLWF
Improvementofspectralselectivity
The
heterogeneous
improve
some
immunoassays
features
of
ELISAs
methodsusedforthispurposesince:
Lowerdetectionlimitsareobtained
Lesserstepsarerequired,sotheyare
simplertoperform.
384
Discussionoftheresults
Newmethodsforantioxidantdetermination
LongwavelengthORACassay
Decrease
of
light
phenomena
methods
Improvementofspectralselectivity
providedbybiosensors
TheuseofTb4O7NPsalsoallows:
reused
fluormeterscannotbe
regionofspectrum
used
other
than
phycobiliproteinscanbeusedtodevelop
LRET assays, which can decrease the
costsoftheseassays
385
Chapter5
References
(1)
(2)
J.Yan,M.C.Estvez,J.E.Smith,K.Wang,X.Ho,L.Wang,W.Tan.
Dyedoped nanoparticles for bioanalysis. Nanotoday (2007) 2, 44
50.
(3)
(4)
(5)
(6)
(7)
(8)
386
Discussionoftheresults
(9)
XL. Chen, JL. Zou, TT. Zhao, ZB. Li. Preparation and
fluoroimmunoassay application of new redregion fluorescent
silicananoparticles.J.Fluoresc.(2007)17,235241.
(10)
W. Yang, CG. Zhang, HY. Qu, HH. Yang, JG. Xu. Novel
fluorescent
silica
nanoparticle
probe
for
ultrasensitive
immunoassays.Anal.Chim.Acta(2004)503,163169.
(11)
(12)
G.A.Saleh,H.F.Askal,I.H.Refaat,F.A.M.AbdelAal.Reviewon
recent
separation
methods
for
determination
of
some
fluoroquinolones.J.Liq.Chromatogr.(2013)36,14011420.
(13)
of
flavonoids
and
their
metabolites
by
chromatographictechniques.TrendsAnal.Chem.(2013)47,4767.
(14)
B.Tang,K.H.Row.Developmentofgaschromatographyanalysis
offattyacidsinmarineorganisms.J.Chromatogr.Sci.(2013)51,599
607.
(15)
(16)
(17)
by
highperformance
387
liquid
chromatography,
Chapter5
(18)
S.Magiera.Fast,simultaneousquantificationofthreenovelcardiac
drugs in human urine by MEPSUHPLCMS/MS for therapeutic
drugmonitoring.J.Chromatogr.B(2013)938,8695.
(19)
R.Ciayadi,G.F.Kelso,M.K.Potdar,S.J.Harris,K.L.Walton,C.
A. Harrison, M. T. W. Hearn. Identification of protein binding
partnersofALK5Kinaseinhibitors.Bioorgan.Med.Chem.(2013)21,
64966500.
(20)
G.Pierri,D.Kotoni,P.Simone,C.Villani,G.Pepe,P.Campiglia,P.
Dugo, F. Gasparrini. Analysis of bovine milk caseins on organic
monolithic
columns:An
integrated
capillary
liquid
(21)
T.F.McGrath,C.T.Elliot,T.L.Fodey.Biosensorsfortheanalysis
of microbiological and chemical contaminants in foods. Anal.
Bioanal.Chem.(2012)403,7592.
(22)
(23)
S.Girotti,S.Ghini,E.Maiolini,L.Bolelli,E.N.Feri.Traceanalysis
of pollutants by use of honeybees, immunoassays, and
chemiluminescencedetection.Anal.Bioanal.Chem.(2013)405,555
571.
388
Discussionoftheresults
(24)
(25)
D.L.Brandon,M.Friedman.ImmunoassaysofSoyProteins.J.Agr.
FoodChem.(2002)50,66356642.
(26)
(27)
(28)
(29)
nanoparticles
used
as
labels
in
solidphase
immunoassays.TrendsAnal.Chem.(2012)31,144156.
(30)
A.GmezHens,J.M.FernndezRomero,M.P.AguilarCaballos.
Nanostructuresasanalyticaltolosinbioassays.TrendsAnal.Chem.
(2008)27,394406.
(31)
D.Knopp,D.Tang,R.Niessner.Review:Bioanalyticalapplications
of biomoleculefunctionalized nanometersized doped silica
nanoparticles.Anal.Chim.Acta(2009)647,1430.
389
Chapter5
(32)
(33)
(34)
X.Zhao,L.R.Hilliard,S.J.Mechery,Y.Wang,R.P.Bagwe,S.Jin,
W.Tan.Arapidbioassayforsinglebacterialcellquantitationusing
bioconjugatednanoparticles.P.Ntl.Aca.Sci.USA(2004)101,15027
15032.
(35)
(36)
HH. Yang, HY. Qu, P. Lin, SH. Li, MT. Ding, JG. Xu.
Nanometer fluorescent hybrid silica particle as ultrasensitive and
photostablebiologicallabels.Analyst(2003)128,462466.
(37)
silica
nanoparticles
probe
for
ultrasensitive
immunoassays.Anal.Chim.Acta(2004)503,163169.
(38)
(39)
390
Discussionoftheresults
(40)
Z.Ye,M.Tan,G.Wang,J.Yuan.Preparation,Characterization,and
timeresolvedfluorometricapplicationofsilicacoatedterbium(III)
fluorescentnanoparticles.Anal.Chem.(2004)76,513518.
(41)
L.LHocine,J.I.Boye,C.Munyana.Detectionandquantificationof
soy allergens in food: study of two commercial enzymelinked
immunosorbentassays.J.FoodSci.(2007)72,C145C153.
(42)
(43)
(44)
(45)
H.Watanabe,A.Satake,M.Matsumoto,Y.Kido,A.Tsuji,K.Ito,M.
Maeda. Monoclonalbased enzymelinked immunosorbent assay
and immunochromatographic rapid assay for monensin. Analyst
(1998)123,25732578.
(46)
Monensin
ELISA
kit
(Cat
KA1422
V.01),
http://www.abnova.com/.
(47)
391
Chapter5
(48)
(49)
M.Ciz,H.Cizova,P.Deneb,M.Kratchanova,A.Slavov,A.Lojek.
Different methods for control and comparison of the antioxidant
propertiesofvegetables.FoodControl(2010)21,518523.
(50)
(51)
(52)
(53)
(54)
(55)
392
Discussionoftheresults
(56)
L.Mller,S.Groyke,A.M.Popken,V.Bhm.Antioxidantcapacity
and related parameters of different fruit formulations. Food Sci.
Technol.(2010)43,992999.
(57)
(58)
B.Lorrain,I.Ky,L.Pechamat,P.L.Teissedre,Evolutionofanalysis
of polyphenols from grapes, wines, and extracts. Molecules (2013)
18,10761100.
(59)
(60)
(61)
(62)
L.M.Magalhes,M.A.Segundo,S.Reis,J.L.C.Lima,A.O.S.S.
Rangel.AutomaticmethodforthedeterminationofFolinCiocalteu
reducing capacity in food products. J. Agr. Food Chem. (2006) 54,
52415246.
393
Chapter5
(63)
(64)
Kizek.
Fully
determination
of
automated
antioxidant
spectrometric
activity:
protocols
advantages
for
and
disadvantages.Molecules(2010)15,86188640.
(65)
(66)
E.Nkhili,P.Brat.ReexaminationoftheORACassay:effectofmetal
ions.Anal.Bioanal.Chem.(2011)400,14511458.
(67)
(68)
(69)
(70)
394
Discussionoftheresults
detectionofphenoliccompoundsinmustandwine.J.Mol.Catal.B
Enzym.(2010)64,189194.
(71)
(72)
(73)
(74)
(75)
J.Robinson(Ed.)Oxfordcompaniontowine3rdedition.(2006)35
36.
(76)
(77)
(78)
K.Kuningas,T.Ukonaho,H.Pakkil,T.Rantanen,J.Rosenberg,T.
Lvgren, T. Soukka. Upconversion fluorescence resonance energy
395
Chapter5
transferinahomogeneousimmunoassayforestradiol.Anal.Chem.
(2006)78,46904696.
(79)
fluorescence
resonance
energy
transfer
homogeneousassays.Anal.Chem.(2007)79,63126318.
396
in
CONCLUSIONES
CONCLUSIONS
Conclusiones
LasinvestigacionespresentadasenestaMemoriahancontribuidoa
expandir la aplicabilidad de la Nanotecnologa al anlisis de alimentos
mediante el desarrollo de diversos mtodos que presentan niveles
adecuados de sensibilidad y selectividad. El uso de microplacas ha
permitido la automatizacin de estos mtodos con un bajo consumo de
muestras y reactivos y una elevada velocidad de muestreo. A partir del
trabajorealizadoseextraenlassiguientesconclusiones:
Sehansintetizadonanopartculasdeslicedopadasconlosfluorforos
delargalongituddeondavioletadecresiloyazulnilo.Lautilizacin
de estos nanomateriales con fines analticos permite mejorar
simultneamentelaselectividadespectral,evitandolasealdefondo
de la matriz de la muestra, y la sensibilidad, consecuencia de su
elevada relacin superficie/volumen. Los principales resultados
obtenidosconestasnanopartculasseresumenacontinuacin:
o
en
micelas
inversas
proporciona
Lainteraccinentrelascargaspositivasdeestosfluorforos
y las cargas negativas de la matriz de slice favorece la
399
Conclusiones
La
versatilidad
aplicabilidad
analtica
de
las
nanopartculasdeslicedopadasconazulnilosehapuesto
de manifiesto mediante su uso para formar marcadores en
fluorinmunoensayo heterogneo. Se ha demostrado su
utilidadparaladeterminacindemacromolculascomolas
protenasdesojaymolculaspequeascomoelantibitico
deusoveterinariomonensn.
o
SehadesarrolladounnuevomtodoORACparaladeterminacinde
lacapacidadantioxidanteenzumosyvinosconelusodelfluorforo
de larga longitud de onda azul nilo como reactivo, mejorando la
selectividad espectral de las determinaciones convencionales basadas
enelusodefluorescenaodeficoeritrina.
400
Conclusiones
401
Conclusiones
402
Conclusions
TheinvestigationsincludedinthisDissertationhavecontributedto
expand the application of Nanotechnology to food analysis by the
developmentofseveralmethodsthatpresentadequatelevelsofsensitivity
and selectivity. The use of the microplate format has allowed the
automation of these methods, also featuring low sample and reagent
consumptionandarelativelyhighsamplethroughput.Bearinginmindthe
resultsobtained,thefollowingconclusionscanbedrawn:
403
Conclusions
Theversatilityandanalyticalusefulnessofnilebluedoped
silica nanoparticles hasbeen shownby their application to
the synthesis of tracers to be used in heterogeneous
fluoroimmunoassays. Their analytical potential has been
demonstratedbydevelopingmethodsforthedetermination
of macromolecules, such as soy proteins, and for small
molecules (haptens), namely the veterinary antibiotic
monensin.
Thetwoimmunoassaymethodsdevelopedcanberegarded
as useful alternatives to the ELISA methods commonly
used for the determination of these species, since lower
detectionlimitsareachievedandtheadditionofasubstrate
tomeasuretheenzymaticactivityofthetracerisavoided.
404
Conclusions
Anewenzymaticmethodforpolyphenoldeterminationinwineswith
fluorometric detection has been developed using the fluorophore 8
hydroxypyrene3sulfonatetrisodiumandterbiumoxidenanoparticles
as new laccase substrate and activator, respectively. This study has
shown that the detection limit obtained using nanoparticles is about
threetimeslowerthanthatobtainedintheirabsence.
405
Conclusions
406
ANEXO
ANNEX
Produccin Cientfica
Scientific Production
ProduccinCientfica
ARTCULOSCIENTFICOS
Tipodepublicacin
Autores
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ndicedeimpacto
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ISSN
ndicedeimpacto
Artculoscientfico
Juan GodoyNavajas, Mara Paz AguilarCaballos,
AgustinaGmezHens
Synthesis and caracterization of oxazinedoped
silicananoparticlesfortheirpotentialuseasstable
fluorescentreagents
JournalofFluorescence
Volumen20(2010),Pginas171180
10530509(Print)15734994(Online)
1.966 (31 posicin en la seccin de Qumica
AnalticadelJournalofCitationReportde2010)
Artculoscientfico
Juan GodoyNavajas, Mara Paz AguilarCaballos,
AgustinaGmezHens
Longwavelength fluorimetric determination of
food antioxidant capacity using nile blue as
reagent
JournalofAgriculturalandFoodChemistry
Volumen59(2011),Pginas22352240
00218561(Print)15205118(Online)
2.816 (12 posicin en la seccin de Qumica
AplicadadelJournalofCitationReportde2011)
Artculoscientfico
Juan GodoyNavajas, Mara Paz AguilarCaballos,
AgustinaGmezHens
Heterogeneous immunoassay for soy protein
determination using nile bluedoped silica
nanoparticles as labels and frontsurface long
wavelengthfluorimetry
AnalyticaChimicaActa
Volumen701(2011),Pginas194199
00032670
4.555(5posicinenlaseccindeQumicaAnaltica
delJournalofCitationReportde2011)
409
Anexo
Tipodepublicacin
Autores
Ttulo
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ISSN
ndicedeimpacto
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ISSN
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Autores
Ttulo
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Artculoscientfico
JuanGodoyNavajas,MaraPazAguilarCaballos,
AgustinaGmezHens
Determinationofmonensininmilksamplesby
frontsurfacelongwavelengthfluoroimmunoassay
usingnilebluedopedsilicananoparticlesaslabels
Talanta
Volumen94(2012),Pginas195200
00399140
3.498(12posicinenlaseccindeQumica
AnalticadelJournalofCitationReportde2012)
Artculoscientfico
JuanGodoyNavajas,MaraPazAguilarCaballos,
AgustinaGmezHens
Automaticdeterminationofpolyphenolsinwines
usinglaccaseandterbiumoxidenanoparticles
EnviadoaFoodChemistry
03088146
Artculoscientfico
JuanGodoyNavajas,TerhiRiuttamki,Tero
Soukka
Evaluationofdifferentdonoracceptorpairsforthe
developmentofhomogeneousbioaffinityassays
usingupconversionluminescenceresonance
energytransfer
Enredaccin
410
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JuanGodoyNavajas
Investigacin del potencial analtico de nuevos
nanomateriales luminiscentes a larga longitud de
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Nuevas metodologas en anlisis de alimentos y
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Longwavelength
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Ttulo
fluoroimmunoassay for the veterinary antibiotic
monensinusingdopedsilicananoparticles
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determinationofantioxidantsinfoodsamples
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