Andres Miranda Martinez

UNIVERSIDAD POLITÉCNICA DE MADRID
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS EN

TELECOMUNICACIÓN
STUDY OF NEW APPLICATIONS OF THE QUARTZ

CRYSTAL RESONATOR (QCR) FOR THE
CHARACTERIZATION OF VISCOELASTIC
PROPERTIES OF BIOLOGICAL FLUIDS OF INTEREST
FOR MEDICAL APPLICATIONS
PhD THESIS
MSc. Andrés Miranda Martínez
2022
DEPARTAMENTO DE TECNOLOGÍA FOTÓNICA Y BIOINGENIERÍA
ESCUELA TÉCNICA SUPERIOR DE INGENIEROS EN

TELECOMUNICACIÓN

PhD THESIS
Author:
Andrés Miranda Martínez

MSc in Biomedical Engineering
Director:
José Javier Serrano Olmedo

PhD in Telecommunication Engineering
2022
PHD THESIS
AUTHOR: Andrés Miranda Martínez

DIRECTOR: José Javier Serrano Olmedo
Tribunal nombrado por el Magfco. y Excmo. Sr. Rector de la Universidad

Politécnica de Madrid el día
PRESIDENTE:
SECRETARIO:
VOCAL:
VOCAL:
VOCAL:
SUPLENTE:
SUPLENTE:
i
Realizado el acto de defensa y lectura de tesis el día .
En el Centro de Tecnología Biomédica de la Universidad Politécnica de Madrid.
Calificación:
EL PRESIDENTE: EL SECRETARIO:
__________________________________ __________________________________
D. D.
EL VOCAL: EL VOCAL:
__________________________________ __________________________________
D. D.
EL VOCAL:
__________________________________
D.
ii
LABORATORIO DE BIOINSTRUMENTACIÓN Y
NANOMEDICINA

PhD THESIS
Author:
Andrés Miranda Martínez

MSc in Biomedical Engineering
Director:
José Javier Serrano Olmedo

PhD in Telecommunication Engineering
2022
iii
Agradecimientos
Todo este trabajo tiene detrás a un gran número de personas sin las cuales no
hubiera sido posible su desarrollo. Primero que nada quiero agradecer a Flor,
mi compañera de vida y mi mejor amiga, quien me motiva y me apoya en los
buenos y malos momentos. Este logro se suma a los que ya tenemos y a los que
vendrán.
Agradezco a mi familia, son quienes siempre me apoyaron en mis objetivos y
me alentaron en todo momento. Agradezco a mis padres, quienes me formaron
y a quienes admiro y les debo gran parte de lo que soy hoy en día. Gracias a mi
mamá por su ejemplo y apoyo, mi constancia y responsabilidad se los debo a
ella. Gracias a mi papá por sus enseñanzas y optimismo, de él he aprendido a
disfrutar cada momento de mi vida. Gracias a mi hermana Edith por ser mi
confidente y compañera de travesuras desde pequeños. Siempre estaremos el
uno para el otro.
Agradezco a Antonio, a Flor de María y a Toño, quienes me recibieron con los
brazos abiertos desde el primer día y me han apoyado siempre que lo he
necesitado.
Gracias a todo el grupo del Laboratorio de Bioinstrumentación y Nanomedicina
(LBN), en especial a Michael, mi vecino de escritorio, y con quién compartí la
experiencia del doctorado desde el comienzo y quien fue parte de mi familia en
Madrid. También agradezco a Nazario por sus consejos desde mis comienzos
en la UPM y por su amistad.
Agradezco a Thomas Crouzier y al grupo de Biopolymers for life de KTH (Real
Instituto de Tecnología, Suecia). Mi recibimiento y estancia fueron
iv
extraordinarios. Sus enseñanzas contribuyeron a mi formación como
investigador.
Gracias, por supuesto, a José Javier Serrano Olmedo, investigador principal del
LBN, amigo y director de mi trabajo de fin de máster y de mi tesis doctoral,
quien me ha enseñado y orientado día a día en este mundo de la ingeniería
biomédica y en el mundo de la investigación a lo largo de estos 5 años.
Muchas gracias a los profesores de la UNAM José María Matías, Salvador
Landeros, Víctor García y Jesús Manuel Dorador, por su ejemplo, orientación y
apoyo que me proporcionaron en México para continuar con mi preparación
profesional.
Gracias a los revisores de calidad de la esta tesis y al tribunal por el tiempo que
le han dedicado a este trabajo.
Finalmente, gracias a mis grandes amistades en México, siempre los llevo
presentes.
v
Abstract
Quartz crystal resonators (QCRs) are widely used as sensors because of their
ability to detect changes in the physical properties of the sample placed on their
surface. In addition, they are inexpensive, sensitive, and have good accuracy. Its
small size benefits the use of small sample volumes, which opens the door to
many possible applications in biomedicine and bioengineering.
The UPM ViSQCT project, which is being developed at CTB, has developed a
prototype sensor based on QCRs. This prototype was updated in the present
work. The sensor is based on the analysis of the conductance curve, generated
by exciting the crystal through a frequency sweep (close to the fundamental
resonance frequency of the crystal). The resonance frequency at the maximum
conductance point is its primary reference when evaluating the samples
deposited on its surface.
Two approaches to the application of QCRs as sensors are proposed. First, the
development of the sensor as a valuable tool for diagnosis or classification
between different conditions of the same fluid is sought. An example of this is
the case of synovial fluid. The viscosity of healthy SF decreases in the case of
pathologies such as rheumatoid arthritis or osteoarthritis. Therefore, measuring
the sample's viscosity can be a parameter that helps the classification of the
sample. Otherwise, a larger sample volume would be required, or large and
expensive devices would be used for the same purpose.
A second approach is to use the device as a tool to characterize the formation of
biopolymer-based hydrogels. Such characterization is carried out using
expensive and large equipment. As an alternative, a low-cost, portable and
straightforward system is proposed to assist in this task. Knowledge of the
mechanical properties of these materials is essential, and the design of the
vi
mechanical properties will determine how well the material will perform
depending on its application. When used as scaffolds for cell growth, stiffness is
a determining factor in cell differentiation. If a hydrogel is sought for
bioprinting, a specific viscosity capable of flowing through the nozzle and yet
stable when forming structures will be required. In the case of implantable gels,
their mechanical properties must match the surrounding tissues. With this in
mind, having a device with the described conditions will help researchers
design hydrogels. Therefore, this work proposes the use of this device for two
biomedical applications.
First, measurements were made with synovial fluid and cerebrospinal fluid
phantoms (with contrasting viscoelastic properties), and differences between
healthy and pathological fluids were observed employing the measured
parameters. Subsequently, measurements were made with synovial fluid
provided by the Hospital Universitario La Paz in Madrid. The samples were
previously diagnosed and classified into two groups: inflammatory pathologies
and infectious pathologies. When measuring the samples, using the parameters
obtained for the differentiation of the groups showed low accuracy; however,
when applying artificial neural network algorithms, the accuracy increased.
Finally, experiments were performed with hydrogels to monitor the formation
of the gel by capturing its viscosity change with the sensor. Two types of bonds
were used for gel formation, the first by covalent crosslinking and the second
by physical crosslinking. In addition, for each case, the agents' concentration
varied to vary the physical properties of the resulting gel. The formation of both
types of hydrogels was captured, the sensor having a different response to each
type of concentration.
The proposed technique presents advantages such as simplicity, reduced
sample volume, low cost, and portability, making it accessible to any laboratory
and generating interest in further development.
vii
This thesis provides new contributions to the development of QCR-based
sensors and opens a possibility for further research lines regarding biomedical
applications.
Key words
Sensor, Quartz Crystal Resonators, Quartz Crystal Microbalance, Synovial
Fluid, Biopolymer based Hydrogels.
viii
Resumen
Los resonadores de cristal de cuarzo (QCR) se utilizan ampliamente como
sensores por su capacidad para detectar los cambios en las propiedades físicas
de una muestra colocada en su superficie. Además, son baratos, sensibles y
tienen una buena precisión. Su pequeño tamaño favorece el uso de pequeños
volúmenes de muestra, lo que abre la puerta a muchas posibles aplicaciones en
biomedicina y bioingeniería.
El proyecto ViSQCT de la UPM, que se desarrolla en el CTB, ha desarrollado un
prototipo de sensor basado en QCRs. Este prototipo ha sido actualizado en el
presente trabajo. El sensor se basa en el análisis de la curva de conductancia,
generada al excitar el cristal a través de un barrido de frecuencias (cercano a la
frecuencia de resonancia fundamental del cristal). La frecuencia de resonancia
en el punto de máxima conductancia es su principal referencia a la hora de
evaluar las muestras depositadas en su superficie.
Se proponen dos enfoques para la aplicación de los QCR como sensores. En
primer lugar, se busca el desarrollo del sensor como una valiosa herramienta de
diagnóstico o clasificación entre diferentes condiciones de un mismo fluido. Un
ejemplo de ello es el caso del líquido sinovial. La viscosidad del SF sano
disminuye en el caso de patologías como la artritis reumatoide o la artrosis. Por
lo tanto, la medición de la viscosidad de la muestra puede ser un parámetro que
ayude a la clasificación de la misma. De lo contrario, se necesitaría un mayor
volumen de muestra o se utilizarían dispositivos grandes y caros para el mismo
fin.
Un segundo enfoque es utilizar el dispositivo como herramienta para
caracterizar la formación de hidrogeles basados en biopolímeros. Dicha
caracterización se lleva a cabo utilizando equipos caros y de gran tamaño.
Como alternativa, se propone un sistema de bajo coste, portátil y sencillo para
ix
ayudar en esta tarea. El conocimiento de las propiedades mecánicas de estos
materiales es esencial, y el diseño de las mismas determinará el rendimiento del
material en función de su aplicación. Cuando se utilizan como estructuras para
el crecimiento celular, la rigidez es un factor determinante en la diferenciación
celular. Si se busca un hidrogel para la bioimpresión, se requerirá una
viscosidad específica capaz de fluir a través de la boquilla y a la vez estable al
formar estructuras. En el caso de los geles implantables, sus propiedades
mecánicas deben coincidir con los tejidos circundantes. Teniendo esto en
cuenta, disponer de un dispositivo con las condiciones descritas ayudará a los
investigadores a diseñar hidrogeles. Por ello, este trabajo propone el uso de este
dispositivo para dos aplicaciones biomédicas.
En primer lugar, se realizaron mediciones con fantomas de líquido sinovial y
líquido cefalorraquídeo (con propiedades viscoelásticas contrastadas), y se
observaron diferencias entre los fluidos sanos y patológicos empleando los
parámetros medidos. Posteriormente, se realizaron mediciones con líquido
sinovial proporcionado por el Hospital Universitario La Paz de Madrid. Las
muestras fueron previamente diagnosticadas y clasificadas en dos grupos:
patologías inflamatorias y patologías infecciosas. Al medir las muestras, la
utilización de los parámetros obtenidos para la diferenciación de los grupos
mostró una precisión baja; sin embargo, al aplicar algoritmos de redes
neuronales artificiales, la precisión incrementó considerablemente. Por último,
se realizaron experimentos con hidrogeles para monitorizar la formación del gel
captando su cambio de viscosidad con el sensor. Se utilizaron dos tipos de
enlaces para la formación del gel, el primero por reticulación covalente y el
segundo por reticulación física. Además, en cada caso se varió la concentración
de los agentes para variar las propiedades físicas del gel resultante. Se logró
x
captar la formación de ambos tipos de hidrogeles, teniendo el sensor una
respuesta diferente a cada tipo de concentración.
La técnica propuesta presenta ventajas como la simplicidad, el reducido
volumen de muestra, el bajo coste y la portabilidad, lo que la hace accesible a
cualquier laboratorio y genera interés para su posterior desarrollo. Esta tesis
aporta nuevas contribuciones al desarrollo de sensores basados en QCR y abre
la posibilidad de nuevas líneas de investigación en cuanto a aplicaciones
biomédicas.
Palabras clave
Sensor, Resonadores de Cristal de Cuarzo, Microbalanza de Cristal de cuarzo,
Fluido Sinovial, Hidrogeles basados en Biopolímeros.
xi
Index
Abstract .......................................................................................................................... vi
Resumen ......................................................................................................................... ix
Acronyms ................................................................................................................... xxiv
Nomenclature ............................................................................................................ xxvi
1. Introduction ............................................................................................................ 1
1.1. Reference framework ..................................................................................... 1
1.2. Problem statement .......................................................................................... 2
1.3. Research methodology ................................................................................... 3
1.4. Thesis organization ......................................................................................... 3
2. Theory and State of the Art ................................................................................... 5
2.1. Viscosity ........................................................................................................... 5
2.1.1. Newtonian and non-Newtonian Fluids ............................................... 6
2.2. Human fluids of interest ................................................................................ 7
2.2.1. Synovial Fluid .......................................................................................... 7
2.2.2. Cerebrospinal Fluid ............................................................................... 11
2.2.3. Pleural Effusions .................................................................................... 14
2.3. Biopolymer-based hydrogels ...................................................................... 16
2.3.1. Mucins ..................................................................................................... 17
2.3.2. Alginate ................................................................................................... 17
2.3.3. Biopolymer-based hydrogels in biomedical applications ............... 18
xii
2.4. The importance of the clinical use of viscosity in human fluids ............ 20
2.5. Viscosity measurement techniques ............................................................ 22
2.5.1. Rotational rheometers/viscometers..................................................... 22
2.5.1.1. Concentric cylinders .......................................................................... 23
2.5.1.2. Cone and plate .................................................................................... 24
2.5.1.3. Parallel plate ....................................................................................... 24
2.5.2. Capillary viscometers............................................................................ 25
2.5.3. Falling body method ............................................................................. 26
2.5.4. Ultrasonics .............................................................................................. 27
2.6. Quartz Crystal Resonators ........................................................................... 27
2.6.1. Butterworth-Van Dyke model ............................................................. 29
2.6.2. Mass deposition ..................................................................................... 32
2.6.3. Fluid deposition ..................................................................................... 33
2.6.4. Penetration depth .................................................................................. 34
2.7. QCRs used as viscometers ........................................................................... 35
2.8. Brief description of artificial neural networks .......................................... 35
2.9. ViSQCT project and previous work ........................................................... 37
2.9.1. AD9850 module ..................................................................................... 37
2.9.2. Analog to digital stage .......................................................................... 38
2.9.3. Power supply ......................................................................................... 38
3. Hypothesis and Objectives ................................................................................. 39
3.1. Hypothesis ..................................................................................................... 39
3.2. General and specific objectives ................................................................... 39
xiii
4. Sensor Development ............................................................................................ 41
4.1. Sensor description ......................................................................................... 41
4.2. PCB design ..................................................................................................... 42
4.3. QCRs an holder cell ...................................................................................... 45
4.4. System control and user interface............................................................... 46
4.5. 3D printed case .............................................................................................. 48
4.6. Final prototype .............................................................................................. 49
5. Protocols and Preliminary Measurements ....................................................... 51
5.1. Measurement protocol ................................................................................. 51
5.2. QCR cleaning protocol ................................................................................. 52
5.3. Open QCM ..................................................................................................... 53
5.4. Volume variation with water ...................................................................... 54
5.5. Conventional fluids measurement ............................................................. 55
6. Experimental Methodology ................................................................................ 62
6.1. Phantoms ........................................................................................................ 62
6.1.1. aCSF ......................................................................................................... 63
6.1.2. aSF ............................................................................................................ 64
6.2. Synovial fluid samples ................................................................................. 65
6.3. Biopolymer-based hydrogels samples ....................................................... 65
6.3.1. Covalently crosslinked mucin hydrogels ........................................... 66
6.3.2. Physically crosslinked alginate hydrogels ......................................... 66
7. Results and Discussion ........................................................................................ 67
xiv
7.1. Phantoms ........................................................................................................ 67
7.1.1. aCSF ......................................................................................................... 67
7.1.2. aSF ............................................................................................................ 70
7.2. SF results ........................................................................................................ 74
7.2.1. Inflammatory and infectious SF .......................................................... 74
7.2.2. Comparison between SF pathologies ................................................. 81
7.2.3. Artificial Neural Networks for the classification of inflammatory
and infectious SF .................................................................................................. 83
7.3. Biopolymer-based hydrogels results.......................................................... 88
7.3.1. Covalently crosslinked hydrogels ....................................................... 88
7.3.2. Physically crosslinked alginate hydrogels ......................................... 90
8. Conclusions and Future Work ........................................................................... 96
8.1. Conclusions .................................................................................................... 96
8.2. Future work ................................................................................................... 97
9. Annex ..................................................................................................................... 99
References ................................................................................................................... 109
xv
List of Figures
Figure 1. System for defining viscosity. __________________________________ 6
Figure 2. Viscosity as a function of shear rate for Newtonian and non-
Newtonian Fluids ____________________________________________________ 7
Figure 3: Tetrazine (Tz) and norbornene (Nb) conjugated to BSM and the crosslinking
reaction _____________________________________________________________ 17
Figure 4: Structure of Alginate reacting with 𝐶𝑎2 +and 𝐵𝑎2 +. _____________ 18
Figure 5. Basic structure of concentric cylinders viscometer _______________ 23
Figure 6. Basic structure of cone and plate viscometer ____________________ 24
Figure 7. Basic structure of parallel plates viscometer _____________________ 25
Figure 8. Basic structure of capillary viscometer _________________________ 26
Figure 9. Basic structure of falling body viscometer ______________________ 27
Figure 10. QCR schematic. ____________________________________________ 28
Figure 11. Shear deformation during oscillation in QCR __________________ 28
Figure 12. Butterworth-Van Dyke model of Quartz Crystal Resonators (QCR)
with liquid load. _____________________________________________________ 29
Figure 13. Frequency response of QCR conductance with and without sample.
___________________________________________________________________ 31
Figure 14. QCR admittance locus ______________________________________ 32
Figure 15. Decay depth of the acoustic wave transmitted to the fluid. _______ 35
Figure 16. Basic ANN model.__________________________________________ 36
Figure 17. Simplified diagram of the signals acquisition system. ___________ 38
Figure 18. Excitation and signal acquisition process in the QCR. ___________ 42
xvi
Figure 19. PCB design ________________________________________________ 43
Figure 20. PCB manufactured _________________________________________ 44
Figure 21. Evaluation of the mounted PCB. _____________________________ 44
Figure 22. QCRs used in measurements. ________________________________ 45
Figure 23. Dimensions (in mm) of the QCRs. ____________________________ 45
Figure 24. Design of the lid required for the holder cell. ___________________ 46
Figure 25. Holder cells manufactured in a 3D printer using PLA. ___________ 46
Figure 26. QCR placed in the holder cell.________________________________ 46
Figure 27. Main window. _____________________________________________ 47
Figure 28. Results window. ___________________________________________ 47
Figure 29. Simplified flowchart of the program algorithm. ________________ 48
Figure 30. Design of the lid and the base of the case. ______________________ 49
Figure 31. 3D printed case. ____________________________________________ 49
Figure 32. ViSQCT sensor _____________________________________________ 49
Figure 33. System elements. ___________________________________________ 50
Figure 34. Complete system operating. _________________________________ 50
Figure 35. Signal acquisition method for Open QCM sensor. ______________ 53
Figure 36. Viscosity measured by varying the volume of water samples. ____ 55
Figure 37. ∆𝑓 measured for water, acetone and alcohol. ___________________ 57
Figure 38. Viscosity measured for water, acetone and alcohol. _____________ 57
Figure 39. Comparison of ∆𝑓 measured for glycerin, sugar, and salt using both
sensors. ____________________________________________________________ 59
xvii
Figure 40. Comparison of ∆Γ measured for glycerin, sugar, and salt using
ViSQCT sensor. _____________________________________________________ 59
Figure 41. Comparison of the viscosity obtained using both sensors and the
viscometer (for glycerin, sugar, and salt). _______________________________ 59
Figure 42. Resonance frequency measurement for water and sugar 30% using:
ViSQCT sensor (left) and Open QCM sensor (right). ______________________ 60
Figure 43. Measurements set-up. ______________________________________ 62
Figure 44. Synovial fluid samples. _____________________________________ 65
Figure 45. ∆𝑓 (black line) and ΔΓ (red line) obtained for aCSF samples. _____ 68
Figure 46. Viscosity obtained with the sensor using aCSF. _________________ 69
Figure 47. Adjustment between the viscosity values measured with the sensor
with the viscosity of the rotational viscometer for aCSF samples. ___________ 70
Figure 48. ∆f (black line) and ΔΓ (red line) obtained for aSF samples. _______ 72
Figure 49. Viscosity obtained with the sensor using aSF. __________________ 73
Figure 50. Adjustment between the viscosity values measured with the sensor
with the viscosity of the rotational viscometer for aSF samples. ____________ 73
Figure 51. ∆𝑓 boxplots. Left: EDTA tube; right: Lithium heparin tube _______ 75
Figure 52. ΔΓ boxplots. Left: EDTA tube; right: Lithium heparin tube ______ 75
Figure 53. Viscosity boxplots. Left: EDTA tube; right: Lithium heparin tube _ 75
Figure 54. Boxplots per sample (EDTA tube) for ∆𝑓 measurements. ________ 76
Figure 55. Boxplots per sample (EDTA tube) for ΔΓ measurements. ________ 76
Figure 56. Boxplots per sample (EDTA tube) for viscosity values. __________ 77
Figure 57. Boxplots per sample (Lithium heparin tube) for ∆f measurements. 77
xviii
Figure 58. Boxplots per sample (Lithium heparin tube) for ΔΓ measurements. 78
Figure 59. Boxplots per sample (Lithium heparin tube) for viscosity values. _ 78
Figure 60. ROC curve for parameters measured with the sensor for: SF in EDTA
tubes (left) and SF in lithium heparin tubes (right). _______________________ 81
Figure 61. Comparison between obtained ∆𝑓 for specific SF conditions. _____ 82
Figure 62. Comparison between obtained ΔΓ for specific SF conditions. _____ 82
Figure 63. Comparison between obtained viscosity for specific SF conditions. 82
Figure 64. ANN algorithm for SF classification. __________________________ 84
Figure 65. Confusion matrices obtained by ANN for SF samples contained in
EDTA tubes. ________________________________________________________ 87
Figure 66. Confusion matrices obtained by ANN for SF samples contained in
lithium heparin tubes. ________________________________________________ 88
Figure 67. Time-dependent evaluation of ∆f (black line) and ΔΓ (red line) for 15
mg/ml Muc-gels. ____________________________________________________ 89
Figure 68. Time-dependent evaluation of ∆f (black line) and ΔΓ (red line) for 25
mg/ml Muc-gels. ____________________________________________________ 90
Figure 69. ∆f (black line) and ΔΓ (red line) change after placing an alginate
solution (15 mg/ml) on the sensor. _____________________________________ 91
Figure 70. ∆f (black line) and ΔΓ (red line) change likely caused by a dilution
effect following exposure of the alginate to a solution of NaCl (0.4 M). ______ 91
Figure 71. Comparison of Δf plateau-like stage for the four cases. __________ 92
Figure 72. ∆f (black line) and ΔΓ (red line) response for alginate gel formation
using 𝐵𝑎𝐶𝑙2 (0.4 M). __________________________________________________ 93
using 𝐵𝑎𝐶𝑙2 (0.2 M). _________________________________________________ 93
xix
using 𝐶𝑎𝐶𝑙2 (0.4 M). _________________________________________________ 93
using 𝐶𝑎𝐶𝑙2 (0.2 M). _________________________________________________ 94
Figure 76. Kinetics of the alginate gelation process with all the cases evaluated.
___________________________________________________________________ 95
Figure 77.Variation of accuracy throughout training with one layer and 100
epochs using SF stored in tubes with EDTA. ____________________________ 99
Figure 78. Variation of losses throughout training with one layer and 100
epochs using SF stored in tubes with EDTA. ___________________________ 100
Figure 79. Variation of accuracy throughout training with one layer and 200
Figure 80.Variation of losses throughout training with one layer and 200 epochs
using SF stored in tubes with EDTA. __________________________________ 101
Figure 83. Variation of accuracy throughout training with two layers and 100
Figure 84. Variation of losses throughout training with two layers and 100
xx
epochs using SF stored in tubes with lithium heparin. ___________________ 104
xxi
xxii
List of Tables
Table 1. Classification parameters for healthy, non-inflammatory, inflammatory,
septic and hemorrhagic SF. ___________________________________________ 11
Table 2. Composition of normal CSF ___________________________________ 12
Table 3. CSF constituents in different disorders. Obtained from [28] ________ 14
Table 4. Water, acetone and alcohol data and results. _____________________ 56
Table 5. Results for glycerin, sugar and salt. _____________________________ 58
Table 6. Artificial Cerebrospinal Fluids (aCSF) compositions. ______________ 63
Table 7. Artificial Synovial Fluids (aSF) compositions. ____________________ 64
Table 8. aCSF data and results obtained with the sensor. __________________ 67
Table 9. aSF data and results obtained with the sensor. ___________________ 71
Table 10. Mean value and p-value per parameter for each classification and
container type _______________________________________________________ 79
Table 11. Area under the ROC curve of the parameters as predictors for
infectious fluid for both tubes. _________________________________________ 80
Table 12. p-value calculation employing Tukey HSD for specific diseases. ___ 83
Table 13. Accuracy obtained for each test. _______________________________ 85
Table 14. Features of the ANN. ________________________________________ 99
xxiii
Acronyms
aCSF artificial Cerebrospinal Fluid
ANN Artificial Neural Network
aSF artificial Synovial Fluid
BM Bacterial Meningitis
BSM Bovine Submaxillary Mucin
BVD Butterworth-Van Dyke
CPPD Calcium Pyrophosphate Deposition
CSF Cerebrospinal Fluid
CTB Centro de Tecnología Biomédica
DDS Direct Digital Synthesis
ECM Extracellular Matrix
EDTA Ethylenediaminetetraacetic acid
EPEs Exudative Pleural Effusions
GelMA Gelatin-methacryloyl
HA Hyaluronic Acid
LBN Laboratorio de Bioinstrumentación y Nanomedicina
LP Lumbar Puncture
xxiv
Muc Mucines
MUS Monosodium urate
Nb Norbornene
OA Osteoarthritis
PCB Printed Circuit Board
PCR Polymerase chain reaction
QCM Quartz Crystal Microbalance
QCR Quartz Crystal Resonator
RA Rheumatoid Arthritis
SDS Sodium dodecyl sulfate
SF Synovial Fluid
TPEs Transudative Pleural Effusions
Tz Tetrazine
UPM Universidad Politécnica de Madrid
VM Viral Meningitis
WBC White Blood Cells
xxv
Nomenclature
Y Admittance
𝛺 Angular velocity (rad/s)
𝐵𝑎2+ Barium ion
𝐻𝐶𝑂3− Bicarbonate ion
𝐶𝑎2+ Calcium ion
𝐶𝑙 − Chloride ion
G Conductance
𝜌 Density (mg/ml)
D Dissipation
𝑓0 Fundamental resonance frequency
Γ Half-bandwidth at half-maximum
ΔΓ Half-bandwidth at half-maximum shift
n Overtone number
δ Penetration depth
𝐾+ Potassium ion
Q Quality factor
xxvi
𝛥𝑓 Resonance frequency shift
𝑓𝑚 Resonant frequency at the maximum admittance point
𝑓𝑠 Series resonance frequency
𝐺𝑞 Shear modulus of quartz
𝛾̇ Shear rate (1/s)
τ Shear stress (Pa)
𝑁𝑎+ Sodium ion
𝜌𝑞 Specific density of quartz
B Susceptance
𝑑𝑞 Thickness of quartz
T Torque (dyne/cm)
𝜂 Viscosity (Pa·s)
𝑣𝑞 Wave speed of quartz
𝑓𝑟 Zero-phase resonant frequency
xxvii
Chapter 1
1. Introduction
1.1. Reference framework
Quartz Crystal Resonators (QCR) are elements that can be used in various
applications. This is mainly due to the piezoelectric effect they possess, which
was first studied in 1880 by the Curie brothers [1], [2]. Other factors that make
them highly usable are their low cost and high accuracy.
The best-known application is as a gravimetric sensor with the well-known
Quartz Crystal Microbalance (QCM), whose discovery dates back to 1959, given
the research of Günter Sauerbrey [3]. Sauerbrey showed the change of
resonance frequency by depositing tiny masses (nanograms) on the surface of
crystals. Later, in 1985, research by J. G. Gordon and K. K. Kanazawa showed
the effect caused by depositing a fluid on the surface of crystals [4]. Now, the
change of resonance frequency of the crystal would be caused by the product of
the density and viscosity of the deposited liquid. This opened the door to new
lines of research, mainly in the areas of biology, chemistry, and biomedicine.
A few years ago (2015), Diethelm Johannsmann published a book entitled "The
Quartz Crystal Microbalance in Soft Matter Research" [2] in which the use of
crystals in contact with samples such as polymers, cells, proteins, etc., is studied
in depth. This provides an overview of the development of these devices from
their discovery to their current applications.
The use of QCRs in the biomedical field has been growing over the years, with
studies using the crystals to detect specific agents and identify diseases such as
influenza [5]–[7], malaria [7], [8], Human Immunodeficiency Virus (HIV) [7],
[9], tuberculosis [10]–[12], Alzheimer's [13] and breast cancer cells [14]. In
addition, experiments are being conducted in [15] to observe the response of
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these sensors when measuring blood, thereby preventing cardiovascular
disease.
Taking into account the broad capabilities of QCRs as sensors, the ViSQCT
project at the Universidad Politécnica de Madrid (UPM) has developed a
functional prototype for fluid characterization using a small sample volume
(tens of microliters) by measuring the physical property of viscosity to broaden
the applications for these devices in the field of bioengineering and
biomedicine. All this is within the Bioinstrumentation and Nanomedicine
Laboratory (LBN) of the Centre for Biomedical Technology (CTB).
1.2. Problem statement
As mentioned above, the use of QCRs as sensors has many advantages. The
possibility of measuring small volumes allows us to explore biomedical cases
where the sample is small. The first case we decided to explore is the distinction
between human fluids, either to differentiate between a healthy and a
pathological fluid or to classify between different conditions of the same fluid.
Otherwise, existing instrumentation is costly, bulky, or incapable of doing so, as
they require a larger sample volume than the existing one.
A second alternative proposed is to characterize the formation of hydrogels.
Monitoring the hydrogel formation process contributes to the hydrogel's design
process, which will define its mechanical properties, which are crucial for its
performance. Like the first case, the existing instrumentation for this type of
analysis is costly and/or bulky.
This work emerges from the need for a sensor that allows the viscosity of a
liquid to be characterized using a small sample volume and whose use is for
biomedical applications. In these cases, the sample volume is usually reduced.
In addition to this development, the aim is to generate an instrument whose
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Chapter 1
cost and dimensions are reduced, which increases its accessibility in
laboratories.
1.3. Research methodology
The methodology followed in this work corresponds to three stages. The first is
the research and study of the state of the art and theory surrounding the
project, including reviewing current journals that serve as a reference. It is also
essential to know the status of the project to contemplate the knowledge
derived from previous work. From this, the hypothesis and objectives of the
work are set out. The second part considers the planning of future
experimentation and the development of a new version of the device. Finally,
the third stage involves experimentation and analysis of the results obtained.
1.4. Thesis organization
The present work is divided into nine chapters, which will be described below.
The first chapter describes the rationale of the thesis. The research
methodology, the problem to be addressed, and the origin and motivation of
the project are illustrated.
The second chapter brings together state of the art and theoretical concepts
surrounding the work. The fundamental concepts of QCRs, viscosity, and
viscosity measurement methods are explained. It also details the human fluids
of interest for the project, why viscosity measurement is interesting from a
medical point of view, and the application of biopolymer-based hydrogels in
biomedicine. Finally, the project's background, the starting point of the present
work, is discussed.
The third section sets out the hypothesis of the work and the objectives, both
general and specific.
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The fourth part begins to talk about the work carried out by describing the
development of the prototype used.
Chapter five mentions the protocols established for the experimental process
and the device's first measurements.
The sixth section develops the experimental methodology and describes the
samples used in the experiments.
The seventh chapter shows the results obtained in each experiment.
Conclusions and proposals for future work are presented in the eighth chapter.
4
Chapter 2
2. Theory and State of the Art
The development of this work begins with the explanation of the concepts that
are parts of it, such as viscosity, quartz crystal crystals, biological fluids of
interest, and viscosity measurement techniques. Subsequently, the focus is
given on why viscosity is a parameter of medical interest and the main
applications of biomaterials such as hydrogels. Finally, examples of advances in
the use of quartz crystal resonators for viscosity measurement are given, and
the context and start of the ViSQCT project in which this work was developed
are introduced.
2.1. Viscosity
Viscosity is a mechanical property of all fluids that can be defined in various
ways, for example, as the fluid's internal friction or the resistance of a fluid to
the movement produced by a force [16]. A couple of parameters, shear stress
and shear rate, must first be defined to describe it adequately in physical terms.
Looking at Figure 1, we have a fluid placed in the middle of two parallel plates
and a force applied to the upper plate, which will cause the displacement of the
fluid layers adjacent to the upper plate in the same direction as the force. The
resulting effect will be a gradient of velocities in the fluid layers as they move
away from the upper plate. Thus, the applied force is the shear stress τ, defined
as a force applied tangentially over an area whose measurement unit is pascals
(Pa); and the resulting strain rate illustrated as the velocity gradient is the shear
rate γ̇ (𝑠 −1 ) [16], [17]. Mathematically, the viscous response of this system
against the shear force is described as:
𝜏 = 𝜂 · γ̇ (1)
where 𝜂 is the coefficient of viscosity, being more specific, considering that a
shear force is being applied, 𝜂 is called the shear dynamic viscosity. In SI units,
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𝜂 is measured in pascals per second (Pa·s), and in c.g.s. units, it is measured in
poise (P). Both can be related as follows: 1 cP = 1 mPa·s.
Figure 1. System for defining viscosity.
2.1.1. Newtonian and non-Newtonian Fluids
The viscosity equation expressed in (1) is valid for so-called Newtonian fluids.
The name is given because it was formulated by Sir Isaac Newton. If the fluid's
viscosity is constant regardless of the shear rate, then it is known as a
Newtonian fluid, water being a clear example of this type of fluid. However,
there are a large number of fluids whose viscosity is shear rate dependent.
These types of fluids are called non-Newtonian fluids [16], [17].
Figure 2 shows the viscosity behavior as a function of shear rate for a
Newtonian fluid and two cases of non-Newtonian fluids: Shear Thickening and
Shear Thinning (Pseudoplastic). It is observed that for a Newtonian fluid, the
viscosity will be the same regardless of the shear rate. In the case of fluids with
shear thickening behavior, the viscosity will increase as the shear rate increases.
A typical example of such a fluid is cornstarch. Conversely, a fluid with shear
thinning behavior will have a decreasing viscosity as the shear rate increases.
Examples of this case can be paint and ketchup.
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Chapter 2
Figure 2. Viscosity as a function of shear rate for Newtonian and non-Newtonian Fluids
2.2. Human fluids of interest
In the context of this work, the analysis of three types of biological fluids whose
viscosity is related to their state of health is proposed. The first is the synovial
fluid which is located in the joints of the body. The second is the cerebrospinal
fluid situated mainly in the brain, and the third is the pleural effusion, an
accumulation of fluid inside the membrane covering the lungs. Synovial fluid
was used for the experimental part; however, the study with cerebrospinal fluid
and pleural effusion are of great interest for future trials.
2.2.1. Synovial Fluid
Synovial Fluid (SF) is a viscous liquid located in the joints whose main
functions are divided into two. The first is the joints' mechanical function,
which consists of lubricating the articular surface and cushioning movements.
The second is to contribute to the nutrition of the articular cartilage by acting as
a nutrient transport medium. It is composed of dialysate of plasma and a high
content of Hyaluronic Acid (HA) [18], [19].
The analysis of this fluid begins with the extraction of the fluid. This process is
called arthrocentesis. The process involves a puncture of the joint to extract the
SF. Then, the sample is collected in a purple-topped tube containing
ethylenediaminetetraacetic acid (EDTA) as an anticoagulant [18], [20].
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Usually, SF analysis is composed of four studies [18], [21]:
1. Macroscopic, where the physical characteristics of the fluid such as color,
viscosity, and volume are analyzed.
2. Microscopic, which includes a leukocyte count plus a scan of crystals and
definition by polarized light.
3. Chemical. Protein, glucose, uric acid, lactic acid and rheumatoid factor
levels should be evaluated.
4. The use of differential stains such as Gram stain, Wright, Red Alizarin,
among others.
The volume required depends on the analysis to be performed (and may vary
between laboratories). For example, for an accurate cell count, approximately 1
ml is required; 2 to 3 ml is an adequate volume to perform the required tests. In
case of obtaining a low volume sample, it should be sent for the analysis of
crystals and culture, which are more diagnostic [20].
2.2.1.1. Macroscopic analysis of SF
Regarding volume, the maximum amount that can be obtained from a normal
joint is between 0.1 and 3.5 ml. The knee can have up to 4 ml. If a larger volume
is obtained, it is an indication of an inflammatory process [18], [21], [22].
Healthy synovial fluid is transparent in color and may have light straw-yellow
tones. Its viscosity is similar to egg white and is included in non-Newtonian
fluids with pseudoplastic behavior. To determine the viscosity, it is usual to
observe the stranding, i.e., to measure the "thread" formed by the liquid when
extended. This can be done by placing the sample drop on a slide and lifting it
with a spatula or using the thumb and forefinger to spread it out. For a healthy
fluid, the "thread" may measure between 3 and 6 cm. SF with poor viscosity will
form a "thread" of less than 3 cm [18], [21], [23]. This method is a subjective
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Chapter 2
form of evaluation and depends on the skills and experience of the operator. It
is possible to use a viscometer or rheometer to measure viscosity; however, they
usually require more sample volume than is available or are expensive and
large to keep within reach.
SF viscosity depends on HA concentration, so at lower concentrations, the
viscosity decreases. Some studies relate the SF’s low viscosity with rheumatic
diseases, such as Rheumatoid Arthritis (RA) and Osteoarthritis (OA) [20], [22],
[24].
An extra test is the mucin clot test, also called the Rope test. This consists of
adding four parts of 2% acetic acid to one part of SF and observing the clot
formation. A clot is categorized as good when it is tight ropy, which correlates
with a normal high viscosity, while a poor clot that breaks easily corresponds to
an inflammatory SF [20].
2.2.1.1. Microscopic analysis of SF
Cell counting should be performed within the first two hours after fluid
extraction, using saline as a diluent in synovial fluids with high cell counts. In
normal SF, the leukocyte count should be less than 200 cells/µl, and in septic SF
the number is greater than 75,000 cells/µl [18], [21].
The differential examination will indicate the percentages of white blood cells.
Normal SF contains few neutrophils and a low number of lymphocytes. The
normal distribution is neutrophils 7%, lymphocytes 24%, monocytes 48%,
macrophages 10%, and synovial lining cells 4% [18].
Crystal identification is performed using a polarized light microscope. With this
study, monosodium urate (MSU) crystals, which are negatively birefringent, are
located. With the help of the polarized light microscope, their thin, needle-like
shape can be precisely observed. The detection of MSU crystals diagnoses gout.
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Other crystals commonly found in SF are calcium pyrophosphate dehydrate
(CPPD) [18], [21] which are present in pseudogout cases.
2.2.1.2. Chemical analysis of SF
These studies include the measurement of protein, glucose, uric acid, and
rheumatoid factor levels.
The normal protein level in SF is in the range of 1 - 3 g/dL. A higher level may
be observed in cases of arthritis, arthropathies, and ankylosing spondylitis. Its
evaluation does not determine between inflammatory and non-inflammatory
fluid [18], [23].
SF glucose should be measured from a fasting specimen whose normal levels
are less than 10 mg/ml. Glucose values greater than 50 mg/ml suggest an
infectious process. As for uric acid, in SF, it usually is in the range of 6 to 8
mg/dl. A high value helps in the diagnosis of gout [18], [20].
Another parameter of interest is Rheumatoid Factor (RF), which is an antibody
against immunoglobulins. RF is present in most patients with RA, although it
may be the result of other diseases, which lends itself to false positives [18].
2.2.1.3. Stains
Staining and culturing makes it possible to identify infections in SF samples and
differentiate the type of cells present. Some of these are Gram's stain, Wright's
stain, and alizarin red stain. Gram staining is performed on the SF sample after
centrifugation. It is a relatively inexpensive and straightforward process,
although it depends on the interpretation of the observer. This study can
provide information on the presence of infection and the type of organism
present. Staining with alizarin red helps detect calcium hydroxyapatite crystals
that are not birefringent and, therefore, difficult to detect by microscopy.
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Staining with Wright's stain makes it possible to differentiate the cells present in
the sample from the SF [18], [20], [21]. Table 1 summarizes the most important
parameters for classifying SF into healthy, inflammatory, non-inflammatory,
septic, and hemorrhagic. Taken from: [18], [20], [21], [23].
Normal Non- Inflammatory Septic Hemorrhagic

inflammatory
Aspect Transparent Yellow, straw- Yellow, turbid Cloudy, Bloody

straw-colored colored and
turbid and
cloudy opaque
Viscosity High Decreased Low Low Absent-Variable
Mucin clot Good Fair Poor Poor Poor
SF < 200 < 2000 2000-50,000 > 50,000 Variable

WBC*/mm3
Glucose 95 – 100 95 - 100 80 - 100 <50 -
(% blood
level)
Protein 1.3 – 1.8 3.0 - 3.5 > 4.0 > 4.0 -
Crystals None None Multiple or None None

None
Table 1. Classification parameters for healthy, non-inflammatory, inflammatory, septic and
hemorrhagic SF.
*White Blood Cells
2.2.2. Cerebrospinal Fluid
Cerebrospinal fluid (CSF) is a clear, colorless fluid produced mainly by the
choroid plexus, whose function is to protect the central nervous system. It
comprises many ions (Na+ , Cl− , 𝐻𝐶𝑂3− , K + , Ca2+ ), vitamins and peptides. In
Table 2, the composition of normal CSF is disaggregated. Its cell count does not
exceed five cells per millimeter [25]–[27]. Adults' average volume of CSF is 150
ml, distributed approximately 25 ml in the ventricles and 125 ml in the cranial
subarachnoid and spinal subarachnoid spaces [25], [26].
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Constituent Concentration (mmol/l)
Sodium 138
Potassium 2.8
Calcium 1.1
Chloride 119
Bicarbonate 22
Table 2. Composition of normal CSF
To study the CSF, it is necessary to have access to it through a lumbar puncture
(LP), an invasive procedure in which the restricted compartment of the
subarachnoid space is accessed. LP involves introducing a needle below the
termination of the spinal cord, allowing access to the subarachnoid space.
Depending on the patient and the purpose, between 1 and 20 ml are extracted
[28].
CSF's routine analysis involves microscopic study (white cell count) glucose
and protein testing. For the glucose study, it is important to have a matched
serum glucose sample to be able to make the comparison between serum and
CSF glucose. Depending on the pathology, other tests such as cell culture,
staining and/or polymerase chain reaction (PCR) will be performed [26], [28].
Healthy CSF will have a clear appearance, low cell count (0 - 5 cells/𝑚𝑚3 ), a
protein concentration of approximately 0.5 g/l, and the glucose is between 60-
75% of that measured in serum [26], [28], [29]. Values may vary depending on
the author.
2.2.2.1. Bacterial Meningitis diagnose
Bacterial meningitis (BM) is an infection that occurs when pathogenic virulence
factors overcome host defense mechanisms. The most common bacteria causing
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Chapter 2
meningitis are S. pneumoniae, Haemophilus influenzae, Neisseria meningitidis,
and Escherichia coli, whose neurotropic potential is related to their ability to
evade host defenses. These types of infections can cross the blood-brain barrier
and survive in the CSF [30], [31].
The gold standard for diagnosing BM is by cell culture. CSF Gram stain, latex
agglutination test, and PCR are other diagnostic tools that can aid in etiologic
diagnosis. Glucose levels (CSF-serum ratio) usually decrease, and proteins
levels increase [29], [32].
Gram staining is an inexpensive and validated diagnostic tool that is used to
identify the causative organism for patients with suspected BM, especially in
cases where cell culture is negative [32].
2.2.2.2. Viral Meningitis diagnose
Viral meningitis (VM) is an infection of the meninges caused by a virus, part of
an aseptic meningitis syndrome. Infections are caused by: Enteroviruses,
Mumps, Lymphocytic Choriomeningitis, Herpes Simplex, Herpes Zoster, and
Arboviruses [33]. Depending on the virus, these viruses enter the central
nervous system by various methods. Once inside the central nervous system,
the viruses spread through the subarachnoid space into the CSF, causing
inflammation that leads to meningitis.
A pleocytosis of lymphocytes is a sign of VM; the white cell count is typically 20
- 500 cells/ml, reaching 1000 cells/ml. Isolation of viruses (in tissue culture) from
CSF is the gold standard for diagnosing many viral pathogens causing
meningitis; however, the procedure is slow, expensive, and not always sensitive
[34]. Currently, the use of PCR to analyze CSF is the most valuable diagnostic
method due to its rapidity, sensitivity, and specificity [35].
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In cases of VM, glucose is usually unchanged. On the other hand, proteins tend
to rise slightly to settle at values between 0.5 - 0.9 g/l [28], [35].
Table 3 shows the main components of the CSF in healthy and infectious cases.
Normal Viral Infection Bacterial Infection Tuberculosis infection
Aspect Clear Clear/opaque Turbid Clear/opaque
White cells 0-5 10-2000 100-60000 50-5000
(cells/mm3)
Protein < 0.5 0.5-0.9 > 0.9 (1.0-5.0) > 1.0 (1-5)
(g/l)
Glucose 60-75% Normal < 40% < 50%
(% serum glucose)
Table 3. CSF constituents in different disorders. Obtained from [28]
The distinction between BM or VM is a challenge, especially in patients with
negative Gram stains and cell culture studies; this leads to the evaluation of CSF
viscosity as a parameter to distinguish between the two types of meningitis [34],
[36].
2.2.3. Pleural Effusions
Pleural effusions are an abnormal accumulation of fluid in the pleural cavity.
The origins are mainly excessive production of the liquid, a lack of drainage, or
both [37], [38]. The causes are varied, being mainly: cancer, heart failure,
pneumonia, and tuberculosis [37].
The key point of pleural effusions is the distinction between transudates and
exudates. A transudate is caused when the reabsorption of pleural fluid is
disturbed. Increased pulmonary hydrostatic pressure or plasma osmotic
pressure are alterations that cause transudates. On the other hand, exudates
arise from inflammation or other diseases of the pleural surface, as in cases of
tuberculosis, pneumonia, pancreatitis, pulmonary infarction, or systemic lupus
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Chapter 2
erythematosus [39]. Differentiation between these two types of effusions is
essential because if a patient has a transudative pleural effusion, only the cause
of the effusion needs to be treated (heart failure, cirrhosis, etc.). However, if it is
an exudative effusion, further testing is necessary to find the cause [40]
2.2.3.1. Diagnosis of Pleural Effusions
The diagnosis begins with an ultrasound or chest x-ray in a decubitus position.
If it is determined that there is a thickness greater than or equal to 2 cm due to
fluid, a thoracentesis will be necessary to study the fluid [37]. The extracted
volume varies depending on the patient's condition, ranging from 5 to 1500 ml,
and, in a few cases, exceeds this maximum [41]. With the extracted sample, the
aim is to classify the fluid between transudate and exudate employing the
"Light's criteria", which contemplates the following:
• Pleural fluid proteins divided by serum proteins > 0.5.
• Pleural fluid lactate dehydrogenase (LDH) divided by serum LDH > 0.6
• Pleural fluid LDH >2/3 (67%) of the upper limit of normal serum LDH
An exudative pleural effusion meets one or more criteria, whereas a transudate
meets none [37], [39].
2.2.3.2. Diagnosis of Pleural Effusions using viscosity
In the works [38], [42] show that viscosity is a parameter that correlates with the
type of exudate. Viscosity is associated with the protein and lipoprotein
concentration of the fluid. By using a viscometer, it was determined that
obtaining the viscosity makes it possible to differentiate between transudates
and exudates, the latter being those of higher viscosity, a result that both
studies agree.
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For this reason, there is interest in measuring the viscosity of pleural effusions
to have an efficient, fast, and simple test to avoid the usual biochemical tests,
which can be more costly and time consuming.
2.3. Biopolymer-based hydrogels
Hydrogels are typically formed by polymeric networks of natural or synthetic
origin, crosslinked by covalent or non-covalent interactions [43]–[45]. New
hydrogel systems are continuously being developed to address their current
limitations and expand into new applications. For instance, hydrogels
bioactivity and rheology are engineered to be used as ink to form scaffold
matrices in 3D bioprinting. In situ application will be improved by enhancing
their injectability, which can be done by monitoring the material's viscous
properties [46], [47]. In the design process of such biomaterials, the material's
mechanical properties are critical. For example, the mechanical properties of
implantable hydrogels are often engineered to match those of the surrounding
tissue. The material stiffness can also direct the behaviour of cells, for instance,
by directing the differentiation of stem cells [48] or the growth kinetics of cancer
cells or biopsies [49]. More practically, the mechanical properties will determine
how the gels can be handled and can be used as a marker of biodegradation.
And in vivo, the mechanical properties of natural hydrogel materials also
define their functions. one example is the mucus gel that covers the lung, gut,
cervix, and which depend on their mechanical properties to properly protect
the epithelial surfaces from shear stresses, pathogen infections, and for their
constant recycling and turnover [50]–[52].
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Chapter 2
2.3.1. Mucins
Mucins are large glycoproteins that coat the surface of many types of cells,
fulfilling functions such as lubrication, hydration, bioactivity, and as a
protective barrier.
The Biopolymers for life group of KTH at the Royal Institute of Technology
(KTH, Stockholm, Sweden) developed and published a novel method to
generate mucins hydrogels by introducing tetrazine (Tz) and norbornene (Nb)
functionalities onto the Bovine Submaxillary Mucin (BSM) molecules [53]. The
protocol leads to the generation of Muc-Tz and Muc-Nb derivatives. When
solutions of Muc-Tz and Muc-Nb are mixed together, they quickly form
covalent bonds and form a hydrogel material (Figure 3). Such hydrogels have
been shown to have interesting immune-modulating properties of interest for
biomedical applications such as encapsulation of cells and microtissue
transplants to dampen the immune reaction and increase treatment efficacy
[54].
Figure 3: Tetrazine (Tz) and norbornene (Nb) conjugated to BSM and the crosslinking reaction
2.3.2. Alginate
Alginate is an algae-derived polysaccharide composed of β-D-mannuronic acid
and α-L-guluronic acid units [46]. This material reacts when cations (𝐶𝑎2+ or
𝐵𝑎2+ ) are added to form an ionotropic gel. This effect generates great interest in
its application in the biomedical field.
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It has been proven to be mucoadhesive, biocompatible, and nonimmunogenic,
and it is often processed into microcarriers for cell encapsulation. For bone
tissue engineering, alginate is often combined with calcium phosphates [46]. On
the other hand, alginate is used in 3D bioprinting because it benefits cell
encapsulation and culture by building structures using biocompatible materials
[55].
As opposed to the Muc-Tz/Muc-Nb system, alginate polymers can be ionically
crosslinked by divalent ions (Figure 4), which present different kinetic and
bond strengths compared to covalent bonds formation [53], [56]. With this
system, the strength of the crosslinking varies by selecting two crosslinking
ions. Calcium, which has been extensively used to crosslink alginate into
hydrogels, and barium, owing to its larger size, has been shown to result in
more stable hydrogels, exhibiting less swelling.
Figure 4: Structure of Alginate reacting with 𝐶𝑎2+ and 𝐵𝑎2+ .
2.3.3. Biopolymer-based hydrogels in biomedical applications
Hydrogels are of great interest in tissue engineering and pharmaceuticals due
to their particular physical properties, such as absorbing large amounts of water
and their high biocompatibility [46], [55]. An example of this is their use as cell
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Chapter 2
encapsulation materials [44], [54], [57]. For the creation of hydrogels, there are
many materials and methods, which will result in a material with unique
mechanical and chemical properties that will define its application, either
medical or pharmaceutical. The following are some of the materials used to
form hydrogels, along with their main applications: Collagen, Gelatin, HA, and
Alginate.
Collagen is a protein present in the extracellular matrix (ECM), of which there
are several types, each one localized in a specific tissue. One of its main
applications is as a scaffold for tissue regeneration such as bone, for which it is
necessary to produce a porous hydrogel by freeze-drying techniques [46]. On
the other hand, by generating scaffolds of cylindrical shapes, collagen can be
used to regenerate blood vessels [58].
Derived from collagen, gelatin has a wide variety of applications, from food to
pharmaceutical. When modified with methacrylamide (GelMA), it has been
used to generate porous scaffolds for bone, cardiac, cartilage, and corneal tissue
regeneration. In addition, due to its electrostatic interactions and structure, it
can be used within drug delivery systems [46], [59].
HA is a polysaccharide that is the main component in the extracellular matrix of
the skin, cartilage, and vitreous humor. It can be used as a material for eye
surgery [60] and is often combined with other materials for nerve regeneration
and blood vessel formation applications [46].
Alginate is another polysaccharide obtained from marine algae. Its soft gelling
behavior allows encapsulation of sensitive bioactive molecules or cells. When
combined with a multivalent ion such as calcium, it forms a hydrogel used for
cell encapsulation [55]. In addition, [61] shows the use of alginate microspheres
for cell immobilization for bone tissue regeneration purposes.
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A recent growing application is in 3D bioprinting, which benefits cell
encapsulation and culture by building structures using biocompatible materials
such as those mentioned above. In this application, the mechanical properties of
the hydrogels (gelation time, viscosity, etc.) must meet specific conditions to
generate the 3D structure [62]–[66].
Another essential element is mucins. Mucins (Muc) coat the surface of many
types of cells, having unique properties and fulfilling functions such as
lubrication, hydration, bioactivity, and as a protective barrier. For this reason,
their use as biomaterials has become widespread, mainly in areas such as tissue
engineering [67]. Recent research shows the use of bovine mucins for the
generation of hydrogels. As mentioned earlier, Muc-Tz and Muc-Nb can be
assembled to form a hydrogel material (whose process is fully detailed in [53])
with interesting properties.
The main idea to highlight about hydrogels is that their production involves
many factors that will result in specific mechanical properties, viscosity being
one of these. Therefore, viscosity is a parameter of interest when studying,
generating, and applying hydrogels and requires precise control and
evaluation.
2.4. The importance of the clinical use of viscosity in human fluids
In the world of medicine, the study of the physical and chemical properties of
the fluids of the human body is essential when identifying pathologies. One
physical property that has been little exploited is viscosity. Correct viscosity
measurement is a helpful factor in the accurate diagnosis of many diseases;
some cases are exposed hereunder.
The distinction between Transudative and Exudative Pleural Effusions (TPEs
and EPEs) in pleural diseases is essential [38], [42], [68]. Currently, this
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discrimination is based on some biochemical tests that are relatively costly and
time-consuming. Lymphocyte-predominant EPE results from many diseases,
with malignancy, tuberculosis being the most common among them. However,
pleural fluid viscosity can reliably differentiate between TPE and EPE [38], [42],
and there is even evidence correlating this viscosity with pathologies such as
tuberculous pleurisy and lung cancer [69]. The measurement of Cerebrospinal
Fluid viscosity is a simple and easy process that can be used as an adjunctive
measure for diagnosing meningitis having a role in the discrimination of
bacterial versus aseptic meningitis [36]. Early diagnosis of acute meningitis has
paramount importance in clinical practice because of mortality and morbidity
of the disease. In arthropathies, synovial fluid exhibits a change in viscosity that
can be linked to an inflammatory response to infection and other causes. For
example, in rheumatoid arthritis cases, the viscosity of the synovial fluid
decreases because of reduced production and polymerization of hyaluronic acid
[20], [22], [24]. Another case of interest is that of blood plasma. Plasma is a
highly concentrated solution of proteins whose viscosity usually remains stable
and, when varying slightly, can provide valuable information for detecting
pathologies. Cases of high viscosity in plasma are correlated with coronary and
peripheral artery diseases [70].
Although viscosity is a property that can be useful for disease diagnosis, it has
been little exploited. On the one hand, in many cases, the volume of fluid
extracted is scarce to measure its viscosity adequately since the existing options
usually require a larger sample volume. On the other hand, in cases where it is
analyzed, this analysis depends on the subjective interpretation of the
examining physician. To these considerations can be added the high cost of
viscosity measuring instruments.
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2.5. Viscosity measurement techniques
The concept of viscosity will be discussed in detail later in this work, but now
we will generally describe the instrumentation currently used for its
measurement. In summary, we can group viscosity measuring instruments into
two categories: viscometers on the one hand and rheometers on the other. In the
first group, the measuring principle is detecting the resistance generated by a
fluid when a flow (preferably isometric) is generated. This will provide a
measure of the fluid's viscosity as a function of the flow velocity generated.
Rheometers are complex instruments that allow characterizing the viscoelastic
properties of fluids. They allow obtaining more parameters of the sample, such
as the elastic modulus and the viscous modulus. A brief overview of the most
used viscometers/rheometers is given below.
2.5.1. Rotational rheometers/viscometers
One of the most common instruments for measuring viscosity, rotational
viscometers base their operation on measuring the resistance to the flow of a
fluid and having the main advantage of operating continuously at one shear
rate. This is achieved when a flow is generated within a space where the sample
is contained. The most common are concentric cylinders, cone-plate, and
parallel plate viscometers.
There are different methods of operation. One is the Searle type viscometer,
where the inner cylinder is the one that rotates (for concentric cylinder
viscometers, for the others, it would be the cone or the upper plate). The other is
the Couette type, where the outer cylinder rotates (the lower plate in other
cases). In addition, it is common to control the shear rate and measure the shear
22
Chapter 2
stress, however, there are instruments where the stress is controlled, and the
shear rate is measured [16], [17], [71].
The advantages of this type of instrument are the possibility of measuring the
viscosity of fluids regardless of their transparency or opacity. It also allows
time-dependent measurements and can make semi-automated measurements.
Furthermore, in some cases, non-Newtonian fluids can be measured. However,
they are expensive devices depending on their complexity (costs vary from
approximately $2000 and can reach up to $10,000 or more), and they become
large and difficult to transport.
2.5.1.1. Concentric cylinders
It consists of an inner cylinder (also known as bob) which has a radius R1 and
an outer cylinder of radius R2 (Figure 5). The liquid to be measured is placed in
the internal space between the two cylinders. As a cylinder rotates at a constant
speed (𝛺 [rad/sec]), the fluid will generate resistance to motion causing the
torque (T [dyne cm]) to be measured. The torque is related to the dynamic
viscosity (𝜂 [Pa·s]) of a Newtonian fluid by the Equation (2) [16], [71]:
4𝜋𝑅12 𝑅22 ℎ𝜂𝛺 (2)

𝑇= = 𝐶𝜂𝛺
𝑅22 − 𝑅12
Where C is a constant specific to the instrument and h is the height of the inner
cylinder that is covered by the fluid.
Figure 5. Basic structure of concentric cylinders viscometer
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2.5.1.2. Cone and plate
This shape allows a uniform shear rate to be generated, which makes it useful
for measuring the viscosity of Newtonian and non-Newtonian fluids. The
uniform shear rate is achieved when the angle between the cone and the plate is
reduced (α< 0.05 rad) [16], [71]. Usually, the angle α is less than 0.1 rad
(approximately 6˚). By obtaining the torque value, the fluid viscosity can be
obtained by Equation (3) [71].
3𝑇𝛼 𝐶𝑇 (3)
𝜂= =
2𝜋𝑅 3 𝛺 𝛺
Where, C is an instrument constant typically provided by de manufacturer, T
is the torque, 𝛺 is the angular speed and R is the radius of the cone. Figure 6
illustrates Cone and Plate structure.
Figure 6. Basic structure of cone and plate viscometer
2.5.1.3. Parallel plate
This geometry is very similar to the cone and plate geometry except that instead
of the cone, another plate is placed (observe Figure 7). In this case, the shear
rate loses uniformity because it depends on the distance between plates h and
the radial distance to the axis of rotation R. The torque T is related to the
viscosity of a Newtonian fluid by equation (4) [16].
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Chapter 2
2𝑇ℎ (4)
𝜂=
𝜋𝑅 4 𝛺
Where 𝛺 is the angular speed.
Figure 7. Basic structure of parallel plates viscometer
2.5.2. Capillary viscometers
This category includes a varied number of instruments; however, they all
operate with the same principle, which is to pass fluid (Newtonian fluid) from
one tube to a smaller one called capillary to measure the volumetric flow rate
along with the time it takes for the entire volume to pass through the
graduation marks on the instrument (Figure 8). The liquid can flow through the
capillary either by the influence of gravity or by an external force such as a
piston [16], [17].
Its operation is usually simple with a relatively low sample volume (a few
milliliters) and has good temperature control and a cheaper cost than the rest.
On the other hand, they have difficulty being cleaned. Depending on the range
of viscosities to be measured will be the instrument's dimensions, which
conditions to have several tubes to measure a wide range of viscosities.
The viscosity calculation using glass capillaries is based on the Poiseuille
equation. The expression is illustrated in equation (5) [71].
𝜋𝑔ℎ𝐷 4 (5)
𝜂= 𝜌𝑡
8𝑙𝑉
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Where rho is the density of the fluid, g is gravity, h is the height of the fluid
surface, D is the diameter of the capillary, l is the length of the capillary, V is the
defined volume of the liquid and t is the time required for the volume to flow
between the graduated marks of the viscometer.
Figure 8. Basic structure of capillary viscometer
2.5.3. Falling body method
The Falling Body Method relates the velocity at which an object (a sphere or a
cylinder) falls through the viscous fluid with the viscosity of the liquid. In
Figure 9, a basic diagram of this method is illustrated. Other parameters related
are the dimensions of the container, the densities of the fluid and the object, and
the force of gravity. This method consists of marking a start and an end on the
container to establish the distance d that the object will travel. When the object
falls between the two marks at its terminal velocity, the displacement time is
measured to calculate the velocity and viscosity. The measuring range can be
adjusted by changing the density or the dimension of the object. It is often used
to measure low or medium viscosities of Newtonian fluids that must be
translucent, and it requires approximately 40 ml sample volume to perform the
viscosity measurement [16], [17].
For the case of a falling sphere, the relationship between the object's velocity
and the fluid's viscosity is seen in Equation (6) [16].
26
Chapter 2
(𝜌2 − 𝜌1 ) (6)
𝜂 = 2𝑔𝑟 2
9𝑈𝑡
Where 𝜂 is the absolute viscosity, r is the radius of the sphere, g is the gravity
force, 𝜌2 is the density of the sphere, 𝜌1 is the density of the fluid and 𝑈𝑡 is the
terminal velocity.
Figure 9. Basic structure of falling body viscometer
2.5.4. Ultrasonics
Viscosity can also be measured using ultrasonic waves (frequencies between
104 and 108 Hz ). This is achieved by detecting the absorbed energy of the
acoustic wave transmitted through the fluid. The measurement method consists
of placing two transducers so that one emits the acoustic wave, which passes
through the liquid and is received by the other transducer, which detects the
decay of the signal.
This method is technically more complex than the others, so it has not been
positioned as a basis within the methods for measuring viscosity [16].
2.6. Quartz Crystal Resonators
Quartz Crystal Resonators (QCR) are elements that take advantage of the
piezoelectricity of quartz, which can be summarized as the ability to generate
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an electrical charge when the crystal is subjected to mechanical stress.
Conversely, when the crystal is subjected to a potential differential, it will
experiment a deformation depending on its physical shape [2], [72], [73]. There
are a great variety of quartz cuts that serve different applications. In this thesis,
we will talk specifically about AT-cut crystals, which work in thickness shear
mode.
To excite the quartz crystal, it is usually placed in the middle of two electrodes
(Figure 10). These electrodes are usually made of conductive metals such as
gold, silver, and copper. In this work, we will focus on gold electrodes.
Figure 10. QCR schematic.
As mentioned earlier, when the QCR is excited with an AC signal, it will suffer
a shear deformation (it will vibrate), as shown in Figure 11; and when the
frequency of this signal coincides with one of the acoustic resonance frequencies
of the quartz plate, the amplitude of the oscillation will be large, and so will be
the electric current consumed by the electrode [2]. This frequency is called the
fundamental resonance frequency of the QCR.
Figure 11. Shear deformation during oscillation in QCR
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Chapter 2
The crystal's vibration depends on its thickness; for example, a crystal with 330
µm thickness will vibrate at a frequency of 5 MHz and one with 168 µm at 10
MHz. On the other hand, gold electrodes usually have a thickness of 0.1 µm
[74]. This relationship is illustrated in Equation (7) [2].
𝑣𝑞 (7)
𝑓0 =
2𝑑𝑞
where 𝑓0 is the fundamental resonance frequency, 𝑣𝑞 is the wave speed for
quartz, and 𝑑𝑞 is the thickness of the quartz.
2.6.1. Butterworth-Van Dyke model
A common representation of the crystal is the electrical circuit known as the
Butterworth-Van Dyke (BVD) equivalent circuit (Figure 12) derived from
Mason equivalent circuit [75]. This model explains the crystal behavior near its
resonance frequency and is composed of two arms; the first is the motional arm,
and it has three series components: 𝑅1 , 𝐿1 , and 𝐶1 . 𝑅1 models the dissipation of
the oscillation energy from the medium in contact with the crystal, 𝐿1 is the
inertial component of the oscillation and is related to the displaced mass while
the crystal is vibrating, 𝐶1 models the stored energy and is associated with the
elasticity of the quartz. The second arm contains a parasitic capacitance in
parallel (𝐶0 ); this capacitance is related to the sum of the crystal’s electrode’s
static capacitances. When the crystal is in contact with a liquid load, one
element is added to the first branch, 𝑍𝐿 is the loading from the liquid sample
[76].
Figure 12. Butterworth-Van Dyke model of Quartz Crystal Resonators (QCR) with liquid load.
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The circuit elements can be calculated theoretically with the Equation (8) for a
specific resonance frequency [77]:
𝜀22 𝐴 (8a)
𝐶0 = ,
𝑑𝑞
8𝐾02 𝐶0 (8b)
𝐶1 = ,
(𝑁𝜋)2
1 (8c)
𝐿1 = ,
𝜔𝑠2 𝐶1
𝜂𝑞 𝜔 2 (8d)
𝑅1 = ( ) ,
𝑐66 𝐶1 𝜔𝑠
where 𝜀22 is the quartz permittivity, A is the electrode surface area, 𝑑𝑞 is the
quartz thickness, 𝐾0 is the electromechanical coupling constant for quartz, N is
the overtone number =1, 3, 5, …, 𝜔𝑠 is the angular series resonant frequency for
the unperturbed QCR = 2π𝑓𝑠 , 𝑓𝑠 is the series resonant frequency in hertz, ω is
the angular excitation frequency = 2πf, 𝜂𝑞 is the effective quartz viscosity, and
𝑐66 is the quartz elastic constant.
Under certain conditions, Equation 8 may be inaccurate; therefore, it is
proposed to use another type of characterization based on impedance analysis
[78], [79].
According to Figure 12, the equivalent admittance at BVD circuit can be written
as [80]:
1 (9)
𝑌= + 𝑗𝜔𝐶0 ,
1
𝑅1 + 𝑗𝜔𝐿1 + + 𝑍𝐿
𝑗𝜔𝐶1
where 𝑍𝐿 is the viscosity from the liquid load:
𝑡2 𝜔𝜂𝐿 𝜌𝐿 𝜔𝜂𝐿 𝜌𝐿 (10)

𝑍𝐿 = 2 [√ + 𝑗√ ],
4𝑒35 𝑆𝑒 2 2
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Chapter 2
2
where t is the thickness of the quartz plate, 𝜔 is the angular frequency, 𝑒35 is the
piezoelectric constant, and 𝑆𝑒 is the electrode area.
Also, the conductance is expressed as:
𝑅1 + 𝑅𝑒 [𝑍𝐿 ] (11)
𝐺= ,
1
(𝑅1 + 𝑅𝑒 [𝑍𝐿 ])2 − (𝜔𝐿1 + 𝐼𝑚 [𝑍𝐿 ] − 𝜔𝐶 )2
1
From the equivalent circuit, we obtain 𝑓𝑠 , which occurs at the maximum
conductance point [81]. According to the electrical model, when the crystal is in
contact with a fluid, the peak of conductance is displaced in frequency to a
minor frequency and decreasing its magnitude (Figure 13); this maximum
conductance can be located by performing a frequency sweep that encloses the
fundamental resonance frequency of the crystal.
Figure 13. Frequency response of QCR conductance with and without sample.
The frequency bandwidth of the QCR is very narrow, which makes them have
excellent accuracy. This sharpness is quantified by the Q factor where Q = 𝑓/2Γ
with 𝑓 as the resonance frequency and Γ as the half-bandwidth at half
maximum (Figure 13). The shift in half-bandwidth at half-maximum (ΔΓ) is
related to the energy transferred from the crystal to the sample over time and
can provide information on the viscoelastic properties of the sample; also is
used as dissipation D = 1/Q [2].
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Another way to visualize 𝑓𝑠 is through an admittance diagram (Figure 14). On
the horizontal axis is the conductance G and on the vertical axis is the
susceptance B. This diagram shows three resonant frequencies representative of
a QCR: 𝑓𝑚 , 𝑓𝑠 , and 𝑓𝑟 . 𝑓𝑚 is the resonant frequency located at the maximum
admittance point (Y), 𝑓𝑠 is the aforementioned series resonant frequency located
at the maximum conductance point (G), and 𝑓𝑟 is the zero-phase resonant
frequency located at the point where the susceptance value (B) is zero [79], [81].
Figure 14. QCR admittance locus
2.6.2. Mass deposition
One of the main applications of QCRs is as a mass sensor, commonly known as
Quartz Crystal Microbalance (QCM), whose origin derives from research
conducted by Günter Sauerbrey. The relationship between the change of the
resonant frequency of the QCR with the added mass is illustrated in Equation
(12) [3].
2𝑓0 2 (12)
∆𝑓 = − 𝐴 ∆𝑚,
√𝜌𝑞 𝐺𝑞
∆𝑓 = 𝑓𝑠 − 𝑓0 , (13)
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Chapter 2
where ∆𝑓 is the frequency shift, 𝜌𝑞 = 2.648 g·𝑐𝑚−3, and 𝐺𝑞 = 2.947 x 1010 𝑁 ·
𝑚−2 are the specific density and the shear modulus of quartz respectively, A is
the piezoelectrically active crystal area, and ∆m is the thin film of mass
deposited.
Strictly speaking, a QCM sensor measures an areal mass density in units of
mass per area beyond measuring mass, and instead of measuring “micro”
masses it senses units near to “nano” masses. An important note is that the
equation described by Sauerbrey only applies when the sample is rigid and free
of a liquid environment (in this case, ∆𝛤 << ∆𝑓). In gravimetric cases where
there is contact with liquids, two frequency changes will accumulate due to: a
mass deposition and the contact with liquid [2].
2.6.3. Fluid deposition
When the sample placed on the QCR is a liquid, the crystal surface movement
generates a shear wave that propagates into the deposited fluid; consequently,
this wave will suffer a damping effect. The connection between the frequency
shift and the fluid's density-viscosity product contact with the crystal is given
by the Gordon-Kanazawa relationship, Equation (14) [4]. Although the equation
is attributed to authors J. G. Gordon and K. K. Kanazawa, the equation was
described in Mason's earlier work [82].
3 𝜌𝐿 𝜂𝐿 (14)
∆𝑓 = −√𝑛 𝑓0 2 √ ,
𝜋𝜌𝑞 𝐺𝑞
where 𝜌𝐿 is the fluid's density, 𝜂𝐿 is the fluid's viscosity, and n is the overtone
number; in this work, the fundamental frequency of the crystal is used; thus, n =
1.
However, this relationship has been derived theoretically for an infinite quartz
plate without interfacial slip and for the case of a perfectly smooth surface. In
addition, some effects make viscosity measurement difficult, such as roughness
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and nanoscopic air bubbles; consequently, the use of this equation has not
proved to be reliable when measuring the density-viscosity product of
Newtonian fluids [83], [84].
As Johannsmann mentions in his work ([34], [47]), it is essential to clarify that
the 𝜂𝐿 viscosity obtained by the QCR (MHz viscosity) is different from the
steady shear viscosity. Moreover, QCR doesn't measure the high shear viscosity
unless it is claimed that the high frequencies mimics the behavior at high shear.
2.6.3.1. Complex viscosity
To obtain the viscoelastic properties of the sample, Johannsmann determines
the relationship between the complex viscosity with ∆𝑓 and ΔΓ [2]:
𝐺´´𝐿 𝜋𝜌𝑞 𝐺𝑞 𝛥𝑓𝛥𝛤 (15)

𝜂´𝐿 = 𝜔
=− 𝜌𝐿 𝑓𝑠 𝑓02
,
𝐺´𝐿 1 𝜋𝜌𝑞 𝐺𝑞 (𝛥𝛤 2 −𝛥𝑓2 ) (16)

𝜂𝐿´´ = 𝜔
=2 𝜌𝐿 𝑓𝑠 𝑓02
,
∆𝛤 = 𝛤𝑠 − 𝛤0 , (17)
where 𝜂´𝐿 and 𝜂𝐿´´ are the real and the imaginary components of the viscosity
respectively, 𝐺´ and 𝐺´´ are the real and the imaginary components of the shear
modulus for the liquid, ω is the angular frequency, and ΔΓ is the half-
bandwidth at half maximum shift. For Newtonian fluids, ∆𝑓 and ΔΓ are equal
and opposite (𝜂´𝐿 = constant, 𝜂𝐿´´ = 0 ). For viscoelastic fluids: 𝜂´𝐿 = 𝜂(𝜔), 𝜂𝐿´´ ≠ 0.
2.6.4. Penetration depth
It is important to note that QCRs measure in the region closest to the crystal
surface. The shear wave decays as it penetrates the fluid as follows (Figure 15)
[84]:
2𝜂 (18)
𝛿 = √𝜔𝜌𝐿 ,
𝐿
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Chapter 2
Where δ is the penetration depth. In water, the penetration depth for a crystal
with 𝑓 = 10 MHz is about 178 nm.
Figure 15. Decay depth of the acoustic wave transmitted to the fluid.
2.7. QCRs used as viscometers
The use of QCR's as tools for measuring viscosity has only recently begun to be
explored, and there are few examples of their use for this purpose. One such
case is illustrated in [85], where QCRs are used to measure the viscosity of
droplets of various industrial oils to determine the operating condition of the
oils. A further case is documented in [86]. Here the authors measure the
viscosity of battery acids using QCRs to assess their state of charge. On the
other hand, in [87], the authors estimate the viscosity of different solutions such
as sucrose, glucose, or ethylene glycol using a volume of the order of microliters
and based on an impedance analyzer.
2.8. Brief description of artificial neural networks
Artificial neural networks (ANNs) are a segment of Artificial Intelligence (AI)
that, based on examples, can induce concepts. They are data processing systems
whose operation is based on the networks of neurons in the brain [88], [89].
These tools help find relationships between data and also in classification and
prediction. They can also improve their performance by using information
obtained from previous tasks.
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Its use has been increasing in many areas of engineering, and biomedical
engineering is no exception. Some examples of ANN applications in
bioengineering can be found in: [88]–[91].
The basic model of these ANNs (known as the multilayer perceptron model) is
shown in Figure 16. It is composed of three types of layers: an input layer, an
output layer, and hidden layers. This type of model allows information to flow
in one direction, from input to output and is known as a feedforward neural
network. In this way, data will enter the network's input nodes, then be
processed in the hidden layers, and finally deliver the processed data to the
output layer [89].
Figure 16. Basic ANN model.
There are three main categories for ANN learning [88]:
1. Supervised learning. Expected outputs are delivered to the network, and
the network is trained to respond appropriately.
2. Unsupervised. The expected outputs are not provided in advance, and it
is expected to find structures present in the inputs.
3. Reinforcement. Expected outputs are not delivered, and instead,
performance indicators are provided periodically.
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Chapter 2
For the case of the present work, a supervised learning technique will be used
with backpropagation algorithm for training. A brief comparison of the results
will be made by modifying the number of hidden layers (between one and two)
and the number of training epochs (100, 200, and 300). A more extensive study
with a more significant number of configurations is possible as the field of
ANNs is vast; however, it is beyond the intended scope of this thesis.
2.9. ViSQCT project and previous work
The ViSQCT (Viscosity Detection by Quartz Crystal Techniques) project arose
in the LBN of the CTB of the Universidad Politécnica de Madrid (UPM) in 2017
as the continuation of a previous project where the theory for the application of
QCRs to measure the viscosity of biological samples with reduced volume was
put forward. Positive results showing the feasibility of using QCRs for this
purpose were obtained from this previous project along with a first prototype
of the sensor, two journal publications [92], [93], and a Ph.D. thesis [94].
Essential aspects of the prototype design are the sweep signal generation
module and the electronics for obtaining and converting the voltage, current,
and susceptance signals from analog to digital. Together with the power supply
of the system, both concepts are explained below.
2.9.1. AD9850 module
The Analog Devices AD9850 module was used to generate the frequency
sweep. This device uses DDS (Direct Digital Synthesis) technology in
conjunction with a high-speed digital-to-analog converter to create
programmable digital waveforms. An Arduino Due board was used to program
and adjust the AD9850, which will be discussed later. The module is adjusted to
perform the desired frequency sweep through serial communication. The
sweep range of the device is from 1 Hz to 40 MHz. Usually, the sweep used is
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50 kHz at frequencies close to 10 MHz, which is the fundamental resonance
frequency of the QCRs used.
2.9.2. Analog to digital stage
The analog voltage, current, and susceptance signals are converted to digital
signals by an Arduino Due board, which will create a data frame of each and
send them to the LabView software on the computer via serial. On the other
hand, Figure 17 shows a diagram illustrating how the analog signals are
obtained. The left branch indicates the frequency sweep generation stage, and
the right branch shows the signals measured after QCR excitation.
Figure 17. Simplified diagram of the signals acquisition system.
2.9.3. Power supply
The circuit is designed to work with two 9 volt batteries in conjunction with
two voltage regulators at 5 and -5 volts (LD1117 and MC7905, respectively).
These will power the board and the AD9850 module. The Arduino due board is
powered with the same USB cable with which it communicates with the
computer. With these elements, the operating time of the sensor is
approximately three hours.
With this previous work, the ViSQCT project begins with designing and
constructing a new version of the sensor prototype for further evaluation as a
clinical tool.
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Chapter 3
3. Hypothesis and Objectives
3.1. Hypothesis
There is evidence on the importance of viscosity as an essential factor in the
characterization of biological fluids, either to determine the health status or to
develop new biocompatible materials. On the other hand, Quartz Crystal
Resonators demonstrate their applicability as sensors that allow determining
the viscosity of a low volume sample of a fluid, which helps the medical field
by generating small, inexpensive, and accurate equipment with clinical and
research and development applications.
As mentioned in the introduction of this work, current methods to determine
viscosity are expensive, bulky, or slow and often require sample volumes not
available in several biological cases. For this reason, this work proposes that it is
possible to design and develop a portable, low-cost QCR-based sensor that,
together with a methodology, can be used to measure the viscosity of a scanty
volume sample to assist in medical applications as a diagnostic and biomaterial
characterization tool.
3.2. General and specific objectives
The main objective of this doctoral thesis is to develop and validate an
enhanced low-cost QCR-based sensor within its methodology to measure the
viscosity of a sample with reduced volume.
To meet the proposed objective, the device must have a space where the QCR
can be easily placed, allowing the sample to be deposited by pipetting. Finally,
the QCR must be removed from its holder and cleaned adequately at the end of
the measurement.
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This development involves four parts that are considered as specific objectives:
design, development, phantoms development, and validation.
1. Design the sensor electronically and mechanically, being the electronic
part the sensor circuit (with its control system), and the mechanical part
the holder that allows isolating the samples. This part seeks to update the
work previously done within the project to enhance the sensor, reduce its
size, and improve its performance.
2. Physical construction of the device at the electronic and hardware level.
The measurement protocol and the crystal cleaning protocol will also be
defined.
3. Search and (or) development of samples. This objective is divided into
two. The first is the development of phantoms that mimics de viscous
properties of human fluids, such as Cerebrospinal Fluid and Synovial
Fluid. The purpose of this is to evaluate the sensor's performance for its
possible application in cases where the measurement of this type of
fluids is required. The second is to search for biomaterials whose
viscosity characterization is relevant for its development. This is
intended to test the capabilities of the sensor in the area of materials.
4. Finally, the validation of the device is required by measuring real
samples. The results of measuring human fluids will allow an evaluation
of the sensor's capabilities as a diagnostic tool. On the other hand, the
results as a biomaterial characterization tool will provide the possibility
of a second approach to the sensor, venturing into the area of biomaterial
development.
40
Chapter 5
4. Sensor Development
In this chapter, the sensor design is detailed. It is important to clarify that this
design is based on a previous prototype that was outdated.
In general, the hardware was improved by reducing its size and optimizing the
signal amplification stages by replacing the analog potentiometers with digital
potentiometers. These changes influenced the software, which was enhanced.
Portability and low cost are essential system requirements. On a technical level,
the ability to measure the resonant frequency of the QCR (sampled and
unsampled) by means of a frequency sweep was maintained. In addition, the
prototype must allow the static measurement of the sample while complying
with the facilities for fluid deposition and subsequent washing of the crystal.
4.1. Sensor description
As discussed in Chapter 2, the series resonance frequency (𝑓𝑠 ) of the QCR (along
with the half-bandwidth) changes when the crystal is in contact with a fluid.
Once the 𝑓𝑠 is located (when G is maximum), a viscosity value can be obtained
using equation 14 . This is by measuring 𝑓𝑠 without sample and then 𝑓𝑠 with the
sample to calculate ∆𝑓. Such ∆𝑓 can also be used as a characterization
parameter. As for sensor operation, the way to locate 𝑓𝑠 is by mapping G at
frequencies close to 𝑓0 . For this, it is necessary to excite the crystal with a sweep
of frequencies close to 𝑓0 . The response of the QCR to this stimulus must be
obtained to obtain G at each frequency of the sweep and thus observe the
maximum point of G. The signals measured as crystal response at each
frequency are voltage (V), current (I), and susceptance (B). A diagram of this
process can be observed in Figure 18.
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Figure 18. Excitation and signal acquisition process in the QCR.
Considering the above mentioned, we can reduce the elements that make up
the prototype to three: electronic, software, and mechanical parts. The electronic
part consists of three parts, and the first one is the frequency sweep generation
that will excite the QCR. Subsequently, there is the signal acquisition stage,
where the signals measured from the crystal response must be treated and
digitized for subsequent analysis and storage. The last stage is the power
supply of the device. The software used to control the prototype is LabView
from National Instruments®, which is used to configure the device to perform
the required measurements and subsequently store the data obtained.
4.2. PCB design
The design of the electronic board was made with Altium software. The
objective was to have the smallest possible dimensions and allow the Arduino
due board and the AD9850 module to be coupled. For this purpose, four
X9C103 digital potentiometers were added to control the amplification stage of
the signals, replacing the analogue potentiometers used in previous versions.
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Chapter 5
Figure 19 shows the digital design of the board, the upper image is the top side
of the circuit (where the AD9850 circuit is coupled), and the lower image is the
bottom side (where the Arduino due board is mounted).
Figure 19. PCB design
Once we had the design, the next step was to manufacture the board. Figure 20
shows the PCB used without the components mounted. Again, the upper image
is the top side of the circuit, and the lower is the bottom.
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Figure 20. PCB manufactured
The last stage of the PCB was to solder the components. After this, the correct
operation of the circuit was checked by testing the correct generation of the
frequency sweep and the correct obtaining of the signals of interest. Figure 21
shows this circuit evaluation stage.
Figure 21. Evaluation of the mounted PCB.
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Chapter 5
4.3. QCRs an holder cell
This work was developed using QCR with 𝑓𝑠 = 10 MHz, gold electrodes,
electrode dimensions of 5 and 11 mm, roughness < 1 nm, and mounted in HC-
51 holder. The crystals were purchased from Krystaly (Hradec Králové, Czech
Republic). Figure 22 shows the QCRs employed. The specific dimensions
(provided by the supplier) are illustrated in Figure 23.
Figure 22. QCRs used in measurements.
Figure 23. Dimensions (in mm) of the QCRs.
On the other hand, it was necessary to manufacture an enclosure where the
crystal could be placed during the measurements and where the liquid to be
measured could be deposited. The design of this holder cell is based on the one
used in the first Open QCM® (Novaetech S.r.l., Naples, Italy) device. The lid
was modified and adapted to the needs of this work by creating an area that
allows access to the crystal, and where the sample to be measured is kept static
for measurement. Two silicone rings contain the crystal, one located in the lid
and the other in the base. Figure 24 shows the 3D design of the lid. The finished
enclosure can be seen in Figure 25, fabricated from PLA using a 3D printer.
Figure 26 illustrates the QCR placed in the holder cell.
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Figure 24. Design of the lid required for the holder cell.
Figure 25. Holder cells manufactured in a 3D printer using PLA.
Figure 26. QCR placed in the holder cell.
4.4. System control and user interface
The system is controlled by a program developed in the LabView environment
of National Instruments®. The program was produced before this work and was
adapted for the new device. It allows configuring the values required by the
AD9850 for the frequency sweep through user input. After the sweep, it
receives the digital signals of voltage, current, and susceptance to plot the
received signals and provide the 𝑓𝑠 and Γ data. This process can be repeated N
number of times, where the user sets N. On the other hand, the program allows
the activation of the gain increase in the amplification stage of the measured
signals. This is important because the measured signals are attenuated when
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Chapter 5
measuring the 𝑓𝑠 of the sampled crystal, so it is necessary to amplify them for
accurate detection.
The main program window is shown in Figure 27. In this window, a first
configuration is observed before being executed. A directory must be
established where the information is stored, and the communication port where
the Arduino board is connected must be identified. The "Sample" button is also
activated for the sample measurement. At the end of the measures, a results
window is displayed showing the conductance and susceptance curves
obtained in each cycle, as shown in Figure 28. A flow chart of the program is
illustrated in Figure 29.
Figure 27. Main window.
Figure 28. Results window.
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Figure 29. Simplified flowchart of the program algorithm.
4.5. 3D printed case
Once the device's functionality was demonstrated, a case was elaborated to be
placed for protection from the outside. It was designed for the sensor's
dimensions with ventilation openings and spaces where the cables for the
batteries, the QCR, and USB can pass. The case material is PLA and was built
with a 3D printer like the QCR holder cell. Figure 30 shows the digital design of
the lid and base, and Figure 31 shows the case once printed.
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Chapter 5
Figure 30. Design of the lid and the base of the case.
Figure 31. 3D printed case.
4.6. Final prototype
The final device (named as ViSQCT sensor) can be seen in Figure 32 without the
holder cell, batteries, and case. It weighs less than 100 g, and costs less than
200€. Figure 33 exhibits the sensor components in operation, and Figure 34
reveals the complete system working.
Figure 32. ViSQCT sensor
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Figure 33. System elements.
Figure 34. Complete system operating.
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Chapter 6
5. Protocols and Preliminary Measurements
Once the ViSQCT sensor has been developed, the experimentation stage
follows. For this, it is necessary to define the measurement and cleaning
protocols to be used in the measurement stage. With the protocols established,
preliminary measurements were made with common fluids to observe the
sensor's response.
It is important to note that, in these first measurements, QCR’s from a first
batch purchased were used. The measurements shown in the following
chapters were performed with a new batch of QCR’s.
This chapter will present the mentioned protocols and the results of the first
measurements with the sensor.
5.1. Measurement protocol
The development of the experiments was carried out with the following
measurement protocol.
1) Connect the sensor to the computer using a USB. Ensure that you select
the appropriate communication port in the program to enable the
connection with the sensor.
2) Place the QCR in the holder cell, carefully close the lid, and connect it to
the sensor.
3) Connect the two batteries to the sensor.
4) Configure the software to measure without sample. Start the
measurement and obtain the 𝑓𝑠 .
5) Transfer the data obtained for analysis.
6) Add the sample to be measured on the crystal. Usually, the sample
volume is 50 µl, but this may vary.
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7) Repeat the measurement by configuring the software to measure with
the sample.
8) Transfer the data obtained for its analysis. With the data obtained from
point 4, get viscosity values using Equation (14).
9) The entire process must be repeated to have several data from the same
sample.
5.2. QCR cleaning protocol
After each experiment, the QCR must be carefully removed from its container
for cleaning.
For cleaning, it is necessary to use nitrile gloves and prepare the following
solutions.
First solution. Weigh 1 gram of Sodium Dodecyl Sulfate (SDS) and add it to 50
ml of distilled water. This mixture will serve as a detergent to clean the glass
removing residues.
Second solution. Make a 70% ethanol mixture. This solution will be used to
disinfect the QCR.
Once the above solutions are ready, we apply the following cleaning protocol.
First, rinse the QCR with the SDS solution until the residues are removed. If the
crystal is still dirty, it will be necessary to moisten the nitrile glove and rub the
glass very gently. Then rinse with distilled water. If performing measurements
with biological fluids, it is essential to sterilize the QCR. For this purpose, it can
be rinsed with 70% ethanol. Onetime disinfected, it is rinsed with distilled
water and dried with air to be used again.
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5.3. Open QCM
As we began to evaluate the performance of the ViSQCT sensor, we considered
it interesting to compare results with a similar sensor that is available on the
market. With this comparison, we will be able to observe if there are similarities
in the results and advantages and disadvantages in the use of both sensors.
The selected sensor is Open QCM Q-1® (Novaetech S.r.l. Italy), an open-source
sensor designed for continuous measurement [95].
This sensor can measure the resonance frequency by making a frequency sweep
and using a gain-phase detector that compares the exciting signal entering the
crystal with the signal at the crystal output (Figure 35). The device was adapted
to be connected to the QCRs used by adding a pair of clip wires to the device's
output.
Although both devices can measure the resonance frequency with a frequency
sweep, the range covered by this sweep is different. In the ViSQCT sensor, the
frequency range is 50 kHz, allowing longer measurement times and a higher
resolution when measuring larger ∆𝑓 (high viscosities). On the other hand, the
Open QCM sensor has a frequency sweep range of 11 kHz, making the
measurement capture faster but difficult when measuring huge ∆𝑓.
Figure 35. Signal acquisition method for Open QCM sensor.
This sensor has its software developed in Python programming language. Once
the measurement is started, it will only stop until the user indicates it. Once the
experiment is finished, all the data obtained are stored.
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The measurement protocol is maintained except for a slight variation. As it is a
continuous measurement, the software visualizes the resonance frequency in
real-time. Thus, the user should consider that the desired resonance frequency
(without or with sample) has been measured once a stable signal is observed.
As the protocol states, the unsampled resonance frequency is obtained first.
When the signal stability is observed (and without stopping the process), the
sample is placed in the QCR. Again, a stable signal must be observed to
consider the resonance frequency localized. Upon completion, the generated
data are transferred to calculate the viscosity.
The cleaning protocol remains the same.
5.4. Volume variation with water
The first experiment was to observe the sensor's response when measuring
water samples by varying the sample volume between 30 and 70 µl. These
values were determined by observing that with 30 µl of water, it is possible to
completely cover the QCR area (without spaces exposed to air). On the other
hand, since no significant changes in viscosity were observed, the
measurements were concluded using 70 µl. Figure 36 shows the viscosity value
obtained (using equation 14), which ranges between 2.8 and 2.9 mPa·s
approximately.
With this, we can conclude that, by increasing the sample volume (completely
covering the crystal's surface), the sample's weight will have a negligible effect
on the viscosity measurement obtained by frequency shift.
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Chapter 6
Figure 36. Viscosity measured by varying the volume of water samples.
5.5. Conventional fluids measurement
Distilled Water, Acetone, Alcohol, and different dilutions of mixtures of
glycerin, sugar, and salt were employed to study the measuring characteristics
with simple fluids. The dilutions used were 10, 20, and 30 wt% to increase the
density-viscosity of the fluid. An analytical balance (Discovery dv215,
OhausTM, Nänikon, Switzerland) was used to weigh the solute of the samples
and to obtain the density by weighing 1 mL of sample volume.
As a reference, a rotational viscometer (Searle-type, Alpha series, Fungilab,
Barcelona, Spain) was used. LCP adapter was employed; this allows viscosity
measures up to 2 Pa·s using 16 mL of the fluid. The viscosity results obtained
with the sensors were compared with those obtained with the rotational
viscometer.
In Table 4, the results for water, alcohol, and acetone are presented. Measured
∆𝑓 and the theoretical values of density and viscosity of the fluids are shown.
The three liquids have different viscosities, with acetone the less viscous and
isopropyl alcohol the most. It can be noted that the frequency difference
increases along with the density-viscosity of the fluid. The difference between
the theoretical viscosity value and the viscosity obtained by each sensor is large
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and superior in the ViSQCT sensor; for example, the viscosity of water is
around 1 mPa·s (@ 20˚C), and the results show a value of 2.86 mPa·s for the
ViSQCT sensor and 2.29 mPa·s for the Open QCM sensor.
Water Alcohol Acetone
Isopropanol
Theoretical Density 1000 786 791
(mg/ml)
Theoretical Viscosity 1.00 2.10 0.32
(mPa·s)
∆𝑓 -3416 -4601 -2467
(Hz) ±62 ±218 ±179

ViSQCT sensor
Viscosity 2.86 6.60 1.88
(mPa·s) ±0.10 ±0.62 ±0.27
Viscosity difference 186 214 489
vs Theory (%)
∆𝑓 -3062 -3634 -1676
(Hz) ±54 ±196 ± 95

Open QCM
Viscosity 2.29 4.11 0.87
(mPa·s) ±0.08 ±0.44 ±0.10
Viscosity difference 129 96 171
vs Theory (%)
Table 4. Water, acetone and alcohol data and results.
Figures 37 and 38 show the values of ∆𝑓 and viscosity measured with both
sensors. The theoretical value of viscosity is also shown.
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Chapter 6
Figure 37. ∆𝑓 measured for water, acetone and alcohol.
Figure 38. Viscosity measured for water, acetone and alcohol.
The results of each dilution are shown in Table 5. Figures 39, 40, and 41
illustrate each parameter measured. In this case, ∆𝑓 and viscosity were
measured with both sensors and ΔΓ only with the ViSQCT sensor; in addition,
the viscosity for each dilution was measured with the viscometer to compare it.
The dashed line represents the data obtained with the Open QCM sensor, and
the continuous line represents the ViSQCT sensor data; the value measured
with water is also shown in the figures. As expected, as viscosity increases on
each fluid, both ∆𝑓 and ΔΓ increases. There is a similar trend in ∆𝑓 increase
between both sensors, this is reflected in the viscosity results where an offset of
approximately 1.5 mPa·s is observed. The differences between the viscosity
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obtained with the sensors and the obtained with the viscometer are significant ;
however, what is crucial for our purpose is the possibility of detecting viscosity
variations using ∆𝑓, which is shown in the results. The ΔΓ value obtained also
increased as viscosity increased. Its usefulness will be evaluated later since it
could be another parameter to help discriminate between samples.
Glycerin Sugar Salt
wt % 10% 20% 30% 10% 20% 30% 10% 20% 30%
Density (mg/ml) 1034 1064 1091 1045 1096 1139 1088 1145 1194
Viscosity (mPa·s) 1.11 1.50 2.00 1.16 1.66 2.94 1.14 1.33 1.40
∆𝑓 -4115 -4789 -5475 -4144 -4949 -6469 -4150 -4557 -5249
(Hz) ±34 ±20 ±106 ±46 ±34 ±65 ±64 ±149 ±52
ΔΓ 2248 2532 2901 2309 2649 3472 2293 2519 2726

ViSQCT sensor
(Hz) ±179 ±288 ±512 ±115 ±93 ±174 ±306 ±272 ±315
Viscosity 4.01 5.28 6.73 4.02 5.47 9.00 3.88 4.44 5.65
(mPa·s) ±0.06 ±0.04 ±0.26 ±0.08 ±0.07 ±0.18 ±0.12 ±0.29 ±0.11
Viscosity difference 261 252 236 247 230 206 240 234 304
vs Viscometer (%)
∆𝑓 -3367 -4091 -4702 -3432 -4296 -5398 -3502 -3855 -4317
(Hz) ±95 ±96 ±67 ±64 ±74 ±135 ±25 ±56 ±76
Open QCM
Viscosity 2.68 3.85 4.96 2.76 4.12 6.27 2.73 3.18 3.82
(mPa·s) ±0.15 ±0.18 ±0.14 ±0.10 ±0.14 ±0.30 ±0.04 ±0.09 ±0.13
Viscosity difference 142 157 148 138 148 113 142 139 173
vs Viscometer (%)
Table 5. Results for glycerin, sugar and salt.
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Chapter 6
Figure 39. Comparison of ∆𝑓 measured for glycerin, sugar, and salt using both sensors.
Figure 40. Comparison of ∆Γ measured for glycerin, sugar, and salt using ViSQCT sensor.
Figure 41. Comparison of the viscosity obtained using both sensors and the viscometer (for
glycerin, sugar, and salt).
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Figure 42 shows the ∆𝑓 measurement process (normalized) using water (black
line) and sugar 30% (blue line) samples with both sensors. In both cases, the
frequency drop is observed when the sample is added. Also, stability is noted
when measuring the resonance frequency. The Open QCM sensor has a more
stable curve when measuring sugar 30% (the sample with the highest viscosity
of the set).
Figure 42. Resonance frequency measurement for water and sugar 30% using: ViSQCT sensor
(left) and Open QCM sensor (right).
We can observe that there is a difference between the measured viscosities (each
sensor) and the reference viscosity (rotational viscometer),
At the end of the preliminary experiments, we can conclude that the sensor and
its methodology are valid for detecting viscosity changes of a fluid with a small
volume (50 µl) placed on the surface of the crystal. A second validation of this is
the comparison of the methodology using a commercial sensor with electronic
methods other than ours, obtaining similar results in the trend of the values as
the viscosity increased. Despite an offset between the two sensors (and a
notable difference between the two with the theoretical values), we can say that
both can respond with an increase in resonance frequency as the viscosity of the
sample increases.
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As mentioned in previous sections, the viscosity measured at high shear rates
can be different from that measured in steady share mode. The equation
obtained by Gordon and Kanazawa (Equation (14)) were obtained for an ideal
case without roughness. Considering all these points, the difference between
the sensors and the viscometer is comprehensible.
In chapter 7, tests using more complex fluids will be shown, whose response
will be vital to achieving the objectives proposed in this work.
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6. Experimental Methodology
This chapter describes the measurement setup together with the experimental
samples. First, liquid phantoms were developed to simulate the viscous
properties of human fluids to test the sensor's capabilities in complex fluids.
Subsequently, measurements were performed with SF from several patients
provided by the Hospital Universitario La Paz (HULP). For this, approval was
obtained from the UPM ethics committee and the HULP ethics committee.
To explore further applications of the sensor, the response of the sensor was
observed when measuring the gelation of hydrogels using two different
gelation phenomena and varying the concentration.
The experimental setup is illustrated in Figure 43; the QCR is placed inside the
holder cell where the liquid sample is dropped. The volume of sample used is
50 µl since it allows to avoid the complete evaporation of the liquid.
Experiments were performed at room temperature.
Figure 43. Measurements set-up.
6.1. Phantoms
Chapter 2 mentioned that two fluids of interest in this project are SF and CSF.
The interest in both arises from studies showing that their viscosity change is
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associated with specific pathologies [20], [22], [24], [36]. Thus, for each case,
artificial fluids were developed to simulate the fluid's viscosity for both a
healthy and a pathological case. The experimentation with artificial
cerebrospinal fluid (aCSF) and artificial synovial fluid (aSF) expose an
interesting challenge for the sensor since each fluid has different behavior from
a rheological point of view. One presents a non-Newtonian behavior (aSF), and
the second shows a Newtonian behavior (aCSF).
6.1.1. aCSF
CSF is a complex fluid mainly composed of Na+ ,Cl− , and HCO−

3 with lesser
amounts of K + and Ca2+ [25], [26]. Some aCSF are reported in [96], [97], where
NaCl and NaHCO3 are the elements with higher concentrations. According to
this, healthy aCSF was developed with the concentrations listed in Table 6.
Albumin was used to increase viscosity in aCSF and simulate abnormal fluid
since it is the protein with a higher concentration in pathological CSF [98], [99].
The cases for viral meningitis (VM aCSF) and bacterial meningitis were
developed; also, two extra samples were created to have more information on
the detection abilities of the sensors.
NaCl 𝐍𝐚𝐇𝐂𝐎𝟑 Albumin
Fluid concentration concentration concentration
(mg/ml) (mg/ml) (mg/ml)
Healthy aCSF 7.25 2.18 0.0
VM aCSF 7.25 2.18 0.5
aCSF1 7.25 2.18 1.0
aCSF2 7.25 2.18 2.0
BM aCSF 7.25 2.18 3.0
Table 6. Artificial Cerebrospinal Fluids (aCSF) compositions.
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6.1.2. aSF
SF is a mixture of plasma and HA, whose concentration determines the fluid's
viscoelastic properties. Arthritic diseases are associated with the reduction of
HA [21], [22], [24], [100]. For this reason, HA dilutions were made to generate
artificial SF (aSF) and emulate physical properties such as viscosity. In healthy
SF, the concentration is around 3.5 mg/ml, while in OA, the HA concentration
decrease to 1.3 mg/ml and in RA to approximately 0.84 mg/ml [101].
Six dilutions were made, one for healthy fluid two for abnormal fluid using the
aforementioned concentrations. Three extra dilutions were created to test the
sensor's detection capabilities. The mixtures were made with ultrapure water
and weighed the necessary amount of HA with an analytical balance. After
adding HA (Mw = 1.5 MDa; Acros Organics Inc.) into the water, gently stirring
was required. Finally, the sample is stored in the refrigerator for a few hours to
eliminate bubbles formed when mixing. Table 7 shows the compositions of the
synovial fluid phantom produced.
H. A.
Fluid concentration
(mg/ml)
Healthy aSF 3.5
aSF3 3.0
aSF2 2.0
OA aSF 1.3
aSF1 1.0
RA aSF 0.84
Table 7. Artificial Synovial Fluids (aSF) compositions.
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6.2. Synovial fluid samples
The Hospital Universitario La Paz (Madrid, Spain) provided synovial fluid
samples. These were the remnants of samples sent to the Emergency Laboratory
(in EDTA and lithium heparin containers). Thirty-three samples from different
patients were provided in EDTA containers, of which 28 were additionally
submitted in lithium heparin containers. The SFs were classified into two main
groups: inflammatory pathology (rheumatoid arthritis, gout, psoriatic arthritis,
etc.) and infectious pathology (septic arthritis and prosthetic infections).
In addition, a sample of healthy synovial fluid was obtained from Hospital
Clínico San Carlos (Madrid, Spain). It is essential to mention that the healthy SF
sample was delivered in a container without additives. In this case, the pre-
treatment consisted of centrifuging the sample at 3500 rpm for 10 minutes.
Subsequently, the supernatant of the sample was used for measurement.
A few samples are observed in Figure 44.
Figure 44. Synovial fluid samples.
6.3. Biopolymer-based hydrogels samples
The monitoring of hydrogel gelation was carried out with covalently
crosslinked mucin hydrogels and physically crosslinked alginate hydrogels.
With this, the gelation kinetics of two hydrogel systems will be explored. The
first is composed of two components formed via covalent bonds, and the
second is formed by alginate hydrogels (crosslinked through divalent ions).
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For this type of experiment, it was necessary to use a smaller sample volume for
optimal detection and seal the QCR holder cell with parafilm paper to avoid
sample evaporation.
6.3.1. Covalently crosslinked mucin hydrogels
Muc-Tz and Muc-Nb were used at two concentrations: 15 and 25 mg/ml using
PBS. Then, a volume of 15 µl of each component was mixed (30 µl in total) and
added immediately to the crystal surface to start the measurement. The
duration of the experiment was 25 minutes to locate the frequency stability
zone (plateau-like zone).
6.3.2. Physically crosslinked alginate hydrogels
Alginate (60% G/M ratio) was stirred overnight at 15 mg/ml using saline
solution (154 mM NaCl). 𝐵𝑎𝐶𝑙2 and 𝐶𝑎𝐶𝑙2 were also diluted at 0.4 and 0.2 M
with NaCl. In the experiments, 20 µl of alginate was deposited on the crystal,
causing it to spread over the entire surface. Then, the resonance frequency of
the crystal was measured until it remained at a stable value. Once a stable
frequency was reached, 4 µl of 𝐵𝑎𝐶𝑙2 (or 𝐶𝑎𝐶𝑙2 ) was placed on the alginate to
start the gelation process. For this case, the measuring time was 15 minutes for
𝐵𝑎𝐶𝑙2 addition and 13 minutes for 𝐶𝑎𝐶𝑙2 addition.
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7. Results and Discussion
This chapter shows the results obtained from the experimental part of this
work. First, the results of the measurements with the CSF and SF phantoms are
detailed, then the analysis with real SF is shown. Finally, the results of the
experimentation with biopolymer-based hydrogels are illustrated. For all
graphs presented in this section, the shaded area denotes the standard
deviation as obtained from measurements of, at least, three independent
samples
7.1. Phantoms
7.1.1. aCSF
The aCSF analysis shows the sensor's response as the albumin concentration
increases, increasing fluid viscosity simulating CSF with a specific pathology, as
mentioned in previous sections. The results obtained are illustrated in Table 8.
The values of density and viscosity obtained with the rotational viscometer are
also shown.
Albumin concentration 0.0 0.5 1.0 2.0 3.0
(mg/ml) (aCSF) (VM aCSF) (BM aCSF)
Density (mg/ml) 1008 1010 1012 1013 1017
Viscosity (mPa·s) 1.01 1.02 1.03 1.05 1.06
∆𝒇 (Hz) -2957 -3112 -3215 -3474 -3587
±45 ±23 ±52 ±32 ±23
ΔΓ (Hz) 1466 1503 1548 1602 1688
±69 ±74 ±66 ±131 ±108
Viscosity measured 2.12 2.35 2.50 2.92 3.10
(mPa·s) ±0.06 ±0.03 ±0.08 ±0.05 ±0.04
Table 8. aCSF data and results obtained with the sensor.
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The first parameters to be observed are ∆𝑓 and ΔΓ (Figure 45). As expected, ∆𝑓
decreased as the albumin concentration increased; in total, the frequency
dropped from around 600 Hz taking into account aCSF healthy (no albumin) to
BM aCSF (higher albumin concentration); a stable dispersion of the data is also
observed as the albumin concentration increases. On the other hand, ΔΓ
increased around 200 Hz with a more significant data dispersion than ∆𝑓.
The ∆𝑓 response makes it possible to distinguish between the measured
samples by having a threshold of about 100 Hz, thus distinguishing between
healthy aCSF, VM aCSF, and BM aCSF cases.
Figure 45. ∆𝑓 (black line) and ΔΓ (red line) obtained for aCSF samples.
Using ∆𝑓, we obtain the viscosity values by equation (14). The graph of the
measured viscosity as the albumin concentration increases is shown in Figure
46.
Again we can see that as the albumin concentration increases, the viscosity
increases. In this case, the measured viscosities range goes approximately from
2 to 3 mPa·s. The distinction between the three samples of interest is possible,
especially with VM aCSF, the sample with the highest viscosity. The dispersion
in each sample spans little more than 0.1 mPa·s (in general), which benefits the
possible detection of each sample.
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Figure 46. Viscosity obtained with the sensor using aCSF.
The last part of the aCSF study evaluates the viscosity measured with the
device with the viscosity obtained with the rotational viscometer. This can be
seen in Figure 47. It is clear that the resulting curve is almost straight, which is
expected for a Newtonian fluid. This can be beneficial in case of a calibration of
the system for this specific fluid is required. The equation of the straight line
(shown in red in the graph) corresponds to:
𝜂𝑄𝐶𝑅 = (19.5 · 𝜂𝑟𝑜𝑡 ) − 17.57, (19)
Where 𝜂𝑄𝐶𝑅 is the viscosity obtained with the sensor and 𝜂𝑟𝑜𝑡 is the viscosity
obtained with the rotational viscometer. The fit of the measured points to the
line presents an RMSE = 0.02.
In the case of aCSF, the viscosity obtained from the ∆𝑓 value is efficient for
distinguishing between samples with different viscosities. ΔΓ shows an
increase, which is related to the rise in the sample's viscosity. However, it
presents a high dispersion in the measured values, which indicates that its
independent use is inadequate for differentiation between samples.
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Figure 47. Adjustment between the viscosity values measured with the sensor with the viscosity
of the rotational viscometer for aCSF samples.
7.1.2. aSF
The results obtained when measuring aSF, which has different physical
properties to aCSF as a non-Newtonian fluid, will now be presented. The
results obtained, together with the density and viscosity measured with the
rotational viscometer are presented below (Table 9).
Figure 48 shows the ∆𝑓 and ΔΓ values measured for each concentration of A.H.
It is clear that ∆𝑓 decrease and the ΔΓ increase as the A.H. increases; however,
they do not change linearly. This is due to the non-Newtonian fluid with
pseudoplastic behavior. The curves present a behavior that resembles an
exponential curve, with a sudden increase for low concentrations and a
progressive one at high concentrations.
The ∆𝑓 curve has a stable and low dispersion, while the ΔΓ curve has a
moderately higher and unsteady dispersion. For the frequency case, the
frequency decreased roughly 225 Hz from the sample with the lowest viscosity
(AR aSF) to the one with the highest viscosity (healthy aSF).
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H.A. 0.84 1.00 1.30 2.00 3.00 3.50
concentration (RA aSF) (OA aCF) (Healthy
(mg/ml) aSF)
Density 1007 1008 1009 1013 1022 1028
(mg/ml)
Viscosity 94 109 141 342 716 1133
(mPa·s)
∆𝒇 (Hz) -3004 -3074 -3138 -3166 -3201 -3228
±29 ±24 ±10 ±09 ±21 ±12
ΔΓ (Hz) 1722 1732 1776 1827 1825 1830
±22 ±15 ±28 ±07 ±15 ±34
Viscosity measured 2.19 2.29 2.39 2.42 2.46 2.48
(mPa·s) ±0.04 ±0.03 ±0.01 ±0.01 ±0.03 ±0.02
Table 9. aSF data and results obtained with the sensor.
When looking at the measurements of the fluids of interest, their differentiation
is remarkable, having a dispersion range of approximately 50 Hz for each
sample. On the other hand, ΔΓ increases by about 100 Hz, and dispersion of the
values compromises the differentiation between the samples of interest, having
values up to 70 Hz approximately.
The viscosity graph is shown in Figure 49. As the amount of A.H. increases, the
viscosity increases. As the frequency increases, the increase in viscosity starts
abruptly, and as the concentration increases, the increase in viscosity becomes
progressively almost linear. It is noticed that the viscosity increase starts at 2.2
mPa·s and ends at 2.5 mPa·s, i.e., about 0.3 mPa·s is the viscosity variation
measured with the sensor with these samples. Despite this reduced range, the
dispersion reaches 0.08 mPa·s, and the samples of interest can be identified.
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Figure 48. ∆f (black line) and ΔΓ (red line) obtained for aSF samples.
Comparing the viscosity measured with the sensor with the viscosity measured
with the viscometer gives the curve shown in Figure 50. In this particular case,
the sensor's viscosity values are lower than those measured with the rotational
viscometer. Since aSF samples are a non-Newtonian fluid with pseudoplastic
behavior, the viscosity will decrease at the higher shear rate. The rotational
viscometer measures at 0.3 rpm; thus, the viscosity will be higher since the fluid
is measured with a low share rate. On the other hand, the crystals vibrate at 10
MHz which can be considered a high shear rate; thus, the viscosity will be
minor. Comparison between low share rate viscosities with high share rate
viscosities is beyond the scope of this work; however, the differentiation
between healthy aSF, OA aSF, and RA aSF is possible using this method.
In case a device calibration is required and using the rotational viscometer as a
reference, the curve that best fits the data is of the exponential type (with two
terms) shown in red in Figure 50. The equation representing this curve is:
𝜂𝑄𝐶𝑅 = 2.4 · 𝑒 (2.88𝐸−5)·𝜂𝑟𝑜𝑡 − 14.1 · 𝑒 (−0.04)·𝜂𝑟𝑜𝑡 , (20)
The fit of the measured points to the line presents an RMSE = 0.006.
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Figure 49. Viscosity obtained with the sensor using aSF.
Figure 50. Adjustment between the viscosity values measured with the sensor with the viscosity of the
rotational viscometer for aSF samples.
Despite the non-Newtonian fluid condition, identifying healthy aSF, OA aSF,
and RA aSF samples is possible with the methodology followed. ∆𝑓 is shown to
be the main parameter to make this distinction. On the other hand, ΔΓ is not
observed as a fundamental parameter but a secondary one that complements
the viscosity information, remembering that its increase is related to the rise in
viscosity, which is observed in these results.
It is clear that the sensor can discriminate pathological phantoms from healthy
phantoms efficiently. This leads us to speculate that, with real fluids, the
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distinction between healthy and pathological fluids is uncomplicated; however,
the discrimination between pathologies could present difficulties. Differences in
glucose, hematocrit, cell, and protein levels in each sample obtained can be a
significant complication in differentiating between pathologies and even
between healthy and unhealthy fluid.
7.2. SF results
7.2.1. Inflammatory and infectious SF
When comparing the SF samples by classification (1 = inflammatory fluid, 2 =
infectious fluid, 3 = healthy fluid), differences can be seen between the first two
with type 3 (for both container tubes). However, in the absence of sufficient
healthy fluid samples, we cannot consider these results to be statistically
significant. Regardless, as a preliminary measure, it is optimistic about
observing what is expected in terms of a healthy SF showing a higher viscosity
than a pathological one. This will have to be worked on in the future.
Figures 51 - 53 show the boxplot and distribution of the measurements of each
parameter (∆𝑓, ΔΓ, and viscosity, respectively) between each type of
classification for the both types of tubes. Figures 54 - 59 show the distribution of
the measurements for each sample for each parameter in both type of tubes. In
all figures, it can be seen that the box on the far right constitutes the healthy SF
sample. Table 10 shows the average values of each measured parameter for
each classification and tube, together with their standard deviation.
The subsequent analysis will be carried out without considering the healthy SF
sample due to the scarcity of samples.
From the graphs, it can be seen that, for samples stored in lithium heparin gel
tubes, all three parameters measured had higher values than those held in
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EDTA tubes. For example, viscosity had a mean value of 3.46 mPa·s for the
EDTA tube samples, and for the samples contained in lithium heparin tubes,
the mean value was 3.74 mPa·s.
Figure 51. ∆𝑓 boxplots. Left: EDTA tube; right: Lithium heparin tube
Figure 52. ΔΓ boxplots. Left: EDTA tube; right: Lithium heparin tube
Figure 53. Viscosity boxplots. Left: EDTA tube; right: Lithium heparin tube
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Figure 54. Boxplots per sample (EDTA tube) for ∆𝑓 measurements.
Figure 55. Boxplots per sample (EDTA tube) for ΔΓ measurements.
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Figure 56. Boxplots per sample (EDTA tube) for viscosity values.
Figure 57. Boxplots per sample (Lithium heparin tube) for ∆f measurements.
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Figure 58. Boxplots per sample (Lithium heparin tube) for ΔΓ measurements.
Figure 59. Boxplots per sample (Lithium heparin tube) for viscosity values.
Disregarding the healthy SF sample, a study was conducted using the Mann-
Whitney-U statistical test to calculate the p-value. SPSS software was employed
for this purpose. The results are shown in Table 10. Other values such as WBC,
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neutrophil percentage, protein, and glucose are also detailed in the table. The
hospital provided these data.
There are significant differences (p < 0.05) in ∆𝑓 and ΔΓ between inflammatory
SF and infectious SF for the lithium heparin tubes. However, no statistically
significant differences were observed in the measurements of ∆𝑓 in the EDTA
tubes, and viscosity for both containers. On the other hand, the WBC value
shows significant differences in distinguishing the type of SF in both tubes.
Inflammatory Infectious p-value
Age (yr) 55.52 ± 27.53 72.75 ± 15.27 0.08
WBC (/𝑚𝑚3 ) 9060 ± 12526 52575.62 ± 75126.19 0.02
Neutrophils (%) 57.28 ± 36.39 85.50 ± 12.43 0.02
Glucose (mg/dl) 99.23 ± 32.11 64.37 ± 35.97 0.05

EDTA
Proteins (g/dl) 3.87 ± 0.82 4.15 ± 0.49 0.23
∆𝑓 (Hz) -3665.36 ± 135.34 -3675.87 ± 104.57 0.25
ΔΓ (Hz) 1787.47 ± 66.97 1810.47 ± 53.34 0.04
𝜂 (mPa·s) 3.46 ± 0.21 3.43 ± 0.30 0.11
Age (yr) 64.66 ± 18.96 71.85 ± 16.27 0.29
WBC (/𝑚𝑚3 ) 9032.76 ± 13478.73 57789.28 ± 79560.83 0.03

Lithium heparin gel
Neutrophils (%) 63.11 ± 36.80 84.00 ± 12.62 0.16
Glucose (mg/dl) 99.23 ± 32.11 59.57 ± 35.98 0.01
Proteins (g/dl) 3.87 ± 0.82 4.11 ± 0.52 0.29
∆𝑓 (Hz) -3775.40 ± 106.55 -3812.91 ± 109.05 0.03
ΔΓ (Hz) 1861.21 ± 95.89 1908.10 ± 72.09 < 0.01
𝜂 (mPa·s) 3.76 ± 0.31 3.67 ± 0.18 0.13
Table 10. Mean value and p-value per parameter for each classification and container type
The predictive abilities of each parameter are shown in the Receiver Operating
Characteristic (ROC curve) in Figure 60 and Table 11, which illustrate the area's
value under the ROC curve (AUC), Confidence Interval (CI), and Standard
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Error (SE). Also shown for reference are the WBC, serum procalcitonin (PCT),
and SF PCT parameters obtained in a different study [102].
In Figure 60 we can see that the viscosity calculation obtained does not
discriminate well the infectious SF. On the other hand, ∆𝑓 and ΔΓ had a better
result in the samples contained in lithium heparin, although they do not
become a test that stands out.
AUC CI 95% SE
Talebi-Taher
WBC (/𝑚𝑚3 ) 1.00 1.00 – 1.00 0.00
[102]
PCT serum 0.82 0.71 – 0.92 0.05
PCT SF 0.65 0.51 – 0.78 0.06
WBC (/𝑚𝑚3 ) 0.78 0.60 – 0.97 0.09
Neutrophils (%) 0.76 0.58 – 0.94 0.09
Glucose (mg/dl) 0.26 0.03 – 0.49 0.12

EDTA
Proteins (g/dl) 0.64 0.44 -0.85 0.10
∆𝑓 (Hz) 0.55 0.46 – 0.65 0.04
ΔΓ (Hz) 0.60 0.51 – 0.69 0.04
𝜂 (mPa·s) 0.42 0.31 – 0.52 0.05
WBC (/𝑚𝑚3 ) 0.80 0.61 – 0.99 0.09
Neutrophils (%) 0.68 0.46 – 0.91 0.11

Lithium heparin gel
Glucose (mg/dl) 0.20 0.00 – 0.43 0.11
Proteins (g/dl) 0.62 0.39 – 0.85 0.11
∆𝑓 (Hz) 0.61 0.51 – 0.72 0.05
ΔΓ (Hz) 0.65 0.55 – 0.74 0.04
𝜂 (mPa·s) 0.42 0.33 – 0.50 0.04
Table 11. Area under the ROC curve of the parameters as predictors for infectious fluid for both
tubes.
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Figure 60. ROC curve for parameters measured with the sensor for: SF in EDTA tubes (left)
and SF in lithium heparin tubes (right).
The obtained parameters showed promise in samples stored in tubes with
lithium heparin. However, for SF held in EDTA, the data were not helpful for
discrimination between infectious and inflammatory SF. This may be due to the
change generated by the type of tube in the physical properties of the sample,
with samples in EDTA being less viscous. In [102], the study shows that
procalcitonin (PCT) is used as a marker to discriminate infectious SF. The study
shows that the WBC value is the most accurate when distinguishing infectious
SF (AUC = 1). When evaluating the value of PCT in serum and PCT in SF, they
show that PCT in serum was better (AUC = 0.82) than PCT in SF (AUC = 0.65).
This last value is comparable with the ∆𝑓 (AUC = 0.61) and ΔΓ (AUC = 0.65)
obtained in this work (lithium heparin tubes). Neutrophils, glucose, and
proteins showed no significant relevance in distinguishing between the two
fluids.
7.2.2. Comparison between SF pathologies
On the other hand, samples with similar pathologies were compared to observe
cases where significant differences were obtained and, with this, to discriminate
between diseases. The selected conditions were: psoriatic arthritis, rheumatoid
arthritis, infectious SF, and monoarthritis, as these were samples from two
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different patients. The results are shown below. Figures 61 - 63 show the points
obtained for ∆𝑓, ΔΓ, and viscosity, respectively. The results for samples
contained in EDTA tubes are on the left side, and on the right side are the
results for samples collected in lithium heparin gel tubes. Table 12 includes the
p-value obtained between each sample.
Figure 61. Comparison between obtained ∆𝑓 for specific SF conditions.
Figure 62. Comparison between obtained ΔΓ for specific SF conditions.
Figure 63. Comparison between obtained viscosity for specific SF conditions.
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There are some cases where there are significant differences (p < 0.05) between
the selected pathologies. There are few coincidences of significant differences
for the same cases in both SF containers, highlighting that, among the instances
of RA-infection and RA-monoarthritis, ∆𝑓 and ΔΓ had a p-value < 0.05. In
addition, ∆𝑓 obtained significant differences when evaluating samples with RA
in all cases and both tubes. This indicates that, for the RA case, differentiation
between SF samples might be possible measuring ∆𝑓; however, the number of
samples evaluated is reduced to draw early conclusions.
EDTA Lithium heparin
∆𝒇 ΔΓ Viscosity ∆𝒇 ΔΓ Viscosity
P. arthritis-RA < 0.001 0.104 < 0.001 < 0.001 0.031 0.910
P. arthritis-infection 0.036 0.307 0.279 0.946 0.114 0.114
P. arthritis-monoarthritis 0.865 0.023 0.162 < 0.001 0.004 < 0.001
RA-infection 0.002 0.002 0.036 < 0.001 0.002 0.744
RA-monoarthritis < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.825
Infection- monoarthritis 0.002 0.487 0.018 0.003 0.055 < 0.001
Table 12. p-value calculation employing Tukey HSD for specific diseases.
7.2.3. Artificial Neural Networks for the classification of inflammatory
and infectious SF
As an alternative to increasing the sensor's capabilities, we have tested the use
of Artificial Neural Networks (ANN) to train them with the data obtained and
achieve the classification between inflammatory and infectious SF samples. For
this purpose, we generated the following algorithm (Figure 64), whose variable
parameters were the number of epochs (100, 200, 300) and the hidden layers (1
and 2). Parameters such as the optimizer, activation function, biases, etc., were
left constant since it is beyond the scope of this work to go into this topic in
more detail.
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Figure 64. ANN algorithm for SF classification.
The ∆𝑓, ΔΓ, and viscosity values are used as inputs to the network, and the two
SF conditions provided by the hospital (inflammatory and infectious) are used
as outputs. To be used optimally, they are normalized to locate the data in a
range of values between 0 and 1. The data are then randomly selected for the
training, validation, and testing phases, with the training phase requiring the
most significant number of data. Later, when calculating the accuracy, the
"testing data" isolated at this stage will be used exclusively for testing the
algorithm.
When defining the neural network, we set the following:
• Three input neurons (one for each input parameter; Relu activation
function),
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• Two output neurons (classification; Softmax activation function),
• One or two hidden layers (each with 50 outputs; Relu activation
function),
• Number of training epochs and,
• Adam-type optimizer.
After training, the algorithm tests the generated classification using the data set
aside for this purpose. In this way, it calculates the accuracy of the algorithm.
Finally, the confusion matrix is obtained, where the performance of the
classification is observed.
The accuracy values obtained for each ANN configuration are shown in Table
13.
More specific details of the ANN and the loss and accuracy curves for each
training can be found in the appendix of this work.
EDTA Lithium
heparin
Hidden Layers: 1 0.774 0.921
Epochs: 100
Epochs: 200
Epochs: 300
Epochs: 100
Epochs: 200
Epochs: 300
Table 13. Accuracy obtained for each test.
The results obtained with both tubes were compared, and it was observed that
using the samples stored in lithium heparin gave a higher accuracy in the
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classification algorithm. The highest accuracy (accuracy = 98.3%) was obtained
using two hidden layers and 300 training epochs and the samples contained in
lithium heparin tubes. The lowest accuracy (accuracy = 77.4%) occurred when
using samples stored in EDTA tubes, 1 hidden layer, and 100 training epochs.
The confusion matrices are shown in Figures 65 and 66, the first showing the
matrices for the case of SF from EDTA tubes, and the second for SF samples
from lithium heparin tubes. For the best case, it is observed that it correctly
classified 774 samples and incorrectly 13. For the worst case, it correctly
classified 577 samples and poorly 168.
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Figure 65. Confusion matrices obtained by ANN for SF samples contained in EDTA tubes.
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Figure 66. Confusion matrices obtained by ANN for SF samples contained in lithium heparin
tubes.
7.3. Biopolymer-based hydrogels results
7.3.1. Covalently crosslinked hydrogels
In testing the sensor's capabilities in measuring the gelling process of a
hydrogel system composed of two components formed via covalent bonds, we
obtained the following results.
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The measurements show a change in resonance frequency of the sensor
approximately 5 minutes after mixing the two components (Figures 67 and 68),
irrespective of the concentrations. The sensor's resonance frequency continued
to drop until reaching a plateau-like state in approximately 25 minutes for
higher concentration. After 25 minutes of gelation, the frequency drop was
clearly high for the more concentrated Muc-gels system.
Speaking of ΔΓ, this parameter did not have a significant change for the lower
concentration Muc-gel. The values remained close to zero during the gelation
process and grew around 100 Hz. On the other hand, growth was observed
after 2.5 minutes for the highest concentration, stabilizing at around 600 Hz. It
can be said that both ∆𝑓 and ΔΓ underwent a minimum change for the Muc-gel
concentrated at 15 mg/ml and, for the Muc-gel at 25 mg/ml, both parameters
experienced a more significant increase. Such changes in both parameters could
be correlated with the physical properties of each material.
Figure 67. Time-dependent evaluation of ∆f (black line) and ΔΓ (red line) for 15 mg/ml Muc-
gels.
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Figure 68. Time-dependent evaluation of ∆f (black line) and ΔΓ (red line) for 25 mg/ml Muc-
gels.
These results clearly demonstrate the potential to use QCR measurement to
validate gelation events in covalently crosslinked hydrogel systems easily and
follow the gelling kinetics and assess differences in the mechanical properties of
the gelled system due to polymer concentration.
7.3.2. Physically crosslinked alginate hydrogels
We then tested whether QCR could also measure the gelling kinetics of alginate
hydrogels, crosslinked through divalent ions.
First, Figure 69 shows the measurement of 20 µl of alginate solution (1.5%,
wt/v) loaded on the QCR before exposure to calcium or barium and its gelation.
The curve shows the gelation process until the frequency stabilizes after
approximately 10 minutes.
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Figure 69. ∆f (black line) and ΔΓ (red line) change after placing an alginate solution (15
mg/ml) on the sensor.
The addition of a similar volume of NaCl solution (0.4 M) to alginate caused a
frequency increase (Figure 70), indicating that the samples were diluted by the
addition of the solution and formed a less viscous fluid. Indeed sodium, which
is used as a counterion to alginate, does not induce its gelation. We can also
observe that, when adding sodium to the alginate, the frequency takes
approximately 2 minutes to reach equilibrium, so we consider this time a
stabilization period caused by factors such as the damping produced when
changing the physical properties of the sample.
Figure 70. ∆f (black line) and ΔΓ (red line) change likely caused by a dilution effect following
exposure of the alginate to a solution of NaCl (0.4 M).
Once the plateau with alginate was reached, the gelation started by adding a
small volume (4 µl) of 𝐵𝑎𝐶𝑙2 or 𝐶𝑎𝐶𝑙2 solutions. When adding the divalent ions,
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the frequency of the QCR dropped, reflecting the increases in the viscous
response of the material. The frequency shift values shown in Figure 71 were
calculated from average frequency values from 12 minutes from adding barium
ions and from 10 and 6 minutes after adding calcium at 0.4 M and 0.2 M,
respectively. The frequency drop was sensitive to two parameters known to
impact alginate gelation: the type of ion used and the molar ratio of divalent
ions to alginate.
Figure 71. Comparison of Δf plateau-like stage for the four cases.
In the following graphics, the kinetics of each gelation are shown. Barium ions
led to a higher frequency drop than calcium at both concentrations (Figures 72 -
75). Decreasing the concentration of each ion by half also reduced the frequency
drop. The concentration of ionic crosslinker affects how many of such
crosslinking points are occupied on the alginate, which would directly affect the
resulting gelling kinetics and mechanical properties. These results indicate that
the QCR can indeed sense the ionic crosslinking of alginate into a hydrogel.
Barium ions (0.195 nm) have been reported to crosslink alginate into more
stable gels than calcium, owing to its larger size than calcium ions (0.097 nm).
Barium ions are a tighter fit within the alginate egg-box configuration that it
adopts around such divalent ions [103]. Thus, stiffer gels can be obtained by
crosslinking alginate solutions with barium ions compared to calcium ions at
the same concentration.
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Figure 72. ∆f (black line) and ΔΓ (red line) response for alginate gel formation using 𝐵𝑎𝐶𝑙2 (0.4
M).
Figure 73. ∆f (black line) and ΔΓ (red line) response for alginate gel formation using 𝐵𝑎𝐶𝑙2 (0.2
M).
Figure 74. ∆f (black line) and ΔΓ (red line) response for alginate gel formation using 𝐶𝑎𝐶𝑙2 (0.4
M).
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Figure 75. ∆f (black line) and ΔΓ (red line) response for alginate gel formation using 𝐶𝑎𝐶𝑙2 (0.2
M).
There are also clear differences in the gelation kinetics of the gels. A sharp
initial decrease in frequency immediately after exposure to the divalent ions for
𝐵𝑎𝐶𝑙2 and 𝐶𝑎𝐶𝑙2 at the highest concentrations could result from mechanical
strain onto the crystal due to the sudden gelation of the alginate solution
(Figures 72, 74). This is also observed for 𝐵𝑎𝐶𝑙2 at the lowest concentration
(Figure 73), indicating that Barium generates a strong and impulsive
crosslinking with both concentrations. The lower concentration of 𝐶𝑎𝐶𝑙2 did not
induce this initial drop (Figure 75), perhaps due to the lower crosslinking extent
and strength compared to the other conditions. To assess whether the initial
sharp decrease in frequency could be due to the development of the ionic
crosslinking within the gel, we estimated the diffusion times of both ions. For
𝐶𝑎𝐶𝑙2, considering a diffusion constant D = 0.79 [104] and a thickness = 0.502
mm, the diffusion time was 2.7 min. For 𝐵𝑎𝐶𝑙2 , maintaining the thickness and D
= 0.84 [104], the diffusion time results in 2.5 min. These diffusion times are
within the timescale of these sharp frequency increases that took about one
minute to develop.
Following the initial drop in frequency, a progressive evolution than
stabilisation was seen, which could be due to further diffusion of the ions into
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the gel. It should also be considered that QCRs measure in the region closest to
the crystal surface. Recalling, the penetration depth is 178 nm for a QCR with 𝑓0
= 10 MHz in water. It is, therefore, possible to consider a slower stabilisation
time in this region.
Figure 76 shows the time response of the complete experiment, from alginate
alone to gelation with the different ions.
Figure 76. Kinetics of the alginate gelation process with all the cases evaluated.
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8. Conclusions and Future Work
8.1. Conclusions
A method is proposed to characterize fluid viscosity (mainly from a biological
source) using a small sample volume (tens of microliters). Phantoms of SF and
CSF were developed and measured, and the results show that distinction
between fluids with different viscosity could be made. The ViSQCT sensor, at
least when being applied to phantoms fluids, can distinguish between healthy
aSF and pathological aSF. Furthermore, the sensor distinguished between the
two pathological phantoms of each fluid type. As aSF is a non-Newtonian fluid,
the viscosity results obtained with the sensor had a minor increase than those
obtained with the reference viscometer. The results obtained with aCSF show
the possibility of detecting the small viscosity changes between the healthy
aCSF, VM aCSF, and BM aCSF. Calibration curves were suggested for each
phantom to adjust the sensor data with the rotational viscometer information.
Interesting results were obtained when comparing the SF samples with specific
diseases and the healthy fluid sample, as differentiation is possible, at least
from the healthy SF and SF cases with monoarthritis and RA. However,
statistically significant results are not available due to the lack of considerable
samples. On the other hand, when comparing the two classifications of SF
(inflammatory and infectious), it isn't easy to distinguish between them when
looking at their distribution. Nevertheless, it was shown that there are
significant differences in viscosity measured in samples stored in lithium
heparin tubes.
As the distinction between classifications by direct comparison of the measured
parameters was not clear, the use of ANN was proposed, which showed that,
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with the help of this technology, it is possible to achieve an efficiency of 98.3%
when classifying between inflammatory and infectious SF.
The sensor demonstrates that can be used to follow the gelling process and
compare the resulting mechanical properties of hydrogels. Using two hydrogel
systems with covalent and physical ionic crosslinking, we demonstrate the
versatility of the approach.
The kinetic of the frequency drop, and the frequency drop at equilibrium, can
be used to compare hydrogel systems and indicate their final mechanical
properties. Such a sensor could be used to assess (quickly) whether crosslinking
has occurred and compare between strategies immediately. Overall this work
provides the proof of concept for this class of sensors to be used as hydrogel
formation monitors.
Finally, it is proven that developing a low-cost, portable sensor is possible by
using QCRs. The results obtained with the device (and its associated
methodology) show its great functionality in biomedical applications, both in
the study of human fluids and the characterization of materials such as
hydrogels. Nevertheless, the device deserves further development to become an
instrument that can assist physicians in analysis (diagnosis inclusive) and a
complete laboratory tool for monitoring mechanical properties in hydrogel
formation.
8.2. Future work
The following developments are proposed for future work:
- The prototype can be reduced in size by including the signal generation and
analog-digital communication modules on the board. Doing without batteries
would help to achieve this goal. Including wireless communication between the
computer and the sensor would improve the sensor's capabilities by allowing it
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to be placed in spaces such as a work hood or incubator for biological
experimentation.
-Improve the system by adding the possibility of dynamic measurements using
a microfluidic system, which should include a temperature sensor, which will
expand the opportunities for experimentation.
-Explore experimentation by the biofunctionalization of the QCR. As with the
previous point, this will open the door to new projects.
-Increase the number of real samples measured to have, on the one hand, a
larger number of data to analyze, and on the other hand, to generate a more
extensive database to help ANN training.
-Getting real samples of CSF or pleural effusions will open new lines of
experimentation that could extend the sensor's functionality.
-It is necessary to carry out a quantitative comparison between the proposed
method and a rheology measurement for its validation in hydrogel
characterization.
-Improve methodology when measuring hydrogels. The high viscosity of the
measured samples makes it difficult to place the precise sample volume by
pipetting. Using better instrumentation to control the volume deposited on the
glass will improve experimentation with these materials.
-Further study of the relationship between measurements obtained with
rheological measurements.
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Annex
9. Annex
This section shows in more detail the main characteristics of the ANN network
(Table 14). Subsequently (Figures 77 - 100), each trained configuration's
accuracy and loss curves are presented.
Parameter Features
Input Layer Neurons: 3
Activation function: Relu
Hidden Layers 1 or 2 hidden layers
Neurons: 50
Activation function: Relu
Output Layer Neurons: 2
Activation function: Softmax
Training Epochs 100, 200, 300
Optimizer Type Adam
Table 14. Features of the ANN.
Figure 77.Variation of accuracy throughout training with one layer and 100 epochs using SF
stored in tubes with EDTA.
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Figure 78. Variation of losses throughout training with one layer and 100 epochs using SF
Figure 79. Variation of accuracy throughout training with one layer and 200 epochs using SF
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Figure 80.Variation of losses throughout training with one layer and 200 epochs using SF
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Figure 83. Variation of accuracy throughout training with two layers and 100 epochs using SF
Figure 84. Variation of losses throughout training with two layers and 100 epochs using SF
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stored in tubes with lithium heparin.
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Annex
105
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Annex
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UNIVERSIDAD POLITÉCNICA DE MADRID

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS EN

STUDY OF NEW APPLICATIONS OF THE QUARTZ

MSc. Andrés Miranda Martínez

ESCUELA TÉCNICA SUPERIOR DE INGENIEROS EN

UNIVERSIDAD POLITÉCNICA DE MADRID

STUDY OF NEW APPLICATIONS OF THE QUARTZ

Andrés Miranda Martínez

José Javier Serrano Olmedo

AUTHOR: Andrés Miranda Martínez

Tribunal nombrado por el Magfco. y Excmo. Sr. Rector de la Universidad

En el Centro de Tecnología Biomédica de la Universidad Politécnica de Madrid.

UNIVERSIDAD POLITÉCNICA DE MADRID

STUDY OF NEW APPLICATIONS OF THE QUARTZ

Andrés Miranda Martínez

José Javier Serrano Olmedo

mi compañera de vida y mi mejor amiga, quien me motiva y me apoya en los

Agradezco a mi familia, son quienes siempre me apoyaron en mis objetivos y

me alentaron en todo momento. Agradezco a mis padres, quienes me formaron

mamá por su ejemplo y apoyo, mi constancia y responsabilidad se los debo a

ella. Gracias a mi papá por sus enseñanzas y optimismo, de él he aprendido a

disfrutar cada momento de mi vida. Gracias a mi hermana Edith por ser mi

confidente y compañera de travesuras desde pequeños. Siempre estaremos el

uno para el otro.

Agradezco a Antonio, a Flor de María y a Toño, quienes me recibieron con los

brazos abiertos desde el primer día y me han apoyado siempre que lo he

Gracias a todo el grupo del Laboratorio de Bioinstrumentación y Nanomedicina

(LBN), en especial a Michael, mi vecino de escritorio, y con quién compartí la

experiencia del doctorado desde el comienzo y quien fue parte de mi familia en

en la UPM y por su amistad.

Agradezco a Thomas Crouzier y al grupo de Biopolymers for life de KTH (Real

Instituto de Tecnología, Suecia). Mi recibimiento y estancia fueron

LBN, amigo y director de mi trabajo de fin de máster y de mi tesis doctoral,

quien me ha enseñado y orientado día a día en este mundo de la ingeniería

biomédica y en el mundo de la investigación a lo largo de estos 5 años.

Muchas gracias a los profesores de la UNAM José María Matías, Salvador

Landeros, Víctor García y Jesús Manuel Dorador, por su ejemplo, orientación y

apoyo que me proporcionaron en México para continuar con mi preparación

le han dedicado a este trabajo.

Finalmente, gracias a mis grandes amistades en México, siempre los llevo

many possible applications in biomedicine and bioengineering.

by exciting the crystal through a frequency sweep (close to the fundamental

resonance frequency of the crystal). The resonance frequency at the maximum

conductance point is its primary reference when evaluating the samples

deposited on its surface.

development of the sensor as a valuable tool for diagnosis or classification

between different conditions of the same fluid is sought. An example of this is

pathologies such as rheumatoid arthritis or osteoarthritis. Therefore, measuring

sample. Otherwise, a larger sample volume would be required, or large and

expensive devices would be used for the same purpose.

A second approach is to use the device as a tool to characterize the formation of

biopolymer-based hydrogels. Such characterization is carried out using

expensive and large equipment. As an alternative, a low-cost, portable and

straightforward system is proposed to assist in this task. Knowledge of the

mechanical properties of these materials is essential, and the design of the

a determining factor in cell differentiation. If a hydrogel is sought for

phantoms (with contrasting viscoelastic properties), and differences between

healthy and pathological fluids were observed employing the measured

parameters. Subsequently, measurements were made with synovial fluid

provided by the Hospital Universitario La Paz in Madrid. The samples were

previously diagnosed and classified into two groups: inflammatory pathologies

when applying artificial neural network algorithms, the accuracy increased.

Finally, experiments were performed with hydrogels to monitor the formation