IEEE TRANSACTIONS ON MICROWAVE THEORY AND TECHNIQUES, VOL. 60, NO.

12, DECEMBER 2012

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Accurate Nanoliter Liquid Characterization Up to 40 GHz for Biomedical Applications: Toward Noninvasive Living Cells Monitoring
Tong Chen, David Dubuc, Member, IEEE, Mary Poupot, Jean-Jacques Fourni, and Katia Grenier, Member, IEEE
AbstractThis paper demonstrates an accurate liquid sensing technique, from 40 MHz to 40 GHz, which is suitable for the detection and quantication of very small contents of molecules, proteins, and for the noninvasive and contactless microwave investigation of living cells in their culture medium. The sensor is based on an interdigitated capacitor (IDC) with a microuidic channel to conne the nanoliter-range liquid and is integrated with microtechnologies to be fully compatible with a massive parallelization at low cost. Both alcohol and biological aqueous solutions are precisely characterized, identied, and quantied in terms of capacitance and conductances contrasts with respect to pure de-ionized water. Mixtures from 20% down to 1% of ethanol in water exhibit large capacitances values of 110 and 7 fF at 11 GHz, respectively. Based on the high accuracy of such characterizations, the detection of very small traces of ethanol (down to 100 ppm) can be envisioned. As far as biomedical applications are targeted, we also demonstrate the potential of fetal bovine serum detection in aqueous solution down to 5% v/v. Finally, the sensor is evaluated with living B lymphoma cells suspension in their traditional biological medium. The in-liquid microwave measurement of less than 20 living cells is successfully performed and corresponds to a capacitance contrast of 5 fF at 3 GHz relative to the reference bio-medium. For low cells concentration, the sensor response is proportional to the number of cells on the IDC, which permits to envision cells quantication and proliferation monitoring with this microwave sensing technique. Index TermsBiology, biomedical transducers, human cells, interdigitated capacitor (IDC), microwave spectroscopy.

I. INTRODUCTION

HE integration of analytic sensing techniques of liquids is of prime importance for applications in chemistry, biochemistry, biology and medicine [1]. During the past years, intensive research are indeed on going to miniaturize biosensors as well as to reduce assays time without compromising selectivity and sensitivity to molecules or cells under analysis [2]. Among all the miniaturizable physical transductions methods (optical, mechanical, electrochemical), the electrical one and

Manuscript received July 10, 2012; revised September 21, 2012; accepted September 25, 2012. Date of publication November 15, 2012; date of current version December 13, 2012. This work was supported by under a French RITC Foundation Grant. This paper is an expanded paper from the IEEE MTT-S International Microwave Symposium, Montreal, QC, Canada, June 1722, 2012. T. Chen, D. Dubuc, and K. Genier are with the National Scientic Research Center, Laboratory of Analysis and Architecture of Systems (LAAS), 31400 Toulouse, France (e-mail: grenier@laas.fr). M. Poupot and J.-J. Fourni are with the Cancer Research Center of Toulouse, Toulouse, 31000 France (e-mail: poupot@inserm.fr). Color versions of one or more of the gures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identier 10.1109/TMTT.2012.2222660

more specically the dielectric spectroscopy is one of the most promising techniques, since it does not require any bio-functionalization or labeling protocols. This ability avoids any targetlabel coupling reactions or main assay alteration, as well as it assures inexpensive and time-efcient operation [3]. Moreover, dielectric spectroscopy analysis techniques operating in the gigahertz frequencies reveal a rich set of bio-information [4] based on the molecular content of the medium under investigation: dielectric polarization and relaxation phenomenon and interactions between molecules notably. In the case of living cells analysis, the operation in the microwave range translates into a full electromagnetic (EM) waves penetration inside the intracellular content [5], which makes the probing able to discriminate physiological state of cell (living/ dead, normal/tumorous, ). This paper presents a biosensor resulting from the combination of both the microwave-based liquid sensing technique [5][7] with microuidics capabilities [8] and able to detect and quantify very small contents of molecules, proteins, and living cells in their culture medium. The device operates in the nanoliter range, which assures very low consumed volume of liquids required for high throughput analysis. The developed technique provides an accurate identication and quantication of very low content of molecules (down to 100 ppm in the case of ethanol) in aqueous solution. Moreover, a population of only 20 living cells has been accurately characterized in their liquid microenvironment. In addition to the label-free ability of the technique, its capability to operate with biological medium (i.e., featuring a high ionic content) opens new and innovative way for noninvasive cell analysis and real-time monitoring. This paper is organized as follows. Section II is dedicated to sensor description and simulations. Section III deals with the experimental validation of the approach, whereas Section IV discusses the experimental results obtained with references binary liquid mixtures based on aqueous solutions, which feature various solutes concentrations. Finally, Section V deals with the evaluation of the sensor in term of cells concentration with living human lymphoma cells in suspension. II. SENSOR DESCRIPTION AND SIMULATIONS The considered nanoliter liquid sensor is based on an interdigitated capacitor (IDC) embedded into a coplanar waveguide, as presented in Fig. 1. The dielectric sensing area is, in this design, a square of 150 150 m and can be easily down-scaled. The liquid connement is assured with a microuidic channel composed of polydimethylsiloxane (PDMS), which features a height

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Fig. 1. Schematic of the IDC with a narrowed microuidic channel placed on top.

Fig. 3. Calculated distribution of the electric-eld magnitude of the IDC loaded with liquid for two congurations. (a) Top view. (b) Cross section.

Fig. 2. Photograph of the fabricated microuidic IDC with a PDMS channel.

of 40 m. This elastomer has the main advantages of being biocompatible, cheap, and simply molded. Its transparency allows an easy observation of the biological material placed in the channel, and notably living cells. However, its high losses in the microwave and millimeter-wave ranges impose to minimize the sidewalls width of the channel, which are located in the direct vicinity of the EM elds. The walls of the uidic channel are thus limited to a width of 200 m on top of the coplanar device. The main liquid passage is 980- m wide to facilitate the injection of the uid, whereas the narrow one in the sensing area is about 150 m, as presented in Fig. 2. The volume of the analyzed liquid is consequently about 0.9 nL. The IDC presents strips and slots of 10- m width. The microwave electrodes are made of 300-nm-thick titanium and gold layers on top of a quartz wafer to minimize additional dielectric losses due to the substrate. The fabrication of the biosensor is based on standard microtechnological processes. It may be divided in two main parts, which are: 1) the elaboration of the IDC on a quartz wafer and 2) the polymer microuidic channel elaboration and assembling. All the corresponding details may be found in [9]. To evaluate the electric-eld distribution in the sensing area of the IDC, a 3-D nite-element method (3-D FEM) analysis is performed using Ansofts High Frequency Structure Simulator (HFSS). Fig. 3(a) and (b) presents a top view and a cross section of the calculated eld, respectively.

As expected for such a component, the electric eld is strongly concentrated in the IDC region and in the vicinity of the coplanar slots. The PDMS channel is consequently limited to the centered area of the IDC to enhance the interaction between the EM wave and the liquid sample in this preferred area. As far as the cross section is concerned, the eld is more intense the rst 15 m above the capacitor [represented in red (in online version) in Fig. 3(b)]. The eld, however, extends to the top of the microchannel (in green in online version), which means that the entire volume of liquid is fully analyzed with the IDC device in the sensing area. Based on these preliminary design considerations, a protocol to extract the complex admittance parameters of the IDC is dened. III. EXTRACTION OF THE COMPLEX ADMITTANCE PARAMETERS AND VALIDATION WITH DE-IONIZED WATER (DI WATER) The electrical characterization of the IDC is performed on wafer from 40 MHz to 40 GHz. The liquid injection inside the microuidic channel is precisely controlled with a syringe pump from Harvard Apparatus, Holliston, MA. Measurements are then treated to extract the complex admittance parameters of the IDC loaded with different types of liquids. The protocol of extraction is based on the IDCs electrical model, which is given in Fig. 4. It includes two access transmission lines (TLs) at the input and output, two capacitors with one related to the

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IV. EVALUATION OF THE SENSOR WITH REFERENCED BINARY LIQUID MIXTURES The nanoliter liquid IDC is evaluated to accurately characterize referenced binary liquid mixtures. Two types of solutions are investigated. The rst one is related to alcohol-based aqueous solutions, whereas the second one corresponds to fetal bovine serum (FBS) in water in order to assess biological liquids in agreement with a realistic environment of cells in suspension. To better dene the sensitivity of the sensing technique for aqueous-based solutions, we extract the IDC complex admittance values contrasts of the mixtures relative to pured DI water, which is the main constituent of the solutions. They are dened with (3) and (4), respectively, as follows: (3) (4) These contrasts of capacitance and conductance better refer to a variation of a chemical proportion in the referenced DI-water liquid. A. With Alcohol-Based Aqueous Solutions Four concentrations of ethanol in DI water are measured: 20%, 10%, 5%, and 1%. The corresponding capacitive and conductive contrasts versus frequency are presented in Fig. 6(a) and (b), respectively. All concentrations lead to signicant contrasts on both - and -parameters, even for the lowest tested concentration of 1% of ethanol and with nanoliter volumes of liquids. These results are explained by the strong value difference that exists between the respective dielectric constants of ethanol and DI water. Moreover, the use of contrasts spectra also permits to establish the most appropriate frequency range for which the sensitivity for such solvent-based binary mixtures is maximal on both and readouts. In the case of ethanol in DI water, the range related to the largest capacitance contrast is located from 10 to 13 GHz for the investigated concentrations. A maximal capacitance contrast of 112 fF is obtained at 11 GHz for a 20% concentration of solvent in water. Fig. 7 indicates the obtained capacitive contrast versus the composition of the mixture. In the considered concentration range, the capacitance contrast is proportional to the ethanol concentration in water. Due to the large capacitance variation of 34 fF at 11 GHz (for 5% of ethanol volume content) and by considering the sensitivity level close to 0.1 fF, the presented IDC structure lets one envision distinguishing very small solute contents in aqueous solution, as low as a few 100 ppm. Such a rationale may also be performed with the second accessible readout of the microwave sensing technique, the conductance. As shown in Fig. 6(b), the maximal contrast of this parameter is reached at 40 GHz, the highest measured frequency of this study, for all investigated ethanol concentrations. A 16-mS conductance contrast is obtained for the 20% concentration of ethanol. To briey conclude this section, the microwave sensing technique permits to analyze nanoliter volumes of liquids and gives access through two readouts ( and ) to the accurate composition of binary solvent-based mixtures in aqueous solution.

Fig. 4. Electrical model of the IDC.

Fig. 5. Capacitance and conductance of the uidic part of the IDC.

quartz substrate and the other one associated to the uid, and a conductance related to the losses induced by the uid. First, a de-embedding procedure is applied to subtract the effect of the coplanar access lines. Both capacitance and conductance related to the uid are then calculated. Fig. 5 presents the corresponding extracted IDC values versus frequency (solid line), when the structure is loaded with DI water. In order to validate the extraction procedure, an EM simulation of the structure is also performed. The relative complex permittivity of water is taken into account with the Debye relaxation model, which is given by (1) and (2) as follows:

(1)

(2)

, , and correspond to the real parts of the where relative permittivity at very low and very high frequencies and to the Debye relaxation frequency, respectively. These parameters are optimized with the technique previously developed [10]. The predicted capacitor, extracted from the simulation, is presented in Fig. 5 via diamond marks. A good agreement between measurements and simulations is reached, which validates the correct operation of the developed nanoliter liquid sensor.

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Fig. 8. Contrast spectra of the: (a) capacitance and (b) conductance of biological-based aqueous solutions. Fig. 6. Contrast spectra of the: (a) capacitance and (b) conductance of alcoholbased aqueous solutions.

This measurement method is also applied to biological-based aqueous solutions. The corresponding results are presented in Section IV-B. B. With Biological-Based Aqueous Solutions The chosen investigated biochemical corresponds to FBS, which is an important constituent of culture cell media. It provides important required nutrients to living cells for their survival and proliferation. Concentrations traditionally used by the biologists are in the range of 5%10% in water. The extraction procedure of the complex admittance parameters, which was previously presented in Section IV-A, is applied and the results are presented in Fig. 8(a) and (b) for both capacitance and conductance contrasts, respectively. Similar to the solvent-based aqueous solutions, the contrasts on both and for the two concentrations of FBS are noticeable. The maximal constrasts for and are also obtained in the lower frequency part of the spectrum or in the highest part correspondingly. Their value ranges are, however, smaller than for ethanol. Whereas 10% of concentration of ethanol in water induces a maximal of 70 fF, the equivalent one for FBS (also for a concentration of 10% in water) is close to 9 fF. This

Fig. 7. Impact of the ethanol concentration in aqueous solution on the capacitance contrast, at 11 GHz.

Compared to planar TL-based sensors [10][15], our proposed technique efciently gives access to liquid discrimination with a high level of accuracy, while being highly down-scalable to the nanoliter range and even further.

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Fig. 10. Cells distributions on the IDC for low, medium, and high concentrations of cells.

Fig. 9. Biological protocol to assure living cells incorporation in the IDC.

remark is also valid for the conductance contrast at 40 GHz. FBS composition is indeed strongly constituted of water with numerous ions, proteins, molecules, and other bio-constituents. The respective dielectric constant of DI water and FBS are consequently much closer than for ethanol. This result is all the more remarkable taking the ionic content into account. It reinforces the interest of using microwave sensors for biological analysis instead of lower frequency-based sensors, which are screened with conductive solutions. Based on a similar sensitivity approach as for ethanol, the detection of 0.1% in volume of FBS in an aqueous solution may be expected. As previously demonstrated for ethanol-based aqueous solutions, both and contrasts are proportional to the solutes concentration. Moreover, compared to the ethanol-based mixtures, the maximal contrasts on the capacitance are obtained at 3 GHz instead of 11 GHz (for ethanol-water mixtures). This is due to the different intrinsic relaxation frequency of each liquid. The spectra constitute consequently specic signatures of the analyzed liquids, which allow their identication. This part consequently demonstrates the ability with an IDC structure to probe biological material in liquid, in the nanoliter range with a sufcient accuracy. It opens the door to cells investigation in a conned microenvironment with appropriate conditions for cells living and even proliferation. With this purpose, we have investigated the impact of concentration of living cells in suspension in the IDC device. V. EVALUATION OF THE SENSOR IN TERM OF CELL CONCENTRATION WITH LIVING LYMPHOMA CELLS IN SUSPENSION Living B lymphoma cells suspensions are used in this study. They constitute a reference model for blood cancer investigations. To ensure the correct living pathological state of the cells during the experiments and reproducible investigations, we use the following biological protocol, which is described in Fig. 9. After the cells culture in a traditional incubator, the cells are collected and placed into an Eppendorf tube. They are then centrifuged to allow the elimination of the supernatant (consumed biological medium), which is replaced by a new and well-controlled culture medium. This one is composed of

Fig. 11. Contrast spectra of the: (a) capacitance and (b) conductance for different concentration of living RL lymphoma cells in their biological culture medium.

Roswell Park Memorial Institute (RPMI) medium with 10% of FBS. The quantity of added medium permits to control the concentration of cells. The living cells are then injected into the device for immediate measurements. Measurements are performed for three cell concentrations. The cells distributions in the sensing area of the IDC are given in Fig. 10. An optical observation permits to evaluate the number of cells for each condition. Consequently, values of about 20, 70, and 150 cells are obtained for low, medium, and high concentrations, respectively. Both capacitance and conductance contrasts are presented in Fig. 11. The reference liquid used for the calculation corre-

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REFERENCES
[1] R. Baw and J. Finkelsetein, Lab on chip, Nature, vol. 442, no. 7101, pp. 367418, Jul. 2006. [2] R. Bashir, BioMEMS: State-of-the-art in detection, opportunities and prospects, Adv. Drug Del. Rev., vol. 56, pp. 15651586, 2004. [3] J. S. Daniels and N. Pourmand, Label-free impedance biosensors: Opportunities and challenges, Electroanalysis, vol. 19, no. 12, pp. 12391257, 2007. [4] S. Gabriel, R. W. Lau, and C. Gabriel, The dielectric properties of biological tissues, Phys. Med. Biol., vol. 41, no. 11, pp. 22312293, 1996. [5] H. P. Schwan, Electrical properties of blood and its constituents: Alternating current spectroscopy, Blut, vol. 46, pp. 185197, 1983. [6] F. Kremer, Dielectric spectroscopyYesterday, today and tomorrow, J. Non-Cryst. Solids, vol. 305, no. 13, pp. 19, Jul. 2002. [7] F. Duhamel, I. Huynen, and A. Vander Vorst, Measurement of complex permittivity of biological and organic liquids up to 110 GHz, in IEEE MTT-S Int. Microw. Symp. Dig., 1997, vol. 1, pp. 107110. [8] G. M. Whitesides, The origins and the future of microuidics, Nature, vol. 442, pp. 368373, Jul. 2006. [9] T. Chen, D. Dubuc, and K. Grenier, Accurate nanoliter liquid complex admittance characterization up to 40 GHz for biomedical applications, in IEEE MTT-S Int. Microw. Symp. Dig., Montral, QC, Canada, Jun. 2012, pp. 13. [10] K. Grenier and D. Dubuc et al., Integrated broadband microwave and microuidic sensor dedicated to bioengineering, IEEE Trans. Microw. Theory Tech., vol. 57, no. 12, pp. 32463253, Dec. 2009. [11] G. R. Facer et al., Dielectric spectroscopy for bio-analysis: From 40 Hz to 26,5 GHz in microfabricated waveguide, Appl. Phys. Lett., vol. 78, no. 7, pp. 996998, Feb. 2001. [12] J. C. Booth et al., Quantitative permittivity measurements of nanoliter liquid in microuidic channels to 40 GHz, IEEE Trans. Instrum. Meas., vol. 59, no. 12, pp. 32793288, Dec. 2010. [13] S. S. Stuchly and C. E. Bassey, Microwave coplanar sensors for dielectric measurements, Meas. Sci. Technol., no. 9, pp. 13241329, 1998. [14] A. Raj, W. S. Holmes, and S. R. Judah, Wide bandwidth measurement of complex permittivity of liquids using coplanar lines, IEEE Trans. Instrum. Meas., vol. 50, no. 4, pp. 905909, Apr. 2001. [15] S. Seo, T. Stintzing, I. Block, D. Pavlidis, M. Rieke, and P. G. Layer, High frequency wideband permittivity measurements of biological substances using coplanar waveguides and application to cell suspensions, in IEEE MTT-S Int. Microw. Symp. Dig., 2008, pp. 915918.

Fig. 12. Impact of the number of cells on the capacitance contrast at 3 GHz.

sponds this time to the biological medium, which is the host liquid of the cells. Similar to the previous study on binary mixtures, the cells concentrations in their biological medium can be discriminated with the IDC through the two readouts: the capacitance and conductance contrasts. The results are signicant and well above the estimated sensitivity level of the sensor, even for the lowest concentration of cells. The frequency, which corresponds to the maximal contrast on the capacitance, is close to the one obtained with the FBS-based aqueous solution. Finally, the capacitance contrast is proportional to the number of cells in the sensing area. This is illustrated in Fig. 12, which presents the number of analyzed cells versus the extracted capacitance contrast at 3 GHz. The linear regression (dashed line in Fig. 12) of experimental data corresponds to a capacitive contrast per cell of 0.2 fF. Considering a sensitivity level of 0.1 fF, the detection of a single cell with the microwave sensing technique can be envisioned. These results suggest a possible cell counting and then cells proliferation monitoring from one single to hundreds of living cells. VI. CONCLUSION This paper has presented a miniature biosensor based on the interaction of a nanoliter-range aqueous solution with the EM waves at gigahertz frequencies. Besides the accurate detection of traces of alcohol (ethanol) and biological-based FBS in aqueous solutions, the detection and quantication of living lymphoma cells in their traditional culture medium are demonstrated. One of the major ability of such a technique resides in the in-culture medium analysis of cells, which assures a noninvasive operation on living cells. Combined with the markerless characteristic, this permits to analyze long-term bio-reactions without cells alteration by the sensor. It also allows reusing the cells after analysis for further investigations. The other major envisioned capability of the developed biosensor corresponds to the single cell detection and identication. This challenge combined with the already mentioned characteristics of the technique makes the proposed sensor highly attractive in regard to other competitors for laboratory-on-a-chip applications.

Tong Chen received the M.S. degree in electrical engineering from the University of Toulouse, Toulouse, France, in 2009, and is currently working toward the Ph.D. degree at the University of Toulouse. He is currently with the Laboratory of Analysis and Architecture of System [part of the National Scientic Research Center (LAAS-CNRS)], Toulouse, France. His research interests involve the development of miniature microwave-based biosensors for dielectric spectroscopy.

David Dubuc (S99M03) received the Agregation degree from the Ecole Normale Suprieure de Cachan, Paris, France, in 1996, and the M.S. and Ph.D. degrees in electrical engineering from the University of Toulouse, Toulouse, France, in 1997 and 2001, respectively. Since 2002, he has been an Associate Professor with the University of Toulouse, and a Researcher with the Laboratory of Analysis and Architecture of System, National Scientic Research Center (LAAS-CNRS), Toulouse, France. From 2007 to 2009, he was a Visiting Senior Researcher with the Laboratory of Integrated Micromechatronic Systems (LIMMS-CNRS)/Institute of Industrial Science (IIS), University of Tokyo, Tokyo, Japan. His research interests include the development of microwave circuits integrated due to microtechnologies and their application to wireless telecommunication and biology.

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Mary Poupot received the M.R. and Ph.D. degrees in biochemistry from the University of Paul Sabatier, Toulouse, France, in 1993 and 1997, respectively. From 2001 to 2007, she was a Postdoctoral Fellow with the Center of Physiopathology of Toulouse Purpan, Toulouse, France. She was interested in the activation of human lymphoid cells to eradicate cancer cells. Since 2007, she has been a Researcher with the Cancer Research Center of Toulouse, Toulouse, France. Her current research interests are based on the impact of the tumor microenvironment on the survey of cancer cell, particularly in hematopoietic diseases.

Jean-Jacques Fourni received the M.S. and Ph.D. degrees in microbial biochemistry from the University of Toulouse, Toulouse, France, in 1983 and 1986 respectively. From 1988 to 1989, he was a Postdoctoral Fellow with the CIRC (currently the Garvan Institute), Sydney University Australia. In 1986, he joined the National Scientic Research Center (CNRS), Toulouse, France, to study biologically active natural substances with a focus on those activating human cell immunity. In 1990, he settled a cell immunology group focused on nonpeptide antigens, and in 1993, he discovered phosphoT cells, and moved this group to the National antigens stimulating human Institute of Health and Medical Research (INSERM) for pharmacological development of phosphoantigens with therapeutic applications for cancer. In 1999, he cofounded Innate Pharma, which produces new anticancer treatments. Since 2011, he has been heading the Cancer Research Center of Toulouse (CRCT), Toulouse, France. His elds of scientic expertise are biochemistry, pharmacology, immunology, and cancer. The aim of his team is based on the immune argeting of hematopoietic diseases.

Katia Grenier (S99M03) received the M.S. and Ph.D. degrees in electrical engineering from the University of Toulouse, Toulouse, France, in 1997 and 2000, respectively. She was engaged in microelectromechanical systems (MEMS) circuits on silicon. She was a Postdoctoral Fellow with Agere Systems (Bell Laboratories). In 2001, she joined the Laboratory of Analysis and Architecture of System, National Scientic Research Center (LAAS-CNRS), Toulouse, France. From 2007 to 2009, she was with the Laboratory for Integrated Micromechatronic Systems CNRS (LIMMS-CNRS)/Institute of Industrial Science (IIS), Universtity of Tokyo, Tokyo, Japan, where she was engaged in launching research activities on microwave-based biosensors. Her research interests with LAAS-CNRS are currently focused on the development of uidic-based microsystems/nanosystems, notably for biological and medical applications at the cellular and molecular levels. Dr. Grenier is a member of the IEEE MTT-10 Technical Committee on Biological Effect and Medical Applications of RF and Microwave of the IEEE Microwave Theory and Techniques Society (IEEE MTT-S).