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Non-invasive microwave spectroscopy-based biosensor for single cell analysis: toward early diagnostic and personalized medicine

By Katia Grenier, David Dubuc, Mary Poupot and Jean-Jacques Fournié

Traditional analyzing techniques of cells are mainly based on optical detection systems(principally microscopy and flow cytometry). However, such equipments are often costly, and are invasive for the investigated cells due to the implementation of labeling techniques. The development of new biological analysis instruments, is therefore very challenging and of high interest, especially for the early diagnostic and personalized treatments of various diseases. To address this issue, we have developed a RF biosensor able to perform the microwave spectroscopy of a single living cell in its culture medium.

Traditional analyzing techniques of cells are mainly based on optical detection systems. Microscopy and flow cytometry constitute the main important ones due to their high efficiency and specificity. However, such equipments are often costly, and may be considered as invasive for the investigated cells. They essentially involve labeling techniques, through various stains and fluorochromes, which interact on surface or inside the cells. These labels may therefore induce modifications of the cells and lead to variations of the mechanism to detect. Erroneous or unwanted observation may consequently be obtained. The development of new biological analysis instruments at the cellular and molecular levels, which would be either non-invasive or without any induced perturbation, is therefore very challenging and would present high interests for various applications, especially early diagnostic and personalized treatments of various diseases.

Among the developed miniature bio-sensing methods, impedancemetry constitutes a promising candidate, as the technique is label-free, non-destructive, and rapid, without any required contact. It is based on the detection of size and dielectric properties. Until recently, cellular impedancemetry has focused on frequencies up to the MHz frequency range, which corresponds to the β dispersion of the dielectric parameters of the living matter [1] and refers to the phenomenon of cell membrane polarization. It enables to reach information in terms of viability, size and morphology of the cells. The technique is however limited in discrimination, when the cells exhibit similar size and morphology. Labels are then required as in optical detection methods [2].

To extend the analyzing potentialities of the technique, there is a strong interest to increase the used frequency range to the GHz regime. At these frequencies, the interaction of the electromagnetic waves with the biological matter is enhanced due to the penetration of the waves inside the cells. It is also associated to the γ dispersion due to the polarization of dipoles [1]. The most well known one is notably the molecule of water, the major constituent of the living matter. Further discrimination and observation of biological mechanisms may therefore be expected with microwave spectroscopy, to significantly contribute to fundamental researches and biomedical investigations such as early disease diagnostics and treatments evaluation on patient cells.

The development of miniature microwave biosensors for cellular and molecular investigations is very recent and involves consequently defining and demonstrating the potentialities of the technique. Toward the possible early diagnostic of diseases and personalized medicine, several demonstrations require to be done, such as the ability to detect the living cells in their biological medium with sufficient sensitivity and selectivity notably. This has been performed with cells suspensions exhibiting large concentrations in the order of several millions of cells per milliliter, and down to 20 cells in a microwave biosensor [3]. A mandatory step consists also in isolating a single living cell and being able to detect and characterize it non-invasively, preferably in its traditional liquid medium [4].

Figure 1

Figure 1: (a) Schematic of the single cell based microwave biosensor. (b) Photograph of a living RL lymphoma cell trapped in the sensor and surrounded by its culture medium to maintain the cell viability during experiments.

Its feasibility is illustrated here. The schematic of the microwave biosensor, which has been developed for this purpose, is shown in Fig. 1, with a photograph of the sensor with a trapped cell. It is composed of a coplanar transmission waveguide with a capacitive gap located at the center of the sensor. Two tapers placed apart decrease the conductor size to an appropriate dimension compare to the cell. It permits to focalize the electromagnetic fields into the cell area. In this study, human B lymphoma cells of RL type, which constitute a well-known cell model for blood cancer investigations, are used. Their diameter is comprised between 10 and 15 µm. A capacitive gap of 15 µm in width is consequently chosen. Perpendicularly to the coplanar line is placed a microfluidic channel on top, which integrates a mechanical trap located above the capacitive gap. The trap permits to well localize the cell to analyze in the sensing area, whereas the channel enables to keep during experiments the living cell in its traditional liquid environment, with all its required nutrients including salts, ions and proteins. Such a medium traditionally screens the signal to be detected at lower frequencies. However, the use of microwaves prevents this drawback and therefore does not impose to modify the liquid culture medium of the cells.

The developed sensor takes therefore all the benefits provided by the microtechnologies through a miniature RF detection circuit and its combination with a microfluidic channel, which integrates with a cell trap. The principle of trapping is quite simple. A cell suspension is incorporated into the microfluidic channel. As soon as a cell follows the streamline located in front of the trap, the cell may be blocked. All other cells are then deviated due to the fluid flow.

The single cell microwave based sensor has been evaluated on a frequency range starting from 40 MHz to 40 GHz. Several living cells have been individually tested and measurement results are presented in Fig. 2 for three different single RL lymphoma cells. To facilitate the distinction of the cell’s contribution in its medium environment, is evaluated the capacitive contrast between the cell contribution and the sensor only loaded with the medium. The three measured capacitive contrasts present a very similar spectrum. The dispersion between the curves can be attributed to the intrinsic heterogeneity of the living and also to the position of the cell in its proliferation cycle. The maximum capacitive contrast is obtained around 5 GHz with a value close to 0.5-0.6 fF, which is much larger than the estimated measurement resolution of 0.01 fF.

Figure 2

Figure 2: Capacitive contrasts of human RL lymphoma cells measured individually in their traditional culture medium (RPMI with 10% of Foetal Calf Serum) from 40 MHz to 40 GHz

In summary, is now demonstrated the possible and non-invasive measurement of a single living cell in its culture medium through microwave spectroscopy. To achieve such a spectroscopy, a RF biosensor has been specifically defined, realized and successfully evaluated with living B lymphoma cells. Their capacitive contrast compare to the host liquid medium is in the order of hundreds of attofarads, far from the measurement resolution. It allows envisaging further non-invasive biological processes studies at the single cell level, such as testing treatments efficiency and kinetics on living patient cells for instance.

Acknowledgments
The research works mentioned were in collaboration with Dr. Tong Chen.

For Further Reading

1. C. Polk and E. Postow, Handbook of Biological Effects of Electromagnetic Fields. CRC Press, 1996, ch. 1 by K.R. Foster and H.P. Schwan.

2. T. Sun, H. Morgan, “Single-cell microfluidic impedance cytometry: a review,” Microfluid. Nanofluid., vol. 8, pp. 423-443, 2010.

3. T. Chen, D. Dubuc, M. Poupot, J-J. Fournié, K. Grenier, “Accurate nanoliter liquid characterization up to 40 GHz for biomedical applications: toward non-invasive living cells monitoring”, IEEE T-MTT, Vol. 60, Issue 12, Part 2, Dec. 2012, pp. 4171-4177.

4. T. Chen, D. Dubuc, M. Poupot, J-J. Fournié, K. Grenier, “Microwave biosensor dedicated to the dielectric spectroscopy of a single alive biological cell in its culture medium”, IEEE International Microwave Symposium, Seattle, USA, June 2013.


Contributors

Katia GrenierKatia Grenier (S’99 -M’03) received her Ph.D. degree in electrical engineering from the University of Toulouse, France. She is now with the LAAS-CNRS lab, in France, where her research interests are focused on the development of fluidic-based microsystems for biological and medical applications as well as for reconfigurable wireless. Katia 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. Read more

 

David DubucDavid Dubuc (S’99, M’03) received the Ph.D. degree in electrical engineering from the University of Toulouse, Toulouse, France. He is an Associate Professor with the University of Toulouse, and a Researcher with the Laboratory of Analysis and Architecture of System part of National Scientific Research Center (LAAS-CNRS), Toulouse, France. His research interests include the development of microwave circuits integrated due to microtechnologies and their application to wireless telecommunication and biology. Read More

 

Mary PoupotMary Poupot received her PhD degree in Biochemistry from the University of Paul Sabatier, Toulouse, France. She is a Researcher at the Cancer Research Center of Toulouse. Her research interests are based on the impact of the tumor microenvironment on the survey of cancer cell in particular in hematopoietic diseases. Read More

 

Jean-Jacques FourniéJean-Jacques Fournié received his Ph.D. degree in microbial biochemistry from the University of Toulouse, Toulouse, France. He is currently heading the Cancer Research Center of Toulouse (CRCT), France. His fields of scientific expertise are biochemistry, pharmacology, immunology and cancer. The aim of his team is based on the immune-targeting of hematopoietic diseases. Read More

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July 2013 Contributors

J. C. ChiaoJ.-C. Chiao is Greene endowed professor and Garrett endowed professor of Electrical Engineering at University of Texas - Arlington; and an Adjunct Associate Professor in the Internal Medicine Department at UT-Southwestern, Medical Center. Read more

Mohammad-Reza TofighiMohammad-Reza Tofighi is an associate professor of electrical engineering with the Capital College, Pennsylvania State University, Middletown, PA. He conducts research on wireless implants, biomedical antennas, medical applications of microwave radiometry, and complex permittivity measurement of tissues using time and frequency domain methods. Read more

Bill SaltzsteinBill Saltzstein is the President of connectBlue Inc., a leading provider of industrial and medical wireless solutions. Bill is also the Medical Business Development Director and joined connectBlue in February of 2011 with a vision to extend medical care out of the traditional environments through the use of wireless technologies. Read more

Katia GrenierKatia Grenier (S'99 -M'03) received her Ph.D. degree in electrical engineering from the University of Toulouse, France. She is now with the LAAS-CNRS lab, in France, where her research interests are focused on the development of fluidic-based microsystems for biological and medical applications as well as for reconfigurable wireless. Read more

David DubucDavid Dubuc (S'99, M'03) received the Ph.D. degree in electrical engineering from the University of Toulouse, Toulouse, France. He is an Associate Professor with the University of Toulouse, and a Researcher with the Laboratory of Analysis and Architecture of System part of National Scientific Research Center (LAAS-CNRS), Toulouse, France. His research interests include the development of microwave circuits integrated due to microtechnologies and their application to wireless telecommunication and biology. Read More

Mary PoupotMary Poupot received her PhD degree in Biochemistry from the University of Paul Sabatier, Toulouse, France. She is a Researcher at the Cancer Research Center of Toulouse. Her research interests are based on the impact of the tumor microenvironment on the survey of cancer cell in particular in hematopoietic diseases. Read More

Jean-Jacques FourniéJean-Jacques Fournié received his Ph.D. degree in microbial biochemistry from the University of Toulouse, Toulouse, France. He is currently heading the Cancer Research Center of Toulouse (CRCT), France. His fields of scientific expertise are biochemistry, pharmacology, immunology and cancer. Read More

Gustavo GuarinGustavo Guarin is currently pursuing his PhD in Electric Engineering at the University of Erlangen-Nuremberg. He works at the Institute for Electronic Engineering with Prof. Dr.-Ing. Dr.-Ing. habil. Robert Weigel and with Prof. Dr.-Ing. Georg Fischer. The focus of his research is on Ultra-Wideband Microwave Sensors for spectroscopy applications in biomedicine. Read More

Maximilian HofmannMaximilian Hofmann received the Dipl.-Ing. degree in electrical, electronic and communications engineering from the University of Erlangen-Nuremberg, Nuremberg, Erlangen, Germany. He joined the Institute for Electronics Engineering, Erlangen, Germany, as a Research Assistant. Read More

Dietmar KissingerDietmar Kissinger received the Dr.-Ing. degree in electrical engineering from the University of Erlangen-Nuremberg, Erlangen, Germany. He holds a position as Lecturer and Head of the RF Integrated Sensors Group at the Institute for Electronics Engineering. His research interests include silicon-based microwave and millimeter-wave integrated circuits as well as wireless sensors and communication systems for ultra-low power, automotive, industrial, security, and medical applications. Read More

Dr. Cristiano PalegoDr. Cristiano Palego is a senior lecturer in Smart Sensors and Instrumentation at Bangor University, UK and a visiting research scientist at Lehigh University, USA. He received the Ph.D. degree in Microwave Engineering and Optoelectronics from the University of Limoges, France. He is currently a research scientist at Lehigh. His interests include electromagnetic theory, micro/nanotechnology and biomedical research as well as RF-MEMS for reconfigurable frontends, antenna arrays, and high-power applications. Read More

Dr. Caterina MerlaDr. Caterina Merla received the Ph.D. degrees in electronic engineering from the University of Rome "La Sapienza," Italy. She is currently with the Italian National Agency for New Technologies, Energy and Sustainable Economic Development (ENEA), Research Centre in Rome and a Visiting Research Scientist at Lehigh University, Bethlehem, PA. Her research interests are mainly focused on the microdosimetric evaluation of the electromagnetic (EM) field at single cell level, biological sample dielectric measurements, and design and dosimetry of exposure systems oriented to EM protection studies and medical applications. Read More

Yaqing NingYaqing Ning is currently working toward her Ph.D. degree at Lehigh University. She is involved in research on RF MEMS capacitive switches and phase shifters, as well as on biomedical devices for detection purposes. She specializes in 3D electromagnetic simulation using finite element methods. Read More

Caroline MultariCaroline Multari is currently working towards her Ph.D. at Lehigh University in Materials Science and Engineering, focusing on research in point-of-care microfluidic biosensors and developing polymeric biomaterials for bioparticle capture. Read More

Dr. Xuanhong ChengDr. Xuanhong Cheng is the Rossin Assistant professor of Bioengineering and Materials Science and Engineering at Lehigh University. She received her Ph.D. in bioengineering at University of Washington. She joined Lehigh University, where her current research interest is focused on developing new nanomaterials and microfluidic platforms to analyze intact, live bioparticles, such as cells and pathogens at the point of need. Read More

Prof. James C. M. HwangProf. James C. M. Hwang received the Ph.D. degree in materials science from Cornell University, Ithaca, NY. He joined Lehigh University, Bethlehem, PA as a Professor of electrical engineering and Director of the Compound Semiconductor Technology Laboratory. His current research interests include MEMS, microwave and photonic devices and integrated circuits, and bio-electromagnetics. Read More

Marco MercuriMarco Mercuri is currently working towards the Ph.D degree within the TELEMIC research group of the Department of Electrical Engineering (ESAT), KU Leuven, Belgium. His research interests include biomedical applications of microwave/RF, wireless sensors, and microwave/millimeter-wave measurements. Read More

Ping Jack SohPing Jack Soh is currently working towards his Ph.D in the ESAT-TELEMIC Research Division, Katholieke Universiteit Leuven, Belgium. His research interests include planar antennas, flexible/textile antennas, on-body communication, metamaterials, passive microwave components and microwave measurements. Read More

Dominique SchreursDominique Schreurs received the Ph.D. degree in electronic engineering from University of Leuven (KU Leuven), Belgium. She is a Full Professor at KU Leuven. Her main research interests concern the (non)linear characterization and modelling of microwave devices and circuits, as well as (non)linear hybrid and integrated circuit design for telecommunications and biomedical applications. Read More