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  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 .
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 . 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 . 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 .
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: 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.
The research works mentioned were in collaboration with Dr. Tong Chen.
For Further Reading
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.
Katia 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 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 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 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