2016 International Conference on Advanced Technologies for Communications (ATC) Biological Microparticles Detection based on Differential Capacitive Sensing and Dielectrophoresis Manipulation T T H Tran1, N V Nguyen2, N C Nguyen2, T T Bui2, T Chu Duc2 Post and Telecommunication Institute of Technology (PTIT), Hanoi, Vietnam Vietnam National University, Hanoi (VNU), Hanoi, Vietnam electrodes via relays The positive dielectrophoretic response of the HeLa cells is responsible for its transportation to the center of the experimenting microchamber The force applying on HeLa cells has the magnitude of roughly sevenfold that on red blood cells They are, consequently, able to relocate at the center electrode much sooner than normal blood cells Being left behind, red blood cells or other normal blood components are intentionally separated from HeLa cells To observe the experiments, the overall circuit system generating the stepping electric field of cellular concentration was constructed on a printed circuit board (PCB) and the micro-fabricated chip was mounted on the electric module Experimental results were recorded to demonstrate the feasibility of concentrating HeLa cells, when the peak-to-peak voltage from 10 V to 16 V and a frequency of MHz were applied Abstract— The manipulation and detection of living cells are essential requirements for many applications in biology, biotecnology and medicine In this study, we propose a microfluidic device, which facilitated of a differential capacitively coupled contactless conductively detection (DC4D) coplanar capacitor structure and a dielectrophoresis (DEP) concentrator based on circular electrodes for detecting of living cells With the proposed device, first, the target and non-target cells are guided toward the center of the working chamber due to the dielectrophoresis forces Then the target cells are captured by the electrode covered with aptamers, which possess a high affinity to the target cells After being flushed to wash away the non-target cells in the working chamber, the differential capacitance is read to identify the presence of target cells Numerical simulations of the DEP manipulation and DC4D were performed to proof the concept of the proposed design A prototype of the proposed device was fabricated using micromachining technology Manipulation and sensing performances of the device were investigated The evaluation results show that the capacitance output changes up to 3fF corresponding to the presence of about twenty-five cells captured at the electrode In our previous designs, the manipulation of microparticles was observed and recorded using an optical microscope A new approach is required for the fabricated microchip, which not only concentrates but also captures and detects the target cells in the device Among the different physical techniques for detection of objects in a microfluidic channel, capacitive sensing emerged as the best technique, thanks to the simple fabrication and measurement setup, as well as minimization capability [12-14] The differential capacitive sensor with three or four adjacent electrode structure has been developed [15-16] The sensor can be used for detection of strange particle, air bubble in microfluidic flow or cell in medical devices with a high sensitivity and robust operation In this study, a novel design is proposed that two symmetric electrodes are added next to the center electrode for sensing purposes The capacitance between two sensing electrodes increases when the cells are trapped in the top of capture electrode The trapped cells can be detected by monitoring this change This symmetric structure is helpful for constructing detection circuit by removing common noise and parasitic capacitors Keywords— Dielectrophoresis, capacitive sensor, DC4D, HeLa cell, cell manipulation I INTRODUCTION Many biomedical applications require to manipulate microparticles on chips, including cell concentration, separation, isolation and identification, patterning, trapping and positioning [1-2] In recent years, detecting cancer cells or circulating tumor cells (CTCs) is a key application in medical diagnostics and characterization During carcinogenesis, CTCs have been generated due to cancer cells detach from the primary tumor into the blood stream, that can serve as early predictors of the metastatic process [3] One of many different techniques regarding the manipulation of particles, dielectrophoresis (DEP) has been proven as a useful technique and extensively employed in the manipulation a wide range of different biological particles, such as cells, bacteria, viruses, protein and DNA [4-6] II In the recent studies, a handheld electric module has been developed by the authors, which provides stepping electric fields for dielectrophoresis (DEP) to selectively concentrate and isolate cervical carcinoma cells (HeLa) from a peripheral blood sample [7-11] The stepping electric field is created by shifting the applied current to an adjourning pair ofcircular 978-1-5090-2710-1/16/$31.00 ©2016 IEEE MATERIALS AND METHOD A DEP manipulation DEP is the movement of characterizing particles placed in a non-uniform electric field caused by polarization effects When the electric field is computed in the frequency domain, the dielectrophoretic force acting on a spherical particle of radius rp suspended in a liquid medium is given as: 297 2016 International Conference on Advanced Technologies for Communications (ATC) FDEP 2 rp3 m Real fCM Erms C f d , A, r (1) where, εr is the relative static permittivity (dielectric constant) of the material between the electrodes, A and d are the area of each electrode and the separation distance between the two electrodes, respectively where Erms is the root-mean-square of the external electric field, p and m are the represented characters of the particle and the medium, respectively fCM is called the Clausius-Mossotti factor, which relates to the effective polarizability of a particle It depends not only on the complex dielectric properties of the particle and the surrounding medium, but also on the frequency of the applied field (f) For a spherical particle, the ClausiusMossotti factor is given by: fCM *p m* * p 2 m* (5) Each change in one of the parameters can result in the change in capacitance The variation in capacitance can be measured to recognize the presence of certain particles C Method The operational concept of target cell manipulation and detection in a microchip is illustrated in Fig (2) with ε* is the complex permittivity of the material, which can be expressed as: * j (c) DEP turn off (a) Initial (3) Flow In which, ε is the permittivity, σ is the electrical conductivity, j is the imaginary unit and ω=2πf is the angular frequency of the applied AC electric field Therefore, the DEP force varies with the relative polarizabilities of a particle and medium solution, the particles' shape and size, and the frequency of the electric field DEP force is frequently classified into pDEP (positive DEP) and nDEP (negative DEP) pDEP occurs when the particle has a higher permittivity than the fluid, the particle is then attracted to the high field region Meanwhile, the lower permittivity of the particle compared to that of the fluid results in nDEP The particle is attracted to the low field region Signal Input + Signal Output - C Differential Amplifier The shell feature can be added to the dielectrophoretic force node to model the dielectrophoretic force on cells with thin membrane The complex permittivity of the shell can differ from the complex permittivity of the rest of the particle When computing the dielectrophoretic force, the complex permittivity of the particle is replaced by the equivalent complex relative permittivity of a homogeneous particle comprising both the membrane and the interior of the particle: Non-target cell Target cell Aptamer Fig Sketching of the microfluidic device for target cell manipulation and detection (a) Target and non-target cells are distributed randomly in the working chamber (b) Cells are manipulated by DEP to move to the center of the working chamber Target cells are captured by the aptamer biding on top of the designed electrode (c) Non-target cells are washed away, only target cells are remained due to their combination specificity with the aptamer (d) DC4D capacitance is identified to determine the presence of the target cells HeLa cells, which are concentrated onto the capture electrode from peripheral blood, are human cervical cancer cells and also target cells in this study They are widely in biology as a human cell line for experiments Being a cancerous cells, HeLa cells not experience replicative senescence, meaning that the cells can continuously replicate themselves indefinitely In fact, HeLa cells are the first line of human cells to survive indefinitely in vitro Moreover, HeLa cells also have many characteristics in common with normal cells, such as producing proteins, expressing and regulating genes, being vulnerable to infections Thus, HeLa cells make it possible for scientists to comprehensively investigate caner In a sucrose medium, both viable HeLa cells and Red blood cells (RBCs) suspended exhibit a positive dielectrophoretic effect Using Eq 2, the Clausius–Mossotti factor for the HeLa cells and RBCs determined are about 1.0 and 0.91 at a frequency of MHz, respectively [17] It is assumed that RBCs are spheres with a diameter of μm [18] For RBCs, the internal dielectric permittivity and conductivity are 57ε0 and 0.52 S/m, *p s* ro * r p 2 s* * * i eq s * * ro p s * * ri p 2 s (d) Capacitive sensing (b) DEP turn on (4) where r0 and ri are the outer and inner radii of the membrane, * respectively; s is the complex permittivity of the outer membrane B Capacitive sensing A typical capacitive sensor bases on a change of parameters in capacitor structure lead to change its capacitance during the sensing time In general, the capacitance value depends on the dielectric material, distance between the electrodes, and the area of each electrode The capacitance of a capacitor can be expressed in terms of its geometry and dielectric constant as: 298 2016 International Conference on Advanced Technologies for Communications (ATC) respectively [19], the membrane capacitance is 0.9 μF/cm2, and the shell parameters [20] For HeLa cells with a diameter of approximately 10 μm, the magnitude of the DEP force is higher about seven times than that on RBCs under a given electric field distribution in the sucrose solution Therefore, HeLa cells are able to separate from normal blood cells conduct simulations, some electric parameters of living cells are shown in TABLE II Gold electrodes are laid on the glass substrate and covered by a thin PDMS layer in order to avoid the contact between the fluidic and the electrodes The microchannel is filled up with the sucrose medium (εr = 78; σ = 1.76×10-3 S/m) RBC and HeLa cells are simulated objects An electric field is applied to each electrode pair to generate a high-electric-field region Then, the applied electric field is subsequently switched to the adjacent electrode pair, from the outermost electrodes to the central electrodes (from Relay to Relay 8) As a result an inward stepping electric field is generated [7-11] The movement of high-electric-field also results in the movement of cells having a positive dielectrophoretic response Initially, both the HeLa cells and RBCs are distributed randomly on the substrate Although both two kinds of cells have the tendency to relocate at the center, HeLa cells move more quickly to the central electrode than the RBCs As a results, the device has separated and extracted HeLa cells from a particular sample Aptamers were formerly attached to the substrate above the capture electrodes HeLa cells are trapped at the central electrodes The capacitive sensor is then employed to measure the difference in capacitance which has resulted from the presence of target cells The output obtained from the sensor can be used to determine or detect the appearance of the HeLa cells or other target cells III TABLE I Parameters Figure shows the schematic of the modeled geometry and design parameters are given in TABLE I The microdevice is designed with three main parts: the glass substrate, circular electrodes and the insulation layer As given radius, the volume of the microchamber is about 113 nL There are ten circular microelectrodes with the gap between them is 30 µm The electrodes generate eight DEP manipulation electrode pairs with the lollipop-shaped central electrode [9] Combining the center electrode with two symmetrically electrode pairs on either side creates a DC4D structure The DC4D design with electrodes to form pick-up capacitors and the reference capacitor, which can bring a high-sensitivity in detecting the appearance of target cells above the capture electrode Target cells Radius of the microchamber 600 µm Microchamber’s height 100 µm Electrode’s width 30 µm Electrode’s gap 30 µm Radius of circle central electrode 90 µm Capture electrode’s width 30 µm Properties RBC HeLa Inner conductivity (S/m) 0.52 0.84 Inner permittivity (ε0) 57 47.5 Inner diameter (μm) 10 Membrane conductivity (S/m) 10-6 2.5×10-7 Membrane permittivity (ε0) 4.44 Membrane thickness (nm) The Clausius-Mossotti factor IV 3.25×10 2.5×105 0.91 RESULTS AND DISCUSSIONS Either a high-strength electric field or a long time exposure time can result in rupture of the cell membrane and reduce the number of cells The field strength required for the mammalian cell lysis is on the order of magnitude of 106 V/m and in less than 33ms using a 1ms pulse length An AC voltage of 16V peak-to-peak at the frequency of MHz was conscientiously adopted to insure the survival of cells and generate sufficiently strong DEP force to manipulate cells Fig shows the square of the simulated electric fields with the inward stepping electric field It can be inferred from the results that the movement of the high-electric-field-gradient region is the same direction with the stepping electric field In addition, the pattern of the microelectrode affects the size of such region as well Particles, or cells, in particular, with a positive dielectrophoretic response, can be steered towards the center on each change in the electrode combination and thus, concentrated at the center of the micro-chamber A Micro chip A-A Glass substrate Unit The concentrations of cells (cells/mL) Manipulating/ Capture electrode with Sensing Electrodes aptamer trapping A Value TABLE II SIZE AND DIELECTRIC PROPERTIES OF RED BLOOD CELL (RBC) [18-20] AND CERVICAL CARCINOMA CELL (HELA) [9-10] DESIGN AND SIMULATION Manipulating Electrodes PARAMETERS OF MODELED GEOMETTRY [10] Insulation layer Fig Drawing of the designed microchip An electric field simulation around the electrodes of the microchip was performed using COMSOL Multiphysics To 299 2016 International Conference on Advanced Technologies for Communications (ATC) clustered at the center of the model with high density at the final stages of the simulation process proves the working principle of the proposed devices 0.2 0.4 0.6 0.8 1.0 1.2 1.4 2 E (V /m ) 1.8 x1012 1.6 Fig The square of the electric field (E2) on microelectrode pairs with inward stepping electric field in a numerical simulation The applied electric field is 16 V peak-to-peak at frequency MHz Fig Distribution of electric field intensity between the left sensing electrode and the center electrode when a single HeLa cell is located at the capture electrode (a) (b) (c) (d) (e) (f) (g) (h) (i) Fig The simulation results of the manipulation of HeLa cells from a blood sample using inward (16 V peak-to-peak; MHz) stepping electric fields The concentration ratio of HeLa cells to RBCs was 1/13 Fig Differential capacitance output versus particle’s numbers The y-axis, xaxis are the differential capacitance and the number of particles, respectively The numerical simulation results the manipulation of HeLa cells from the blood sample are shown in Fig The concentrations of HeLa cells and RBCs introduced into the microchamber are 2.5 × 105 cells/mL and 3.25 × 106 cells/mL (the ratio of HeLa cells to RBCs was 1/13), respectively Originally, the HeLa cells (target cells) and other blood cells (non-target cells) are randomly distributed on the substrate On alternately applying electric field to pairs of electrodes, both cells are attracted by DEP force to move to the highelectric-field-gradient region However, it is noteworthy that due to Hela cells’ intrinsic properties, the magnitude of such force on HeLa cells is approximately seven times greater than that on other cells under an identical electric field distribution The target cells (HeLa) have a much higher velocity when moving to the innermost electrodes HeLa cells are thus more attracted to the central electrodes than being RBCs when the inward stepping electric field was applied, making it feasible to concentrate HeLa cells from the given specimen Although there are still some non-target cells, the fact HeLa cells The detection and characterization of cells are conducted by the differential capacitive sensor constructed by two symmetric electrodes next to the center electrode Fig.5 presents the electric field profile around the center electrodes with the presence of HeLa cell at the capturing electrode The simulation results in Fig indicated the relation between the difference capacitance and the number of cells It can be observed that the differential capacitance output proportionally increased with the number of particles The concentration of particles contributed to the change in overall capacitance, which primarily depend on the permittivity of the medium between two capacitive sensing electrodes By utilizing appropriate aptamers, having a high affinity to the target cells, the proposed device shows that such cells adhered to the aptamers That is, they are captured and prevented from being washed away in the flushing process The density of target cells over non-target cells is, therefore, augmented and thus, enhances the detecting precision An increase of up to 3.4 pF was achieved and sufficient to possibility of cell detection 300 2016 International Conference on Advanced Technologies for Communications (ATC) detected by a differential capacitive sensor The device has a capability of both selectively isolating, concentrating and detecting the presence of specific target cells of the given sample It is, thus, benefiting to biotechnology, medicine and diagnostics CONCLUSION We proposed a biological microparticles detection platform based on a novel integration between dielectrophoresis manipulation and differential coupled contactless conductivity detection The device employs an array of circular electrodes and dielectrophoresist phenomenal to manipulate the HeLa cells to the sensing region Such cells are captured and ACKNOWLEDGMENT [11] This work was funded by the Asia Research Center (ARC), Vietnam National University, Hanoi, through the project entitled “Development of a living cell microfluidic sensor for biomedical applications" [12] REFERENCES [13] [1] G M Whitesides, “The origins and the future of microfluidics”, Nature, vol 442, pp 368-373, 2006 [2] K Khoshmanesh, S Nahavandi, S Baratchi, A Mitchell, K KalantarZadeh, “Dielectrophoretic platforms for bio-microfluidic systems”, Biosens Bioelectron, vol 26, pp 1800–1814, 2011 [3] P Paterlini-Brechot, and N L Benali, “Circulating tumor cells (CTC) detection: Clinical impact and future directions”, Cancer Lett., vol 253, pp 180-204, 2007 [4] R Pethig, “Review Article—Dielectrophoresis: Status of the theory, technology, and applications”, Biomicrofluidics, vol 4, 022811, 2010 [5] B Yafouz, N A Kadri, and F Ibrahim, “Dielectrophoretic Manipulation and Separation of Microparticles Using Microarray Dot Electrodes”, Sensors, vol.14, pp 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