Electrical Impedance Measurements of Biological Cells in Response

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Electrical Impedance Measurements of Biological Cells in Response to External Stimuli Amin Mansoorifar, Anil Koklu, Shihong Ma, Ganesh V. Raj, and Ali Beskok Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05392 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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Analytical Chemistry

Electrical Impedance Measurements of Biological Cells in Response to External Stimuli Amin Mansoorifar1, Anil Koklu1, Shihong Ma2, Ganesh V. Raj2, and Ali Beskok1,* 1

Department of Mechanical Engineering, Southern Methodist University, Dallas, TX 75205, USA

2

Departments of Urology and Pharmacology, University of Texas Southwestern Medical Center,

Dallas, TX 75390, USA

*

Corresponding author: [email protected]

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Abstract Dielectric spectroscopy (DS) is a non-invasive technique for real-time measurements of the impedance spectra of biological cells. DS enables characterization of cellular dielectric properties such as membrane capacitance and cytoplasmic conductivity. We have developed a lab-on-a-chip device that uses an electroactivated micro-wells array for capturing, DS measurements, and unloading of biological cells. Impedance measurements were conducted at 0.2 V in 10 kHZ - 40 MHz range with six-seconds time resolution. An equivalent circuit model was developed to extract the cell membrane capacitance and cell cytoplasmic conductivity from the impedance spectra. A human prostate cancer cell line, PC-3, was used to evaluate the device performance. Suspension of PC-3 cells in low conductivity buffers (LCB) enhanced their dielectrophoretic trapping and impedance response. We report the time course of the variations in dielectric properties of PC-3 cells suspended in LCB and their response to sudden pH change from a pH of 7.3 to a pH of 5.8. Importantly, we demonstrated that our device enabled real-time measurements of dielectric properties of live cancer cells, and allowed the assessment of the cellular response to variations in buffer conductivity and pH. These data support further development of this device towards single cell measurements.


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The dielectric properties of cells are major indicators of cell health, function, and stage1-4. Dielectric spectroscopy (DS) is widely used for analyzing the electrical properties of biological cells and can be used to examine and discriminate between tumor cells and healthy cells. Using impedance measurement techniques, drug resistant breast cancer cells (doxorubicin resistant MCF-7 DOX) may be distinguished from their parental cells (MCF-7 WT, wild-type)5. Simultaneous measurements of impedance with DS and fluorescence may sort between T-lymphocytes, monocytes, and neutrophils6. Since DS is non-invasive, label-free and amenable to real-time monitoring7,8, quantitative assessments of the growth rate, state of bioreactions, and early indications of catastrophic events such as contamination or cessation of growth, may be obtained9. DS can investigate the real-time effects of external stimuli and drug uptake on membrane and cytoplasm without the need of complex and expensive biochemical and microscopy techniques10. To monitor cellular properties as a function of time in a high-throughput and statistically relevant manner, it is often desirable to localize individual cells in an array format to retain their position and prevent their migration. Many different techniques have been applied for trapping cells, including passive confinement of cells in micro-wells11-13, biochemical patterning14, and direct printing15. Hydrodynamic16,17, mechanical18, optical19,20, acoustic21,22, and dielectrophoretic23,24 forces were also applied for cell trapping/immobilization purposes. Recently, we showed that combination of micro-wells with dielectrophoresis (DEP) provides a high throughput capturing microfluidic system enabling DS measurement of biological cells25. DEP is a label-free technique that exploits differences among dielectric properties of the particles for creating 3d metal structures26 or particles handling27. As an illustration, Tang et al. developed a novel method for creating Galinstan 3d micro-structures using DEP, which can be used to enhance trapping efficiency or improving heat dissipation within a microfluidic channel28. Differences in the polarizability of the particles and the suspending medium determine the direction of the DEP force27. Combined with pressure driven flow, DEP is an effective force that can manipulate the particles as a function of the applied electric field magnitude and frequency.



DEP trapping mostly requires biological cells to be suspended in a low conductivity medium27. Lower extracellular ionic concentration causes stronger polarization at the cell interior than the extracellular medium, which results in collection of cells at high electric field regions. However, low conductivity buffers (LCB) induce changes in biological cells. Cells mainly respond to LCB by pumping out ions followed by shape regulation29,30. This results in time dependent dielectric property response31. Therefore, there is a critical need to consider time dependent dielectric response of cells in LCB. Moreover, extracellular pH plays an important role in cell functions. The rate of cellular growth and metabolism of protein synthesis are strong functions of medium pH32. Direct measurements of cell response also have the advantages of continuous recording of metabolic changes in drug discovery processes33. Many cellular activities are reduced at lower extracellular pH value, including cytosolic and membrane associated enzyme activities, ion transport activity, protein and DNA synthesis, and cAMP (cyclic adenosine monophosphate) and calcium levels34. Further, acidic microenvironments are significant not only in the enhancement of an aggressive tumor phenotype, but they may also play a role in efficacy of the drugs that target tumors35. Using our microfluidic device that combines DEP-based micro-well capture with DS, we have previously demonstrated the capture of yeast cells in 441 30µm×30µm distinct wells, followed by impedance characterization25. Due to the small dimensions of yeast cells, each micro-well contained up to 64 yeast cells. However, the data processing required long computational times, making device operation non-ideal for real-time studies and typical clinical applications25. The current study focuses on real-time measurements of PC-3, a highly metastatic prostate cancer cell line, and its response to changes in the external conductivity and pH. Since limited information about bioelectric characteristics of PC-3 cells was available, we first determined the impedance of PC-3 cell suspension using a parallel-plate electrode configuration, and found the optimum frequencies for DEP assisted cell loading and unloading. Dimensions of PC-3 cells are suitable for single cell capture in the micro-wells allowing simultaneous DS measurement for up to 441 cells. An equivalent circuit model was 4 ACS Paragon Plus Environment

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developed for the current bio-chip geometry to extract cell membrane capacitance and cytoplasmic resistance. After diluting the cell suspension with LCB and capturing the cells inside micro-wells, the impedance spectrum was recorded for two hours with six seconds increments, which enabled a time dependent evaluation of the PC-3 cell membrane capacitance and its cytoplasmic resistance change in LCB. This is followed with studies on cell response to external pH alterations between pH of 7.3 and 5.8. These data could be used to model cellular response to drug therapies. Materials and Methods Chip Fabrication Photolithography supplies (photoresists, developers, and remover) were purchased from Microchem Corp. (Westborough, MA, USA). All other chemicals used were of analytical grade and obtained from SigmaAldrich (St Louis, MO, USA). All solutions were prepared with 18 MΩ·cm ultrapure water obtained from Millipore Alpha-Q water system (Bedford, MA, USA). As previously described25, the glass slides are firstly cleaned in an ultrasonic bath with 1M KOH, Acetone, Isopropyl Alcohol, and deionized (DI) water, respectively. The electrode structures are fabricated using lift-off photolithography method and microwells are created using negative photoresist SU8. The microfluidic channel is made up of double sided tape. The fluidic ports are drilled by a diamond drill bit and copper tapes are used for electrical connections. To assemble the device, the electrode pair is aligned using Karl SUSS MJB3 mask aligner. Finally, they are clamped and put in a convection oven at 75oC to enhance adhesion between the slides. The fabrication process has been explained extensively in our previous study25. A top view of three main layers of the microfluidic device is shown in Figure 1a. The bottom layer is a 1×1 mm square shape gold electrode on glass slide and covered with negative SU8 photoresist except for 30×30×30 µm features as the micro-wells and the electrical connection port. The micro-wells are formed as 21×21 arrays, yielding 441 wells on the sensing area. The microchannel is made up of a 70 µm thick double-sided tape. The top layer has 1×1 mm square shape gold electrode on a glass slide, which also contains the inlet and outlet holes. The top and bottom electrodes are used for capturing the cells inside micro-wells, DS measurements, and



cell unloading purposes. Figure 1a shows the top view of different layers and side view of the assembled microfluidic device along with relevant device dimensions.

Figure 1 a) Top view of the microfluidic device layers and side view of the assembled device; b) Schematics of the experimental setup.

Experimental setup The experimental setup is shown in Figure 1b. A syringe pump (New Era Pump System Inc., NE4000) delivers the cell suspension to the inlet of the microfluidic device and the outlet is collected in a drain. The suspension contains PC-3 cells at 105 cells/mL concentration, and the flow rate is fixed at 1 mL/hr. The electrodes were excited using a function generator (Tektronix AFG3102) to provide sinusoidal AC electric fields at desired amplitudes and frequencies for cell capture and release purposes. The cells were captured inside the micro-wells by applying 2 Vpp at a predetermined frequency to induce DEP response that pulls them into the micro-wells. After the cells were captured, rest of the cell suspension inside microchannel is washed with LCB towards the drain via a polyvinyl chloride (PVC) tube. Then, the microfluidic device is 6 ACS Paragon Plus Environment

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connected to the impedance analyzer to record the impedance spectrum. A high precision impedance analyzer (HP Agilent 4194A) is used for impedance measurement in 10 kHz - 40 MHz frequency range. Using medium integration time setting (i.e., the period over which the analyzer measures the input signal), each impedance measurement sweep takes six seconds for 401 discrete, logarithmically spaced frequencies. The output data of impedance analyzer are transferred to a PC using a General-Purpose Interface Bus (GPIB) cable and then the data are processed using Matlab (R2014b) software. A CCD camera (Hamamatsu Orca, C11440) connected to an inverted microscope (Olympus IX81) is used for cell visualization and images are recorded through an imaging software (CellSens). Once the DS measurements are over, the device is reconnected to the function generator, and the cells are released from the microwells by applying 2 Vpp at a predetermined frequency to induce DEP response that pushes them out of the micro-wells. The released cells are washed to the drain by simultaneously applying pressure driven flow using the syringe pump. A movie showing the whole procedure using yeast cells was shown in our previous study25. Cell Preparation and Viability Test PC-3 cells were obtained from the American Type Culture Collection (ATCC). The PC-3 cells are cultured in RPMI 1640 growth medium (Sigma Aldrich). RPMI growth medium is supplemented with 10% fetal bovine serum, with penicillin (100 IU/ml) and streptomycin (100 µg/ml)36. Cells are grown in an incubator (Thermo Scientific) at 37oC with 5% CO2 atmosphere. For subculture, the cells are washed with PBS and incubated with 0.05% trypsin-EDTA solution (Sigma Aldrich) for 10 min with the same conditions to detach the cells from petri dish. The growth medium is then added to inhibit the effect of trypsin and cells are centrifuged for 5 minutes at 1000 rpm and re-suspended in LCB solution for DS measurements or DEP experiments. The harvested cells were spherical and 22.0 ± 4 µm in diameter. LCB is the most commonly used medium for DEP based particle trapping, manipulation, or separation. It is a mixture of isotonic sucrose/dextrose solution and small amount of salts. Cells are suspended in an LCB solution containing 229 mM sucrose, 16 mM glucose, 1µM CaCl2 and 5 mM Na2HPO4 in type 1 DI water (pH 7.3) for 7 ACS Paragon Plus Environment


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experiments after washing with isotonic buffer. The conductivity of LCB is adjusted by adding PBS and measured using a conductivity meter (Con11, Oakton). The pH value of LCB solution is adjusted by adding sodium phosphate mono/dibasic solution and pH was measured using a pH meter (Orion Versastar, Thermo Scientific). The Trypan Blue exclusion test is used to determine the viability of cells in LCB. After adding PC-3 cells to LCB, the resulting cell sample is diluted in Trypan Blue dye of an acid azo exclusion medium by preparing a 1:1 dilution with 0.4% Trypan Blue solution (Sigma Aldrich). After that, a hemocytometer is filled with the suspension and incubated for 1 minute. Because of cell membrane selectivity, Trypan Blue is not absorbed through a viable cell membrane, while it passes through the dead cells membranes and makes them have distinctive blue color under microscope. After loading with cells, the hemocytometer is placed under the inverted microscope stage, and images are recorded using the CCD camera. Finally, the images are analyzed with ImageJ37 software and cell viability is calculated. DEP Theory and DS DEP is the motion of polarizable particles suspended in an ionic solution and subjected to a spatially nonuniform external electric field. Time averaged dielectrophoretic force generated by constant phase electric field for an isotropic and homogenous sphere surrounded by a conducting medium is given by27:

= 2 ∇

(1)

where is the radius of particle, is permittivity of vacuum, is relative permittivity of the medium,

∇ is the gradient of the electric field squared, and is the real part of the Clausius-Mossotti (CM) factor, which is defined as27,38:

=

∗ ∗ − ∗ ∗ + 2

(2)

where *, p, and m denotes complex value, particle, and medium, respectively. The complex permittivity is ∗ = −

! , "

where is the permittivity, # is the conductivity, $ = √−1 and is the angular frequency. 8 ACS Paragon Plus Environment

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Depending on the sign of , the DEP force will move the particles toward the electric field maxima or minima. Particles more polarizable than the medium are attracted to the regions of high electric field intensity, which is known as positive DEP (pDEP). Particles less polarizable than the medium are repelled to the low electric field regions, and this is known as negative DEP (nDEP)39. DS is an experimental method to characterize biological cell suspensions. In this method, a small AC voltage is applied to a cell suspension and the response current is measured. The electrical impedance is calculated dividing voltage by current. This process is repeated in a desired frequency range, and analyzing impedance spectrum enables determination of the dielectric properties of individual components of the suspension. One of the most common methods to analyze impedance spectra of complex systems is equivalent circuit modeling using basic electrical components. Electrodes in contact with an electrolyte experience Electrode Polarization (EP) effects due to the shielding of the applied electrode potential by the mobile counterions in the electrolyte40. This undesirable effect becomes dominant in high conductivity media and affects impedance measurements typically at 0-100 kHz range. It is common to theoretically model the EP effect with double layer capacitance in series with the whole system41. For practical applications, the constant phase element (CPE) model predicts the non-ideal behavior of EP more precisely and effectively40. CPE impedance model is defined as42:

'( =

1 )$*

(3)

where ) and + are the CPE coefficient and exponent, respectively. The parameter + changes from zero to one corresponding to purely resistive and capacitive interfacial impedance, respectively. For our microfluidic device, the equivalent circuit model depicted in Figure 2a-b is proposed for filled and empty wells.



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Figure 2 Proposed equivalent circuit model for a) filled and b) empty micro-wells. c) The equivalent circuit model for the microfluidic device.

The current between two electrodes has at most three different paths. It can flow through the SU8 part, the solution inside the micro-well, and through the trapped cell for the case of filled wells. These three paths are modeled as SU8 capacitance (,-.), the micro-well solution resistance for empty and filled cases (/,1

and /,2 ), and cytoplasmic resistance (345 ) in series with membrane capacitance (676) as the cell model, respectively. Outside the micro-well, the electric current flows through the solution inside the microchannel which can be presented as a resistor (89 ). In both filled and empty micro-wells, the EP effect is modelled as a CPE element ('( ) in series with the circuit. The impedance of empty ('1 ) and filled ('2 ) micro-wells are calculated as:

'1 = '( + 89 + '2 = '( + 89 +

1

/,2

1

/,1

1

+ $:;.

+ $:;. +

1

(4)

(5)

1

1 + $


1

?

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Electric current inside the SU8 is assumed uniform, and the following analytical formulation is used to calculate the SU8 capacitance:

:;. = ,:;.

@:;. A

(6)

where ,:;. is relative permittivity of SU8 material ( = 3.2), @:;. is the SU8 surface area, and A is the

SU8 thickness. After application of pDEP for cell capture, D2 number of micro-wells will be filled with

cells and D1 wells will be empty. Each micro-well is considered as a parallel element with the rest and a parasitic capacitance (2 ), as shown in Figure 2c, is parallel to the whole system. The total impedance of the proposed equivalent circuit is calculated as follows:

1 '>E> = D D1 2 + + $2 '2 '1

(7)

In order to find the individual electrical components, real and imaginary parts of the experimental results are fitted into the real and imaginary parts of the proposed equivalent circuit model using non-linear least square method. The fitting criterion is finding a minimum to the sum of squares of the following matrix by employing Marquardt-Levenberg algorithm as previously shown43:

∆= ∆ ,2G , … ∆ ,2I , ∆J,2G , … ∆J,2I

(8)

MNO', P QRMSO', P ∆ ,2K = 1 − L L, ∆J,2K = 1 − L L MNO'2J>, P QRMSO'2J>, P

(9)

where ∆ ,2K and ∆J,2K are defined as:

In the above equations, subscripts m and fit denote the measured and fitted data, respectively. This fit will yield CPE parameters () and +), 89 , /,2 , /,1 , 2 , and cell parameters (1 and 8=> ). For consistency, 10 random set of initial conditions were employed in the optimization algorithm. A solution is considered correct only if the results from multiple of initial conditions converge to a single global solution set. Finally, the average values are reported.



Results and Discussion DEP Response Prediction Based on the electrodes and micro-wells configurations, the electric field inside the wells was greater than that in the microchannel and the top electrode. This configuration enabled effective use of pDEP for capturing cells in the wells, as well as for emptying the wells using nDEP by altering the applied electric field frequency. The cross over frequency (T8 ), or the frequency at which DEP force changes its sign, is usually determined through trial-and-error based on the cells’ DEP response. In this study, the optimum pDEP and nDEP frequencies required for loading and unloading the micro-wells with PC-3 cells were determined by measuring the dielectric properties of PC-3 cells in a microfluidic device with parallel plate electrode configuration43, which provided the for PC-3 cell suspensions in LCB, as previously described43. Impedance measurements of suspension were repeated 5 times for each 10 minute period and the averaged data was reported. Fig. 3a shows the time averaged for PC-3 cells measured in 1 kHz

- 10 MHz frequency range at different time periods. The results revealed that the T8 of PC-3 cells increased

with time. The PC-3 cells showed pDEP response for an hour at frequencies above 100 kHz, with an T8 around 40 kHz at the beginning (0-10min) of the measurement period, and an T8 of 100 kHz after incubation in LCB for 60 minutes. Cancer cells are known to go under time-dependent cytoplasmic and membrane re-modelling through shedding of cytoplasm in LCB44. Since the conductivity of cell decreases in LCB because of ion leakage, the became more negative and T8 shifts to higher frequencies, consistent with published studies45. According to the DS measurements with parallel plate electrodes, 5 MHz was chosen to trap the cells in the wells, because it yielded the highest CM factor and consequently the largest pDEP force for a given electric field magnitude. The device was energized at 20 kHz for unloading. The applied voltage was kept at 2 Vpp to reduce the effects of Faradaic reactions, Joule heating, and electroporation46-48.


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Figure 3 a) Real part of CM factor for PC-3 cells suspended in LCB at various times b) captured PC-3 cells after 20s of pDEP application at 2 Vpp and 5 MHz frequency. The inset shows a sample of individual empty and filled microwells.

DS Measurements PC-3 cells with a concentration of 105 cells/mL were introduced into the microfluidic device at 1 mL/hr flow rate. The conductivity and pH of the external medium was 0.05 S/m and 7.3, respectively. In order to trap the cells using pDEP, AC voltage of 5 MHz with 2 Vpp amplitude was applied between the top and bottom electrodes. Once majority of the wells were filled with PC-3 cells, the function generator was turned off, and the floating cells were washed with a continuous flow of LCB solution by increasing the flow rate (5 mL/hr). By using pDEP, 96% of wells were successfully filled with single PC-3 cells in less 13 ACS Paragon Plus Environment


than 20 seconds. Figure 3b shows a picture of the trapped cells in the wells, as well as a zoomed view of empty and filled individual micro-wells. Subsequently, impedance of cell suspension was measured in the 10 kHz - 40 MHz frequency range. In the next step, the recorded impedance data were fitted to the equivalent circuit model using MATLAB software. One representative example of the raw experimental data that fit with the proposed equivalent circuit model is shown in Fig 4. Figures 4a and 4b represent the experimental and fitted impedance values and phase angles of PC-3 cell suspension in LCB with pH of 7.3 in 10 kHz - 40 MHz frequency range, obtained five minutes after the cell suspension process. We found that the impedance spectrum of the circuit model exhibited a similar trend with the experimental data, validating the proposed equivalent circuit model. Small discrepancies between the model and experimental data were due to EP impedance that dominates the impedance data in the low frequency spectrum.

Figure 4 Experimental and fitted a) impedance value and b) phase angle of PC-3 cell suspension in LCB with pH=7.3 in 10 kHz - 40 MHz frequency range obtained five minutes after the suspension of PC3 cells within LCB.


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PC-3 Cells Response to LCB Suspension In order to observe how PC-3 cells respond to suspension in LCB, their impedance spectrum at LCB with pH of 7.3 was measured for two hours with six-second resolution. Each impedance spectrum was fitted to the equivalent circuit model and cell parameters (676 and 8=> ) extracted. Figure 5 shows the changes in membrane capacitance and cytoplasmic resistance after PC-3 cells suspended in LCB. This figure showed that cell membrane capacitance decreased drastically after mixing PC-3 cells with LCB and it reached a steady state within an hour. The decrease in membrane capacitance was attributed to the cell cytoskeletal tension, which caused the cells to round up into a pseudo-spherical shape after being released into the suspension. Since, a sphere has the smallest surface area for any given volume, the membrane capacitance, which is proportional to the membrane surface area, decreased after cell suspension in LCB. Moreover, this figure showed an increase in the cytoplasmic resistance, likely caused by osmotic imbalance generated by efflux of intracellular-ions, and resulting in time dependent increase in cytoplasmic resistance 44.

Figure 5 Time dependent variations in the membrane capacitance and cytoplasmic resistance after suspension of PC-3 cells in LCB.

PC-3 Cells Response to pH change



In order to study how PC-3 cells response to external pH changes, the PC-3 cells were trapped inside micro-wells using LCB solution with pH of 7.3 (normal pH environment). After 1 minute, the external solution was changed to LCB with pH of 5.8 (acidic environment). Finally, the external pH was reversed to 7.3 after 10 minutes. Figure 6 illustrates the extracted cell membrane and cytoplasmic resistance with changing external pH values. The gray shaded region showed the time that extracellular pH was kept at 5.8. In the first minute of pH change, the cells responded rapidly to the sudden stresses applied with pH changes. This response could be related to cytoskeleton changes and ion transport through cell membrane ion channels and pumps. The remodeling, elongation, shortening, and architectural organization of the actin filaments may be a result of signaling cascades set by environmental cues49, including changes in conductivity, pH, nutrition, and etc. Using a theoretical model, Naumowicz et al. demonstrated the pH dependence of lipid membranes formed by 1:1 phosphatidylcholine-phosphatidylserine mixtures50. They concluded that the electrical capacitance of the analyzed bilayer had a minimum value around pH of 4.2 and increased as the pH decreased or increased. Our data (Figure 6) suggested that the PC-3 membrane capacitance was lower with a pH of 5.8 compared with a pH of 7.3, which is consistent with prior studies50. Further, for the first time, we have defined a profile of the changes in cell membrane capacitance and cytoplasmic resistance over time. When the pH was reversed at 10 minutes, the membrane capacitance increased and the cytoplasmic resistance decreased. These changes could be attributed to changes in cytoskeleton remodeling and increased influx of ions. At 14 minutes, the PC-3 cells relaxed and the changes in membrane capacitance and cytoplasmic resistance were reversed to normal conditions. At 20 minutes, the PC-3 cells reached a new state with lower membrane capacitance and higher cytoplasmic resistance compared to the initial state.


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Figure 6 Extracted cell membrane and cytoplasmic resistance with changing external pH values. White region shows pH=7.3 and gray regions indicate pH=5.8.

Viability Tests In order to check the cell activity, viability tests were conducted at two different pH values in LCB (pH of 5.8, and 7.3) using Trypan Blue exclusion method. Figures 7a and 7b show the viable and dead cells at these two different pH values after 1 hour. Since dead cells are permeable to Trypan Blue dye, they can be distinguished as dark particles in the gray scale figure. Figures 7c and 7d represent the percentile viability of PC-3 cells suspended in LCB for pH of 7.3 and 5.8 over 120 minutes period. Based on these figures, after two hours of suspension of PC-3 cells in LCB, less than 10% of the cells were dead at either pH of 7.3 or 5.8. Moreover, these data confirmed that cancer cell viability decreased in acidic conditions51. Viability studies of PC-3 cells in growth medium showed 90-100% viability for 24 hours52, while the current results in LCB at pH of 7.3 and 5.8 show the same viability range during the impedance measurements. These findings indicated that the measured dielectric response was driven by live PC-3 cells, which showed the applicability of the lab-on-a-chip device in biomedical and clinical settings.



Figure 7 Viable and dead PC-3 cells in LCB with a) pH=7.3 and b) pH=5.8 after 1 hour. Percentile viability of PC-3 cells in LCB with c) pH=7.3 and d) pH=5.8.

Concluding Remarks We presented a lab-on-a-chip device for real-time measurement of biological cells’ response to external stimuli such as sudden changes in the buffer conductivity and pH. The device captured cells in a microwell array using pDEP, followed by the DS measurements and nDEP assisted cell unloading processes. Well dimensions determined the number of cells that can be trapped in each micro-well. We defined the device performance for cancer cells using the PC-3 cell line using 30 µm square wells. PC-3 cells suspended in 0.05 S/m LCB exhibited a time-dependent response, where their membrane capacitance and cytoplasmic resistance decrease and increase by time, respectively. Alterations of the extracellular pH between 7.3 and 5.8, changed cellular dielectric parameters, likely related to organization of actin filaments and ion flux regulations as a response to pH alterations but not due to changes in cell viability. These data demonstrated the applicability of our microfluidic chip to measure the dielectric properties of live cancer cells with specialized LCB.


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Based on these data, we intend to deploy the current device for single cell measurements by fabricating separately addressable N×N electrode platform. Such device will allow time-dependent dielectric response measurements for individual cells with the ability of selectively releasing them using nDEP and pressure driven flow. Since DS measurements are noninvasive, selectively isolated single live cells can be used for further studies. A fundamental limitation of DS is the EP effect that overwhelms the measurements in low RF frequencies. The EP effect increases with decreased electrode size and increased buffer conductivity. Our studies along with others have shown that EP can be drastically reduced using electro deposition of fractal gold nanostructures40,53,54, platinum black40, iridium oxide55, PPy:PSS55, and PEDOT:PPS56, which will increase the device sensitivity in high conductivity media at low frequency range, addressing the aforementioned limitations of the DS technique.

Acknowledgements Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institute of Health (NIH) under the award number R21AR063334. The content is solely the responsibility of the authors and does not necessarily represent the views of the NIH. The authors declare no competing financial interest.



References (1) Cheung, K.; Gawad, S.; Renaud, P. Cytometry Part A 2005, 65A, 124-132. (2) Han, A.; Yang, L.; Frazier, A. B. Clin. Cancer Res. 2007, 13, 139-143. (3) Justice, C.; Brix, A.; Freimark, D.; Kraume, M.; Pfromm, P.; Eichenmueller, B.; Czermak, P. Biotechnol. Adv. 2011, 29, 391-401. (4) Heileman, K. L.; Tabrizian, M. Analyst 2017, 142, 1601-1607. (5) Eker, B.; Meissner, R.; Bertsch, A.; Mehta, K.; Renaud, P. PloS one 2013, 8, e57423. (6) Holmes, D.; Pettigrew, D.; Reccius, C. H.; Gwyer, J. D.; van Berkel, C.; Holloway, J.; Davies, D. E.; Morgan, H. Lab Chip 2009, 9, 2881-2889. (7) Caduff, A.; Hirt, E.; Feldman, Y.; Ali, Z.; Heinemann, L. Biosens. and Bioelectron. 2003, 19, 209-217. (8) Daoud, J.; Asami, K.; Rosenberg, L.; Tabrizian, M. Phys. Med. Biol. 2012, 57, 5097. (9) Kustermann, S.; Boess, F.; Buness, A.; Schmitz, M.; Watzele, M.; Weiser, T.; Singer, T.; Suter, L.; Roth, A. Toxicol. in Vitro 2013, 27, 1589-1595. (10) Leung, G.; Tang, H. R.; McGuinness, R.; Verdonk, E.; Michelotti, J. M.; Liu, V. F. JALA: Journal of the Association for Laboratory Automation 2005, 10, 258-269. (11) Ochsner, M.; Dusseiller, M. R.; Grandin, H. M.; Luna-Morris, S.; Textor, M.; Vogel, V.; Smith, M. L. Lab Chip 2007, 7, 1074-1077. (12) Lindström, S.; Andersson-Svahn, H. Biochim. Biophys. Acta (BBA)-General Subjects 2011, 1810, 308316. (13) Hirata, Y.; Morimoto, Y.; Nam, E.; Yoshida, S.; Takeuchi, S. In Micro Electro Mechanical Systems (MEMS), 2017 IEEE 30th International Conference on; IEEE, 2017, pp 13-16. (14) Wang, S.; Wang, H.; Jiao, J.; Chen, K. J.; Owens, G. E.; Kamei, K. i.; Sun, J.; Sherman, D. J.; Behrenbruch, C. P.; Wu, H. Angew. Chem. 2009, 121, 9132-9135. (15) Roth, E.; Xu, T.; Das, M.; Gregory, C.; Hickman, J.; Boland, T. Biomaterials 2004, 25, 3707-3715. (16) Tanyeri, M.; Johnson-Chavarria, E. M.; Schroeder, C. M. Appl. Phys. Lett. 2010, 96, 224101. (17) Che, J.; Yu, V.; Garon, E. B.; Goldman, J. W.; Di Carlo, D. Lab Chip 2017, 17, 1452-1461. (18) Valero, A.; Merino, F.; Wolbers, F.; Luttge, R.; Vermes, I.; Andersson, H.; van den Berg, A. Lab Chip 2005, 5, 49-55. (19) Shaked, N. T.; Roichman, Y.; Habaza, M. In Optical Trapping Applications; Optical Society of America, 2017, p OtM4E. 3. (20) Neuman, K. C.; Chadd, E. H.; Liou, G. F.; Bergman, K.; Block, S. M. Biophys. J. 1999, 77, 2856-2863. (21) Evander, M.; Johansson, L.; Lilliehorn, T.; Piskur, J.; Lindvall, M.; Johansson, S.; Almqvist, M.; Laurell, T.; Nilsson, J. Anal. Chem. 2007, 79, 2984-2991. (22) Lenshof, A.; Johannesson, C.; Evander, M.; Nilsson, J.; Laurell, T. In Microtechnology for Cell Manipulation and Sorting; Springer, 2017, pp 129-173. (23) Khamenehfar, A.; Gandhi, M. K.; Chen, Y.; Hogge, D. E.; Li, P. C. Anal. Chem. 2016, 88, 5680-5688. (24) Chen, X.; Liang, Z.; Li, D.; Xiong, Y.; Xiong, P.; Guan, Y.; Hou, S.; Hu, Y.; Chen, S.; Liu, G. Biosens. Bioelectron. 2018, 99, 416-423. (25) Mansoorifar, A.; Koklu, A.; Sabuncu, A. C.; Beskok, A. Electrophoresis 2017, 38, 1466-1474. (26) Hallfors, N.; Khan, A.; Dickey, M. D.; Taylor, A. M. Lab Chip 2013, 13, 522-526. (27) Pethig, R. Biomicrofluidics 2010, 4, 022811. (28) Tang, S. Y.; Zhu, J.; Sivan, V.; Gol, B.; Soffe, R.; Zhang, W.; Mitchell, A.; Khoshmanesh, K. Adv. Funct. Mater. 2015, 25, 4445-4452. (29) Gascoyne, P. R.; Pethig, R.; Burt, J. P.; Becker, F. F. Biochim. Biophys. Acta (BBA)-Biomembranes 1993, 1149, 119-126. (30) Georgiewa, R.; Donath, E.; Gimsa, J. J. Electroanal. Chem Interfacial Electrochem. 1989, 276, 255-270.


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(31) Asmar, A. J.; Sabuncu, A. C.; Levenstein, M. A.; Stacey, M. W.; Beskok, A. Biophys. J. 2014, 106, 811a812a. (32) Miller, W.; Blanch, H.; Wilke, C. Biotechnol. Bioeng. 2000, 67, 853-871. (33) Naciri, M.; Kuystermans, D.; Al-Rubeai, M. Cytotechnology 2008, 57, 245-250. (34) Lardner, A. J. Leukoc. Biol. 2001, 69, 522-530. (35) Ippolito, J. E.; Brandenburg, M. W.; Ge, X.; Crowley, J. R.; Kirmess, K. M.; Som, A.; D’Avignon, D. A.; Arbeit, J. M.; Achilefu, S.; Yarasheski, K. E. PloS one 2016, 11, e0159675. (36) Ravindranathan, P.; Lee, T.-K.; Yang, L.; Centenera, M. M.; Butler, L.; Tilley, W. D.; Hsieh, J.-T.; Ahn, J.M.; Raj, G. V. Nat. Commun. 2013, 4, 1923. (37) Abràmoff, M. D.; Magalhães, P. J.; Ram, S. J. Biophotonics international 2004, 11, 36-42. (38) Mansoorifar, A.; Ghosh, A.; Sabuncu, A.; Beskok, A. IET Nanobiotechnol. 2017, 11, 874-882. (39) Fernandez, R. E.; Koklu, A.; Mansoorifar, A.; Beskok, A. Biomicrofluidics 2016, 10, 033101. (40) Koklu, A.; Sabuncu, A. C.; Beskok, A. Electrochim. Acta 2016, 205, 215-225. (41) Koklu, A. Fractal gold electrodes for decreasing impedance at the electrolyte/electrode interface. Southern Methodist University 2015. (42) Brug, G.; Van Den Eeden, A.; Sluyters-Rehbach, M.; Sluyters, J. J. Electroanal. Chem. Interfacial Electrochem. 1984, 176, 275-295. (43) Sabuncu, A. C.; Zhuang, J.; Kolb, J. F.; Beskok, A. Biomicrofluidics 2012, 6, 034103. (44) Gascoyne, P. R.; Shim, S. Cancers 2014, 6, 545-579. (45) Sabuncu, A. C.; Asmar, A. J.; Stacey, M. W.; Beskok, A. Electrophoresis 2015, 36, 1499-1506. (46) Lastochkin, D.; Zhou, R.; Wang, P.; Ben, Y.; Chang, H.-C. J. Appl. Phys. 2004, 96, 1730-1733. (47) Squires, T. M. Lab Chip 2009, 9, 2477-2483. (48) Wu, S.-L.; Orazem, M. E.; Tribollet, B.; Vivier, V. Electrochim. Acta 2014, 131, 3-12. (49) Stossel, T. P.; Fenteany, G.; Hartwig, J. H. J. Cell Sci. 2006, 119, 3261-3264. (50) Naumowicz, M.; Figaszewski, Z. A. J. Membr Biol 2014, 247, 361-369. (51) Gu, M.; Zhang, Y.; Zhou, X.; Ma, H.; Yao, H.; Ji, F. Oncol. Lett. 2014, 8, 1739-1744. (52) Mokhtari, M. J.; Motamed, N.; Shokrgozar, M. A. Cell Biol. Int. 2008, 32, 888-892. (53) Koklu, A.; Sabuncu, A. C.; Beskok, A. Electrophoresis 2017, 38, 1458-1465. (54) Koklu, A.; Mansoorifar, A.; Beskok, A. Anal. Chem. 2017. (55) Malleo, D.; Nevill, J.; Van Ooyen, A.; Schnakenberg, U.; Lee, L.; Morgan, H. Rev. Sci. Instrum. 2010, 81, 016104. (56) Cuentas-Gallegos, A.; Rincón, M.; Orozco-Gamboa, G. Electrochim. Acta 2006, 51, 3794-3801.



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Figure 1 a) Top view of the microfluidic device layers and side view of the assembled device; b) Schematics of the experimental setup. 43x56mm (600 x 600 DPI)

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Figure 2 Proposed equivalent circuit model for a) filled and b) empty micro-wells. c) The equivalent circuit model for the microfluidic device. 33x15mm (600 x 600 DPI)


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Figure 3 a) Real part of CM factor for PC-3 cells suspended in LCB at various times b) captured PC-3 cells after 20s of pDEP application at 2 Vpp and 5 MHz frequency. The inset shows a sample of individual empty and filled micro-wells. 56x97mm (600 x 600 DPI)



Figure 4 Experimental and fitted a) impedance value and b) phase angle of PC-3 cell suspension in LCB with pH=7.3 in 10 kHz - 40 MHz frequency range obtained five minutes after the suspension of PC3 cells within LCB. 178x259mm (600 x 600 DPI)


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Figure 5 Time dependent variations in the membrane capacitance and cytoplasmic resistance after suspension of PC-3 cells in LCB. 95x51mm (600 x 600 DPI)



Figure 6 Extracted cell membrane and cytoplasmic resistance with changing external pH values. White region shows pH=7.3 and gray regions indicate pH=5.8. 81x44mm (600 x 600 DPI)


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Figure 7 Viable and dead PC-3 cells in LCB with a) pH=7.3 and b) pH=5.8 after 1 hour. Percentile viability of PC-3 cells in LCB with c) pH=7.3 and d) pH=5.8. 49x45mm (600 x 600 DPI)


Electrical Impedance Measurements of Biological Cells in Response

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