ELECTRICAL IMPEDANCE SPECTROSCOPY OF SINGLE-CELLS IMMOBILIZED IN A SILICON-BASED MICROPORE

Bryan Arroyo Fernndez R.E. Daz-Rivera

Department of Mechanical Engineering University of Puerto Rico, Mayagez Mayagez, PR 00681

ABSTRACT

It often happens in the science of drugs that a drug affects body cells or areas that are not wanted to be attacked in order to attack cells that are malignant, such as cancer. One possible solution to this problem is the use of more precise detection systems in combination with less invasive treatments that can separate cancerous cells from noncancerous cells and treat the first group in isolation, omitting or reducing the adverse effects in the second group of cells. Although this system is expected to have great advantages over the current in terms of treatment and disease detection, this system has not been developed because of the difficulties presented by having high precision systems at the cellular level and the fragility of the cells and percentage of dismemberment of them in the existing selection systems [1]. In this presentation we propose a model of micropore chip that allows a cell to be trapped and then characterize the electrical properties [2] of the cell by means of a study of the impedance and admittance versus frequency. One of the main goals of this experiment is the development of a mathematical model to derive the values of interest for admittance and impedance of the cell from the measured values of these parameters for the system. The results confirm the feasibility of developing a system for the characterization of resistance and capacitance values for the cell, information of great value to develop, ideally, separation methods of malignant from non-malignant cells.
INTRODUCTION

cancer, determine the relevance of investigation focused on this type of method. In the case of cancer, it is known that this disease is treated by surgery, chemotherapy or radiotherapy, methods which are known to be aggressive to both cancerous and non-cancerous cells in the vicinity of the area covered. The incapacity to select which cells are to be attacked and which not is in many cases the cause of an ineffective method of treatment. The development of a system that can distinguish and isolate cancer cells from non-cancerous would take a step forward in development of cancer treatments whose effectiveness rates are expected to be significantly higher. In the development of this paper will delve into using a system developed in our laboratory [2] to isolate individual cells in a micropore system that allows studying the electrical properties of the cell without being invasive to the cell or its surroundings.
MATERIALS AND METHODS

The opportunity to isolate the surrounding from a system of interest for study and manipulation has led to progress in areas such as medicine, general science and others. In the area of medicine we have the example of the use of contact drugs which allow us to treat more intensively one specific part of the body [1]. However, as science moves technology forward, our ability to work with smaller and smaller systems increases, which in turn allows us to develop more specific studies and manipulations, and thus have a greater range of possibilities facing a problem . At this time diseases like

For our system we used a chip of Crystal Silicon covered by a thin layer of silicon nitride. This chip has a micropore of 3m of diameter [2] that connects the upper chamber with the lower chamber. In both chamber, an electrode with a different geometry is fixed. This device was not constructed for this experiment; it is part of the previous works of Diaz-Rivera, R.E. and B. Rubinsky in area of microfluidics [2]. The purpose of this research is to obtain impedance and admittance values for the cellchip system. For this study we proposed the use of Gamry Instruments. The following picture is a visual representation of a front view of the system used for our experiment. As we can see, the cell is trapped in a micropore on a silicon nitride surface that connects the upper chamber with the lower one. It is important to mention that the cell is suspended in a PBS solution [1-2]

 | | cos( ) 2 2 =( (| | cos( ) 2 2 ) (| | sin( ) ( ( 1 1 + (2 + (2 ) ) cos(tan (2() ) ) cos(tan (2() )) )), 1 *

In order to study and manipulate the information obtained for the measurements for admittance and impedance values, an electrical model was developed based on mechanical and electrical considerations using the theory of equivalent circuits. The proposed model was a block system in parallel resistors and capacitors connected in series [3]. Forward is a pictorial representation of the same.

| | sin( ) 2 ( (| | cos( ) 2 2 ) (| | sin( ) +( +( 1 1 + (2 + (2 ) ) sin(tan (2() ) ) sin(tan (2() ))

1 *

), j

As expected this equation consists of a real and an imaginary component, the latter being succeeded by the imaginary unit vector j [4]. In this the ZT corresponds to the magnitude of the measured impedance in Ohms for the system, T is the phase angle of the impedance, RA, CA, are the resistance and capacitance values in Ohms and Farads respectively for element A and finally f is the frequency in s-1. Once we obtained the values for total system impedance and phase angle and using the resistance and capacitance values for the electrodes PBS, Pore and Silicon Nitride we can develop a program to obtain the admittance of the cell as a function of frequency. Thereafter the values for the cell capacitance and resistance can be obtained using the following equations [4].
= = + + = 1 2

As can be observed, according to experimental considerations, in an impedance analysis, the electrical properties of the cell are in parallel with the pore and the silicon nitride, which are the immediate surroundings. This subsystem is in series with the electrodes and the PBS solution. In order to manipulate the results obtained and derive information of the capacitance and resistance of the cell, a mathematical model was developed that made an admitive analysis [4] of the system and ends in a conversion of conductance values (real component of admittance) and susceptance (imaginary component of admittance) to the values of resistance and capacitance of the cell [4]. The equation proposed for the theoretical model of our system, cleared for the cell information is presented next

+ +

= ((

) 2+

For simplicity, the capacitance of the single capacitance configuration; this capacitance [4].

cell membrane capacitance and the cell cytosol were integrated into a value because they are in parallel new variable is called the cells
= +

Similarly, the cells resistance is the equivalent resistance to the resistance of the cell membrane and the resistance of the cytosol of the cell [4].
= +

RESULST AND DISCUSSION

As an undergraduate student, the development and manipulation of these equations and programming them is not a factor of difficulty in achieving the goals of the experiment. However, as seen in the equations and representations of the system, the large number of variables and the idealizations used to arrive at these equations have the obstacle of promoting experimental error describing a complex system without segmented it into parts or smaller subsystems to correct any errors that may appear in the recollection of data. For this reason Dr. Rubn E Daz took the determination to stop the work on the system of interest to develop and study simpler systems to validate the equations that have been developed. This was the priority during the last period of the research this semester (August to December 20122013).

In order to validate the proposed models and equations a simpler system was developed. The system has been developed with parallel capacitors and resistors as illustrated above. This system has been designed with random values for capacitances and resistances. The values of the electrical properties of the system components are presented in the next table.

C1 (Farads) R1 () C2 (Farads) R2 () Ceq (Farads) Req ()

1.52E-09 9.71E+03 6.270E-10 1.000E+05 2.143E-09 8.853E+03

Above is an visual representation of the system proposed by Dr. Rubn E Daz consisting of two capacitors and two resistors in a parallel configuration. For this system a matemathical model for the impedance as a function of frecuency is presented below.
1 = (( + (2 ) ) cos(tan (2 )))

In the next table are shown the analytical and experimental values for the phase angle of the impedance versus frequency. The frequency range used was from 10 Htz to 35,000 Htz. As we can see the percentages of difference between analytical and experimental values were reduced as the frequency increased, reaching a minimum difference and then the difference percentage started to increase again. These differences in the extremes of the frequency range may be due to many factors; however it is worth mentioning that the sensitivity required by the measurement equipment in extreme values is higher, with respect to intermediate values of frequency. This data allows us to infer that the high end differences percentage are probably due to errors in the measurement equipment. More experimentation is required to validate this assumption.
Analytic Data Frequency[s-1] Phase (deg) 359.93 359.32 356.59 353.20 329.20 309.99 299.22 292.76 288.55 Experimental Data Phase (deg) 317.74 359.66 356.63 353.32 329.96 310.79 296.24 288.50 282.66 %Dif 11.72 0.10 0.01 0.03 0.23 0.26 0.99 1.45 2.04

1 (( + (2 ) )

sin(tan (2 ))) j

10 100 500

= +

1000 5000 10000 15000 20000 25000

30000 35000

285.62 283.48 Analytic Data

278.43 274.88 Experimental Data Magnitude () 8750.00 8750.00 8750.00 8700.00 7560.00 5700.00 4460.00 3550.00 2930.00 2490.00 2160.00

2.52 3.03

Frequency[s-1] 10 100 500 1000 5000 10000 15000 20000 25000 30000 35000

Magnitude () 8853.09 8852.47 8837.41 8790.86 7604.76 5689.80 4321.27 3424.33 2816.36 2384.12 2063.48

%Dif 1.16 1.16 0.99 1.03 0.59 0.18 3.21 3.67 4.03 4.44 4.68

In the above table analytical and experimental values for the magnitude of the impedance of the system as a function of frequency is presented. With the purpose of comparing the last column of percentage difference has been included as in the previous table. In this case we observed that the behavior of the percentages of difference showed the same pattern as the case of phase angle percentage difference, being greater at the ends and lower for intermediate values of frequency. The data presented provides the following information: the proposed mathematical model for the total system impedance and phase angle is appropriate to describe the properties of the system; the equipment used for measuring impedance increases its effectiveness as the frequency reaches intermediate values of frequency. The minimum percentage difference occurs at the frequency of 5,000 Htz.
SUMMARY

develop the system because of its feasibility in manufacturing; also because it is designed to allows us to neglect the effects of geometrical considerations of the cells on their electrical properties and because it is not invasive for the cell. The system was modeled by an equivalent circuit system for which a mathematical model was developed. This equation relates the values of interest, in this case, capacitance and resistance values for the cell with the admittance of the cell. The equivalent circuit modeling led to a significantly complex equation that left the door open to experimental errors. For this reason, Dr. Rubn E Daz focused the attention of the experiment to simplified systems experiments with known values for capacitance and resistance within that allowed us to validate the equations developed using the same equivalent circuit method. The latter was achieved by presenting the values for impedance magnitude and phase angle analytical and experimental. These showed difference percentages of 0.59% as a minimum. These results open doors to new research with more elaborate systems, similar to the system of interest, micropore-cell chip. We can conclude that the investigation of this method for study and treatment of cells can give us information that could be used to develop more complex systems that allow measurements of capacitance and resistance of cells flowing through a channel or cell selection systems in a solution [1-2].
REFERENCES

1.

Younghak, C., Hyun S.K., Frazier, A.B., Chen, Z.G., Shin, D.M. and A. Han, Whole-Cell Impedance Analysis for Highly and Poorly Metastatic Cancer Cells, Journal of Microelectromechanical Systems, 2009: p.808-817. Diaz-Rivera, R.E. and B. Rubinsky, Electrical and thermal characterization of nanochannels between a cell and a silicon based micro-pore. Biomedical Microdevices, 2006. 8(1): p. 25-34. Grimnes, S. and M. Orjan-Grottem, Bioimpedance and Bioelectricity Basics. 2000, London: Academic Press. iii, 51 p. Hambley, A.R., Electrical Engineering Principles and Applications. 2011, New Jersey: Pearson Education. v, 218 p.

2.

3.

As discussed above, systems that increase the accuracy of study and manipulation of biological systems would boost our ability to progress in areas of scientific interest such as the study and treatment of cancer. For this case, a system with the characteristics described above, will need to look for the electrical properties cell by cell in a solution in order to be able to count the number of malignant cells in a solution, to take one example. In our research a chip-micropore system was proposed to

4.