Breast tumor cell detection at single cell resolution using an electrochemical impedance technique

Breast tumor cell detection at single cell resolution using an electrochemicalimpedance technique{

Sunil K. Arya,*a Kok Chuan Lee,a Dhiya’uddin Bin Dah’alan,b Daniel a and Abdur Rub Abdur Rahman*ac

Received 29th November 2011, Accepted 12th March 2012

DOI: 10.1039/c2lc21174b

Gold micro-electrodes with various diameters (25, 50, 75, 100 and 250 mm) were manufactured using

standard micro-fabrication techniques and optimized for counting of MCF-7 cells (breast tumor cells)

with single cell resolution. For specific cell capture, anti-EpCAM was immobilized on 11-

mercaptoundecanoic acid (11-MUA)-3-mercaptopropionic acid (3-MPA) mixed self-assembled

monolayer (SAM) modified gold surface of micro-electrodes. Electrodes were characterized using

optical, cyclic voltammetry and electrochemical impedance spectroscopic (EIS) techniques. Cell

capture response recorded using EIS suggested that optimum electrode dimensions should be

analogous to desired cell size. For MCF-7 cells with an average diameter of 18 ¡ 2 mm, an electrode

with 25 mm diameter was established as the optimum electrode size for precise single cell recognition

and enumeration. In EIS investigation, the 25 mm electrode exhibited an impedance change of

y2.2 6 107 V in response to a single tumor cell captured on its surface. On the other hand other

electrodes (250, 100, 75 and 50 mm) showed much less response for a single tumor cell. In future, the

use of high density arrays of such electrodes with surface modifications will result in miniaturized lab

on a chip devices for precise counting of MCF-7 cells with single cell resolution.

1. Introduction

Cancer and its metastasis with malignant cancer cells spreading

to distant sites is a leading cause of mortality and despite

advances in early detection and treatment, patients continue to

suffer from the disease.1–4 Precise counting of tumor cells in the

blood of cancer patients will be the most appropriate approach

for more reliable and effective diagnosis of cancer.5–13 Among

various cancers, breast cancer is the most common type of cancer

in women, and its distant metastasis is regarded as a major cause

of death.3,14–16 Recently, it has been shown that patients with

metastatic breast cancer having more than five circulating tumor

cells (CTCs) per 7.5 ml of blood have a much lower survival rate

than patients with fewer cells.1,7,17 Given that clinical decisions

could be made based on very few CTCs, it is critical to develop a

highly sensitive and unbiased method to enumerate them.

However, the main technical challenge in counting these cells

in peripheral blood lies in the rarity,7,17,18 and non-existence of

technology with high sensitivity to specifically count the low

numbers of tumor cells (y1 per ml of whole blood) that

characterize minimal levels of disease.19

Currently, the FDA-approved CellSearchTM system from

Veridex is in use to measure levels of CTCs in various metastatic

cancers patients.7,16,17 Further, to improve the sensitivity and

detection limit, the search for new and reliable methods has been a

subject of intense research.12,20,21 However, to achieve standard-

of-care status at global level, improvements in precision and

detection reproducibility of these cells at single cell level is

essential.1 In the case of breast cancer, MCF-7 cells derived from

breast adenocarcinoma is the most widely used epithelial cancer

cell line for investigating new detection and counting methods.

Researchers have previously reported various protocols to

enrich CTCs from large volumes of blood.4,17,22–35 However, for

cell recognition and enumeration, the majority of the methodol-

ogies use optical fluorescence.12,17,27–31,33,36 Although this

method has the advantage of being quite mature and well

established, it is labor intensive, costly, time consuming and

suffers from operator variance.37 Recently, non-optical methods

such as conductivity based sensor, impedimetric transduction,

voltammetric transduction etc. have also been used and proved

beneficial for sensitive detection and counting.6,15,38–41

Presently, in non-optical/electrochemical methods, large

macro-electrodes are commonly used to record cell capture

signals.6,15,38,42 However, large area electrodes are limited in

their resolution to the minimum number of cells that can be

precisely detected. Thus, micro-electrodes are desirable for

applications such as in rare cell counting, wherein higher

aBioelectronics Programme, Institute of Microelectronics, A*STAR(Agency for Science, Technology and Research), 11 Science Park Road,Singapore Science Park II, Singapore 117685.E-mail: [email protected] (Sunil K Arya)bSchool of Engineering, Department of Biomedical Engineering, Ngee AnnPolytechnic, SingaporecGenome Institute of Singapore, A*STAR (Agency for Science,Technology and Research), 60 Biopolis street, Singapore 138672.E-mail: mailto:[email protected] (Abdur Rub Abdur Rahman);Fax: +65 64640517; Tel: +65 90702464{ Electronic supplementary information (ESI) available. See DOI:10.1039/c2lc21174b

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resolution is required. Micro-electrodes offer several benefits

over larger area macro-electrodes, such as higher sensitivity with

minimum variability due to radial diffusion profile as opposed to

a planar diffusion profile in macro-electrodes.43–46 The cap-

ability of detecting up to 10 MCF-7 cells per electrode has

already been reported in our prior work.41 Jiang and Spencer

demonstrated the use of micro-electrode arrays for precise

counting of CD4+ cells.47 They utilized densely packed working

electrode pixels comparable to cells in size and achieved single

cell resolution by measuring interfacial impedance change on

each independent pixel.

Asphahani et al. described the response behavior of chlor-

otoxin, an ion channel inhibitor, on the human glioblastoma

cells bound on different size micro electrodes using impedance

change. For cell binding, cultured cells were allowed to adhere to

the electrode for 24 h under standard culture conditions. Results

of their studies indicate that detecting electrode hosting a single

cell exhibited a significant change in impedance, whilst electrodes

hosting a large number of cells showed little or no change in

impedance.48 In other reports on microfluidic based single cell

impedance sensors, cell topography, ion channel activity, degree

of metastatic behavior of cancer cell etc., were recorded for single

positioned cell or cell passing under laminar flow.39,49–53

However, to the best of our knowledge, there are no reports

on the detailed characterization of electrode size effect with the

view of achieving precise counting of cancerous cells with single

cell recognition.

In this study, attempts have been made to optimize the

dimensions and surface chemistry for gold micro-electrodes to

achieve precise counting of breast cancer MCF-7 cells via label

free electrochemical impedance spectroscopy (EIS) based detec-

tion at a single cell level. Investigations were made to record the

response of electrodes with a surface coverage of approximately

0, 25, 50 and 100%. The experimental results revealed that the

larger electrodes are incapable of resolving a single cell, whereas

the use of a 25 mm electrode for a single MCF-7 cell produces a

shift of more than 107 V in the EIS signal, leading to an

unambiguous differentiation between the states of presence and

absence of a single cell. Thus, via label free EIS technique and the

use of a micro-electrode array with an optimized electrode, a

binary ‘‘on/off’’ type response for better understanding and

counting of MCF-7 cells at single cell level can be achieved.

Fig. 1 shows the schematic of an electrochemical current

response of 25 to 250 mm electrodes for the presence of a single

cell.

2. Materials and methods

2.1 Chemicals and reagents

11-mercaptoundecanoic acid 95% (11-MUA, cat# 450561),

3-mercaptopropionic acid 99% (3-MPA, cat# M580),

N-hydroxysuccinimide 97% (NHS, cat# 56480), 1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide hydrochloride (EDAC,

cat# E6383), ferrocenecarboxylic acid 97% (FcCOOH) (cat

#106887), 4-morpholineethanesulfonic acid monohydrate (MES,

cat#69889) and bovine serum albumin (BSA, cat# A3294) were

obtained from Sigma-Aldrich, USA. Phosphate buffer solution

(PBS, pH = 7.4) was from Invitrogen, USA. Epithelial VU-1D9

antibody (anti-EpCAM) (cat# 01420) was from StemCell

Technologies Inc. (Vancouver, Canada). MCF-7 cells, from a

human breast cancer tumor, were purchased from the American

Type Culture Collection (ATCC) (cat # HTB-22) and received as

frozen vials. They were cultured in Minimum Essential Media

(MEM) (Gibco, cat # 11095-080) completed with 10% FBS

(Gibco, cat # 10270106), 1 mM sodium pyruvate (Gibco, cat#

11360-070), and 0.1 mM MEM non-essential amino acids

(Gibco, cat #11140-050).

2.2 Micro-electrode fabrication

Micro-electrodes with varying sensing areas (Fig. 2) were

fabricated on silicon wafer using standard silicon micro-

fabrication techniques. Low stress silicon dioxide (1 mm) was

deposited on the silicon substrate before gold metal micro-

electrode patterning, to provide insulation between various

micro-electrodes. The titanium (Ti; 0.1 mm)–gold (Au; 1 mm)

film stack was deposited on the oxide coated wafers using

electron-beam evaporator (Temescal Inc). The Ti layer acts as

the adhesion promoter for the gold film. The sensing micro-

electrodes, bond pads and connecting route lines were patterned

by a series of processes involving photoresist spin coating (PFI-

A26, Sumitomo Chemical Co., Ltd.), photolithography through

a chrome mask in EVG 5200 Mask Aligner (EVG Group),

and photoresist development in Shipley MF-319 developer

(MicroChem Corp.). The unpatterned metal regions were etched

with acid: Au etchant (Au-600, CLC) for the top Au film, and Ti

Fig. 1 Illustration of electrochemical current response of the 25 to

250 mm electrodes for the presence of a single cell.

This journal is � The Royal Society of Chemistry 2012 Lab Chip, 2012, 12, 2362–2368 | 2363

etchant (Ti-890, CLC) for the adhesion Ti layer. The photoresist

was stripped in solvent and the wafers were cleaned in

H2SO4 : H2O2 solution (3 : 1) at 125 uC to remove residual

polymer contamination. Subsequently, the wafer was passivated

with moisture barrier layers SiO2 (0.8 mm)–Si3N4 (0.2 mm)

through plasma enhanced chemical vapor deposition (PECVD).

PECVD was conducted at 200 uC to prevent inter-diffusion of Ti

into the Au surface. The sensing microelectrodes array and bond

pads were exposed for electrochemical analysis after photolitho-

graphy and reactive ion etching (RIE) with CF4. The fabricated

gold micro-electrodes were characterized for resistance and

roughness and found to possess a sheet resistance of 3.6 mV-cm

and an average surface roughness of 50 ¡ 5 nm.

2.3 11-MUA and 3-MPA mixed self-assembled monolayer (SAM)

formation and immobilization of anti-EpCAM antibodies on

electrode surface

The micro-electrodes were first cleaned using piranha solution

(H2SO4 : H2O2; 3 : 1 in volume) and sonicated in ultra-pure

water several times. The piranha cleaned electrodes were

subjected to plasma oxidation and soaked in a freshly

prepared mixture of 2 mM 11-MUA and 20 mM 3-MPA in

ethanol (MUA : MPA = 1 : 10, v/v) overnight at room

temperature for SAM formation. SAM modified electrodes

were rinsed in ethanol and ultra-pure water and dried under

nitrogen gas.

For antibody immobilization on the electrode surface, the

carboxylic groups on the mixed SAM modified micro-

electrode surface were activated via immersing in a solution

of 150 mM EDAC and 30 mM NHS in MES buffer (0.1 M

MES, 0.5 M sodium chloride, pH 6) and incubating on a

rotator for 45 min at room temperature. The electrodes were

then rinsed in ultra-pure water twice and incubated with 10 ml

of 100 mg ml21 of anti-EpCAM antibodies in PBS for 4 h at

room temperature for covalent binding of anti-EpCAM

antibodies onto the mixed SAM modified surface. The

electrodes were then rinsed with PBS–tween twice and

incubated with 1% BSA for an hour at room temperature to

block nonspecific sites. The electrodes were stored in PBS

under 4 uC when not in use.

2.4 Cyclic voltammetry (CV) and electrochemical impedance

spectroscopy (EIS) characterization of micro-electrodes

For CV characterization, three-electrode electrochemical cyclic

voltammetry was performed using the electrochemical work-

station, CHI660C (CH Instruments Inc., USA). CV was

performed between the various working electrodes and the

counter electrode of gold, with an off chip Ag/AgCl reference

electrode in 1 mM ferrocene carboxylic acid with 1 mM

potassium chloride solution. CV studies were performed using

DC voltage and no AC frequency was applied. For CV data

recording, measurements were carried out at 0 to 0.7 V at a scan

rate of 0.01 V s21.

For EIS testing, the impedance detection set-up consisted of a

test jig, connection wires, and an impedance measurement

system (Autolab Impedance Analyzer, Ecochemie) controlled

by a computer. The impedance was measured from individual

working electrodes and the on-chip counter electrode at 0.35 V

with respect to the Ag/AgCl reference electrode in a 3 electrode

set up at an amplitude of 0.025 V. The frequencies ranged from

1 Hz to 106 Hz and 29 data points (7 points per decade) were

recorded and analyzed for each measurement.

Fig. 2 Design of micro-electrodes of various sizes and their arrangement. Inset on the lower right is the optical image of the actual fabricated

electrode.


2.5 Cell capturing studies

For specific capture of MCF-7 cells, 10 ml of MCF-7 cells of

appropriate concentrations were loaded onto the chips and

concentrated on the electrode surface using patch clamp setup

(World Precision Instruments, USA). The cells were allowed to

interact with surface bound anti-EpCAM for 1 h at 37 uC for

specific cell capture via standard antibody–antigen binding based

chemistry. After cell capture, the chip was washed gently with

PBS to remove any unbound cells. Cell capture was character-

ized using CV and EIS techniques and used for optimization of

the micro-electrode for single cell detection.

3. Results and discussions

3.1 Optical characterization of micro electrodes

Fig. 3a and b show the optical and fluorescent images of blank

micro-electrodes, respectively. The exposed circular area covered

by a ring represents the active gold area and contribute to the

signal. All other areas, including connection wires covered with

an insulating SiO2/Si3N4 layer, do not contribute to any signal.

Fig. 3c and d show the fluorescence images of micro-electrodes

modified with fluorescent tagged antibodies. The bright fluores-

cence coming from the circular area confirms the uniform and

dense binding of antibodies on the MUA/MPA mixed SAM

modified gold surface via EDC/NHS activation. Fig. 3e and f

show the optical and fluorescent images of cells bound on micro-

electrodes and on 25 mm diameter electrode, respectively.

3.2 Electrode characterization using CV and EIS

Fig. 4a shows the cyclic voltammograms of various size

electrodes at a 0.01 V s21 scan rate. It is evident from Fig. 4a

that the 500 mm diameter electrode exhibits diffusion-limited

macro-electrode characteristics, which gradually transits towards

micro-electrode behavior as the electrode diameter decreases.

Fig. 4b shows the impedance magnitude plot (bode diagram) of

500, 250, 100, 75, 50 and 25 mm diameter electrodes in 1 mM

ferrocene carboxylic acid solution. The impedance plot reveals a

systematic increase in impedance with decreasing electrode size.

This can be attributed to the dependence on both solution

resistance and double layer capacitance on the surface area of the

electrode. It is speculated that the distance of the reference and

counter electrodes from various working electrodes might affect

the response, however, to avoid any interference, the size of the

counter and reference electrodes were kept very large compared

to the electrode size. Also, the counter electrode covers all of the

electrodes in the chip design, thus producing a uniform field over

all micro-electrodes. In addition, to further minimize any effects,

the difference in response signals before and after cell capture

was measured and used for analysis.

The transition of diffusion-limited macro-electrode character-

istics for 500 mm to micro-electrode behavior for 25 mm was also

visible in the Randles-Sevcik plot of the peak current densities

plotted with respect to the square root of scan rate for various

electrodes (Fig. 5). In the Randles-Sevcik plot for various

electrodes, peak current density is seen to increase with

decreasing electrode size as a consequence of large currents

supported by radial diffusion. This micro-electrode characteristic

is found to be key to the success of the 25 mm electrode in

providing the binary ‘‘on/off’’ type response to the presence and

absence of cells as opposed to the larger electrode which provides

a less distinct and more ambiguous response to the presence of a

single cell. The radial diffusion profile for smaller electrodes

allows much larger current densities as compared to larger

electrodes and when such an electrode experiences surface

blockage due to the presence of a cell, the reduction in surface

current is dramatic.

3.3 Effect of surface modification on electrode currents

CV spectra were recorded to characterize and confirm the

surface modification at each step. Fig. 6 shows the CV spectra of

a bare electrode, with mixed SAM, with anti-EpCAM and with

cells for 250 mm and 25 mm electrodes, respectively.

Supplementary Fig. 1 (ESI{) shows the CV spectra for other

electrodes. From Fig. 6, it is clear that the CV profile of the

electrodes was substantially altered due to the surface treatment

at each step, thus confirming the modification and binding of

antibodies (anti-EpCAM) for specific capture of MCF-7 cells.

Further, the electrodes showed consistent deviations in current

after cell capture. For surface coverage effect, electrodes

saturated with cells on the surface were tested and analyzed.

The 25 mm electrode holding a single cell exhibited the most

significant change in CV current (Fig. 6). The current for 25 mm

Fig. 3 (a) Optical image of blank micro-electrodes; (b) fluorescent

image of blank micro-electrodes; (c) and (d) fluorescence image of micro

electrodes modified with fluorescent tagged antibody; (e) optical image of

a cell bound on micro-electrodes, inset (e) optical image of a cell bound

on 25 mm diameter electrode; and (f) fluorescent image of cells bound on

25 mm diameter electrode.


electrode dropped to almost zero upon capture of cell, where as

significant current was observed for saturated 250 mm electrode.

It is this effect which suggested that 25 mm diameter electrode is

ideal for single MCF-7 tumor cells detection.

3.4 EIS studies for MCF-7 cell detection

Impedance has been widely used to detect cells on electrodes and

as such is the preferred method due to its non-destructive nature

and its ability to probe live cells.54 Fig. 7a shows the impedance

response of a 100 mm electrode with various cell populations

covering approximately 0, 25, 50 and 100%. The non-linear

dependence change in impedance recorded at 20 Hz was found to

be 1.2 6 105 V, 6.7 6 105 V and 4.0 6 106 V for 25, 50 and

100% coverage, respectively. The non-linearity of impedance

response to capture a varying number of cells may arise from the

changing nature of the electrode diffusion profile from planar to

linear as more and more of the surface area is taken up by the

cells. Supplementary Fig. 2 and 3 (ESI{) show the impedance

spectra for various electrodes for cell capture experiments.

Fig. 7 (b) shows the change in impedance of 25, 50, 75, 100 and

250 mm electrodes for the presence of a single cell. The 25 mm

electrode exhibited an impedance change of 2.2 6 107 V at 20 Hz

in the presence of cells as compared to the other electrodes,

which showed a change between 3.6 6 105 V and 1.1 6 104 V in

response to the presence of a single cell on the surface. The

resolution for bigger electrodes is limited by the change in

surface area coverage brought about by the single cell as well as

the measurement resolution and error of the measuring

instrument. In present study, the best resolution with binary

Fig. 4 (a) CV spectra of the 500 to 25 mm diameter bare electrodes in 1 mM FcCOOH at 0.01 V s21 scan rate, (b) Impedance spectra of 500 to 25 mm

electrodes in 1 mM FcCOOH.

Fig. 5 Randles–Sevcik plots of 500 to 25 mm electrodes in 1 mM

FcCOOH. The peak current densities show consistent scaling with

electrode size.

Fig. 6 CV spectra of (i) bare (ii) with mixed SAM, (iii) with anti-EpCAM and (iv) with cells for 250 mm (a) and 25 mm (b) electrodes, respectively.


‘‘on/off’’ type response upon the presence and absence of a single

MCF-7 tumor cell was achieved with a 25 mm electrode.

Response time, the time taken by the chip to capture cells

(incubation time with cells), and measure response was found to

be about 65 min. The best sensitivity was observed for the 25 mm

electrode which has size analogous to MCF-7 cells. Also, antibody

modified chips were stored at 4 uC when not in use and were found

to be stable and effective for cell capture for more than 1 month.

Conclusion

Cyclic voltammetry (CV) and electrochemical impedance spec-

troscopic (EIS) techniques have been used to analyze gold micro-

electrodes with different diameter size (25, 50, 75, 100 and

250 mm) for breast cancer MCF-7 cell capture response. Cells

were successfully and specifically captured using an anti-

EpCAM modified surface and the optimum electrode size

(25 mm diameter) was established for the precise detection of

single MCF-7 cells with an average diameter of 18 ¡ 2. The

25 mm electrode in the EIS investigation exhibited an impedance

change of 2.2 6 107 V at 20 Hz in response to a single MCF-7

cell captured on its surface, whereas other electrodes (250, 100,

75 and 50 mm) showed responses between 3.6 6 105 V and 1.1 6104 V for a single MCF-7 cell. Such substantial change in

impedance for an electrode with a size analogous to the cell leads

to possibilities for high accuracy, label free and automated

counting of breast cancer MCF-7 cells, for improved cancer

prognosis, diagnosis and therapy monitoring. Based on observed

binary ‘‘on/off’’ type responses upon the presence and absence of

a single MCF-7 tumor cell on 25 mm surfaces, efforts are in

progress to fabricate and use a micro-electrode array of 25 mm

electrodes to sense and count MCF-7 cells over a wide

concentration range with single cell precision. The results of

present investigations have shown the promise for fabrication of

a lab on a chip device for simultaneous counting and testing

of CTCs with single cell precision. Specific electrodes can be

designed for specific cell types having electrode size analogous to

cell size to result in optimum response for their testing at a multi-

cell level in microarrays.

Acknowledgements

We acknowledge Tang Kum Cheong for providing the

fabricated electrode devices for electrochemical testing and

characterization.

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Breast tumor cell detection at single cell resolution using an electrochemical impedance technique

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