ECS Transactions, 19 (33) 61-68 (2009)

10.1149/1.3263135 © The Electrochemical Society

Direct Biopsy Screening of Colorectal Cancer by Electrochemical Biosensor

S. Vernicka, A. Freemanb, J. Rishponb, and Y. Shacham-Diamanda

a
Department of Physical Electronics, Tel-Aviv University, Tel-Aviv, 69978, ISRAEL
b
Department of Molecular Microbiology and Biotechnology, Tel-Aviv University,
Tel-Aviv, 69978, ISRAEL

We propose and demonstrate feasibility of a new electrochemical

assay performed directly on slices of fresh tumor biopsies. An
amperometric biosensor was used to screen for colon cancer on the
basis of electrochemical distinction between cancerous and normal
epithelia. This observation is based on the known down-regulation
of the enzyme Alkaline Phosphatase in cancerous cells. Biopsy
samples removed from tumors induced in mice were compared to
biopsies samples simultaneously collected from the small and large
intestine (colon) of same mice. The samples were suspended in an
especially designed multichamber electrochemical sensor. Upon
the addition of a suitable substrate current signals were recorded.
Significant differences in current signal were observed for biopsies
removed from tumors comparing to samples removed from the
corresponding healthy tissue. Reproducibility was demonstrated.
The new technology allows a direct assay carried out on small
volume biopsies without the need of sample transfer to cell culture,
thus eliminating the need for pretreatment and labor intensive
preparative steps. This approach offers rapid and straightforward
detection directly affected within minutes of biopsy removal.

Introduction

Detection of colon cancer in early stages is expected to have a dramatic impact on

mortality rates. Diagnostic devices integrating biology and microelectronics are
becoming a major factor in the field of cancer diagnostics. Electrochemical biosensors
based on amperometric transducers have been widely studied as cancer diagnostic tools
due to their high sensitivity and wide linear range (1). Many reports describing the
immunological detection of tumor biomarkers by amperometric biosensors were
published. Tumor associated antigens such as CA19-9 (pancreatic, colorectal, gastric and
hepatic carcinoma), CEA (colorectal cancer), PSA (prostate cancer), CA125 (ovarian
cancer), CA15-3 (breast cancer) were detected by amperometric immunosensors (2-10).
Despite of the progress made in this field, these methods had no impact on neither the
incidence nor on mortality rates. Furthermore, the list of commercially available
biomarkers recommended for use in screening for colorectal cancer is exceptionally short
(11). The electrochemical detection of cancer biomarkers via amperometric
immunosensors usually requires the integration of antibodies and immunoconjugates
making these sensors more costly and labor intensive. The current diagnostics trend is
geared, however, towards high throughput systems equipped with user friendly,
inexpensive device and short response time (11). Here we propose and demonstrate the
feasibility of using an electrochemical cell comprised of three screen printed electrodes
and a mixing chamber, previously described by us (12) for other applications, for

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 ECS Transactions, 19 (33) 61-68 (2009)

bioelectrochemical assay performed on fresh biopsy slices. The proposed straightforward

assay provides distinction between cancerous and normal epithelia on the basis of
measuring the enzymatic activity of Intestinal Alkaline Phosphatase enzyme, known to be
down regulated in cancerous cells.

In the intestinal epithelium, the evolution from a pluripotent stem cell to a

differentiated enterocyte is accompanied by secretion of the enzyme intestinal alkaline
phosphatase, solely expressed by differentiated enterocytes (13). The level of expression
of alkaline phosphatase in many poorly differentiated colorectal cell lines is known to be
very low, almost negligible (14) and its activity shown to be as low as < 0.0001 unit/mg
of protein while a differentiated intestine epithelial cell may exhibit activity of > 0.7
unit/mg protein (15). The biochemical cellular pathways involved in alkaline phosphatase
expression in colorectal cell lines were extensively investigated (16, 17). It was
concluded that the down-regulation or absence of this enzyme in colon cancer cells may
be useful for colon cancer detection when compared to its relatively high expression level
in healthy differentiated colon tissue. Such comparative analysis of alkaline phosphatase
levels may be carried out by means of electrochemical assays as this enzyme is
commonly used as enzyme label in many immunoassays (18).

In a previous study from our lab we reported a unique high-throughput

electrochemical system for screening of suspended human cancer cells' response to
differentiation therapy. Cells of the cancer cell line HT-29, known to be sensitive to
differentiation inducing agents such as butyric acid derivatives, were treated and
subsequently exhibited enhancement of alkaline phosphatase activity. The activity of the
enzyme was monitored by chronoamperometry on a 3-electrode chamber by using p-
aminophenylphosphate (pAPP) as substrate. The product of the enzymatic reaction, p-
aminophenol (pAP) was oxidized on the working electrode at 220 mV vs. Ag/AgCl
reference electrode generating oxidation current (19).

The electrochemical detection of cancer biomarkers via amperometric biosensors is

generally carried out by placing cultured cancer cells in the electrochemical system.
Alternatively, the levels of markers secreted into or extracted by the culture medium were
detected. In the case of colon cancer, however, biomarkers are normally displayed on the
tumor tissue and are not secreted, requiring the use of biopsy as inoculum for the growth
of cell culture, involving a labor intensive, costly and time consuming procedure. Direct
detection of biopsy samples as is, is therefore highly envisaged. Here we propose and
demonstrate feasibility of detection method scheme where results are obtained within a
few minutes, by an electrochemical measurement of alkaline phosphatase level directly
from biopsy slices suspended in especially designed electrochemical chamber, enabling
determination of alkaline phosphatase activity for the evaluation of the degree of cell
differentiation in the sample. A scheme of the proposed detection method is presented in
Figure 1:

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 ECS Transactions, 19 (33) 61-68 (2009)

Biopsy
removal
Dissect
and load
onto EC
cells
Multi
plexer
add substrate and
measure

Figure 1. A scheme depicting feasibility demonstration of the proposed detection method.

'Direct diagnosis' is performed on slices of biopsies immediately upon their removal
suspended in EC cell, plugged to a potentiostat via a multiplexer. Substrate should be
added and the resulting current measured.

The added substrate pAPP undergoes dephosphorilation by the secreted enzyme

intestinal alkaline phosphatase, yielding the pAP product. Subsequently, pAP is oxidized
on the working electrode at a potential of 0.22 V vs. Ag/AgCl reference electrode. The
electrochemical reaction is described in Figure 2:

Figure 2. A. The substrate, p-aminophenyl phosphate is dephosphorilated by the enzyme

ALP (Alkaline phosphatase); B. The product p-aminophenol is oxidized on the electrode
at 0.22V generating current.

Experimental

Detection of Alkaline phosphatase in biopsy slices ('direct diagnosis'):

For feasibility demonstration tumors were induced in mice by two cancer cell lines
injected to nude mice, representing two different subtypes of colon cancer. The cancer
cell line and mice were provided by the Department of Gastroenterology, Rabin Medical
Center, Petach Tikva, Israel as follows: HCT116 and HM7 colon adenocarcinoma cell
lines were injected separately to athymic nude mice subcutaneously and abdominally (a
modification of splenic injection as described in [20]).

Tumors were allowed to develop and mice were sacrificed following 6 weeks.
Subcutaneous and abdominal HCT116 and HM7 tumors were collected. As control,

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 ECS Transactions, 19 (33) 61-68 (2009)

healthy tissues were simultaneously collected from the small and large intestine (colon)
of same mice.

Following removal, biopsy samples were stored in culture medium comprising

DMEM 88% (v/v), fetal calf serum 10% (v/v), glutamine 1% (2 mM) and antibiotics 1%
(100 units/ml penicillin, 100 µg/ml streptomycin, 1250 units/mL nystatin), all purchased
from "Beit Haemek Biological Industries", Israel, at room temperature, in sealed 50 mL
tubes. Prior to measurements, biopsy samples were washed 3 times with PBS and
dissected to 2 mm slices. Biopsy samples were weighed and placed in temperature
controlled (37 °C) PBS and tested for electrochemical alkaline phosphatase level within 1
hour.

Chronoamperometry was performed in an eight-channel, highly sensitive multiple

potentiostat (constructed by C. Yarnitzky, Technion, Israel Institute of Technology)
allowing for the simultaneous measurement of eight electrochemical cells. An in-house
apparatus providing electrical contacts of the screen print electrodes combined with
suction–expulsion-based efficient stirring was used (12). The potentiostat was interfaced
to a PC via an A/D converter employing visual basic software (12). The electrochemical
chamber was constructed as a 300 µL chamber equipped with a screen printed electrode
(SPE) (custom made by Gwent, UK) at its bottom. Electrode configuration was
comprised of a gold working electrode, carbon auxiliary and Ag/AgCl reference
electrodes. During measurements continuous mixing was affected.

Each biopsy slice was suspended in 220µl PBS in the electrochemical chamber. All
electrodes were connected via the eight-channel multiplexer, continuously operating
under mixing. A potential of 220 mV vs Ag/AgCl reference electrode was applied.
Following a short equilibration time, allowing the stabilization of the system and
determination of the background signal emerging from background electrochemical and
biochemical reactions (19), the substrate pAPP (p-amino phenyl phosphate) was added
(25 µL to make a final concentration of 0.1 mg/mL).

Results

In order to distinguish between cancer tissue and healthy tissue samples on the basis
of electrochemical detection of the expression level of alkaline phosphatase, the level of
alkaline phosphatase was measured directly on the suspended biopsy samples. Alkaline
phosphatase activity from 2 mm3 biopsy slices was measured by the addition of pAPP
and monitoring of the current generated. Results of the chronoamperometric
measurement of biopsy samples from HCT116 tumors and from healthy tissues
demonstrated significant differences in current signal between samples derived from the
cancerous and healthy tissues, as shown in Figure 4:

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 ECS Transactions, 19 (33) 61-68 (2009)

Average current signal from HCT116 tumor samples

Vs. healthy tissue samples
250

200
Current (nA)

150
pAPP added
100

50

0
0 200 400 600 800
Time (sec)
HCT116 subcut tumor (avg x19)
HCT116 abdomin tumor (avgx11)
Normal Small intestine from HCT116 injected mice(avgx16)
Normal Colon from HCT116 injected mice(avgx8)
Figure 4. A Chronoamperometric measurement of biopsies removed from HCT116
tumors and from healthy colon and small intestine tissues. Current signals correspond to
enzymatic activity of alkaline phosphatase reflecting cell differentiation state
(subcut=subcutaneous, abdomen=abdominal, avgX19=average of 19, etc.)

Samples were allowed to equilibrate at 0.22 V for a period of 5 minutes prior to the
addition of the substrate pAPP. An electrochemical response was clearly visible within a
few seconds following substrate addition, presenting a gradually increasing current. The
data of Figure 4 clearly indicate that while samples derived from healthy small intestine
secreted and affected high concentrations of alkaline phosphatase, in accord with other
reports (13), current signals obtained from tumor tissues were significantly lower and
indicated down regulation of alkaline phosphatase enzyme expression. The current
signals obtained from healthy small intestine tissue were the highest among all samples
indicating the up regulation of alkaline phosphatase. The 'alkaline phosphatase-secreting
nature' of this tissue was thus confirmed, in accord with previous report (13), validating
the feasibility of the proposed detection method enabling our proposed 'direct diagnosis'
of biopsies. However, since colorectal cancer is mostly found in the colon rather than
small intestine, clinically relevant results should compare samples derived from colon, as
shown in Figure 5:

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 ECS Transactions, 19 (33) 61-68 (2009)

Average current signal from HCT116 tumor

samples Vs. healthy tissue samples
90
80
70
60
Current (nA)

50
40 pAPP added
30
20
10
0
0 200 400 600 800
Tim e (sec)
HCT116 subcut tumor (avg x19)
HCT116 abdomin tumor (avgx10)
Normal Colon from HCT116 injected mice(avgx8)
Figure 5. A Chronoamperometric measurement of biopsies removed from HCT116
tumors and from healthy colon tissue, Current signals correspond to enzymatic activity of
alkaline phosphatase reflecting cell differentiation state (subcut=subcutaneous,
abdomen=abdominal, avgX19=average of 19, etc.)

Alkaline phosphatase expression in healthy colon exhibited current signal which may
be easily distinguished from the current observed for samples derived from tumor
biopsies. Standard deviation was calculated from the average current signal obtained
following 800 seconds from substrate addition:
120

100
average current (nA)

80

60

40 78.81
20
15.04 20.52
0
Normal Colon from HCT116 subcut tumor HCT116 abdomin
HCT116 injected (avg x17) tumor(avgx9)
mice(avgx6)

Figure 6: Average current signal and standard deviation obtained at 800 sec.
(subcut=subcutaneous, abdomen=abdominal, avgX6=average of 6, average of 17 etc.)

Despite of differences in cell types of the samples tested, it is evident from our results
that tumor derived samples yielded reproducible results reflecting substantially lower
levels of alkaline phosphatase activity detected by the electrochemical measurements.

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 ECS Transactions, 19 (33) 61-68 (2009)

Thus, within 7.5 minutes following substrate addition HCT116 tumors samples could be
clearly distinguished from healthy tissues samples.

Conclusions

The realization of an electrochemical biosensor for cancer diagnosis is highly desired,

mainly in view of the labor intensive and costly characteristics of currently employed
methods. Here we described the feasibility of a straightforward amperometric biosensing
scheme testing fresh biopsy samples. The use of multiplexer enabled the high capacity
detection scheme resulting in simultaneous multi-sampling. Due to the inherent
sensitivity of chronoamperometry detection based on tiny sliced samples derived from a
small biopsy became possible.

The straight forward simple detection of ALP secretion from intact tumor samples
would allow a rapid detection within minutes from biopsy removal. Results obtained
clearly demonstrated that a rapid electrochemical measurement may provide a distinction
between differentiated healthy intestinal epithelial cells and malignant poorly
differentiated cells. The significantly lower current response from colon cancer cells
clearly reflected the known down regulation of intestinal alkaline phosphatase as
differentiation marker. This form of 'direct diagnosis' may be subsequently automated to
expand the number of samples tested. Within the trend towards point-of-care cancer
diagnostics we anticipate that this kind of high capacity biosensing would pave the way
for the fabrication of a cost effective clinical tools to be used in clinical point-of-care as
well as conventional centralized laboratories.

Acknowledgments

The authors would like to thank Prof. Yaron Niv and Dr, Alex Vilkin from the
gastroenterology Dept. at Rabin Medical center for their vital contribution. Also, Mr.
Klimenty Levkov from Biotechnology Dept. at Tel Aviv University for helping with the
bioelectrochemical measurements.

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