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 ECS Journal of Solid State Science and Technology, 4 (10) S3067-S3070 (2015) S3067

JSS FOCUS ISSUE ON MICRO-NANO SYSTEMS IN HEALTH CARE AND ENVIRONMENTAL MONITORING
Pattern Recognition of HER-2 in Whole Blood Samples
Using Stochastic Microsensors
Iuliana Moldoveanua,b,∗ and Raluca-Ioana Stefan-van Stadena,b,∗∗,z
a Laboratory of Electrochemistry and PATLAB, National Institute of Research for Electrochemistry and Condensed
Matter, Bucharest 6, Romania
b Faculty of Applied Chemistry and Materials Science, University Politehnica Bucharest, Bucureşti 060042, Romania

Three stochastic microsensors were developed and used for analysis of HER-2 (a protein from Epidermal Growth Factor Receptor
(EGFR) tyrosine kinase family) in whole blood samples. Chitosan I (n = 371 - 744), chitosan II (n = 868 - 1365), and maltodextrin
(DE 4–7) were used as modifiers for the design of the new stochastic sensors based on diamond paste. The microsensors proposed
can be used for the analysis of HER-2 in the concentration range between 3 fg/mL to 30 ng/mL. The highest sensitivity (1.47 × 107
s−1 /mg/mL) was obtained for the microsensor based on chitosan II and diamond paste, while the lower limit of quantification (3
pg/mL) was obtained when the microsensor based on chitosan I and diamond paste was used. HER-2 was reliable determined from
whole blood samples with recoveries higher than 93.00%.
© The Author(s) 2015. Published by ECS. This is an open access article distributed under the terms of the Creative Commons
Attribution Non-Commercial No Derivatives 4.0 License (CC BY-NC-ND, http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is not changed in any
way and is properly cited. For permission for commercial reuse, please email: oa@electrochem.org. [DOI: 10.1149/2.0181510jss]
All rights reserved.

Manuscript submitted March 4, 2015; revised manuscript received September 2, 2015. Published September 17, 2015. This was
Paper 2122 presented at the Chicago, Illinois, Meeting of the Society, May 24–28, 2015. This paper is part of the JSS Focus Issue
on Micro-Nano Systems in Health Care and Environmental Monitoring.

Due to its stable structure, diamond can accommodate the best,
nanostructured materials like chitosan, and maltodextrins for the de-
sign of stochastic sensors. Stochastic sensors are excellent tools for
biomedical analysis due to their advantages like: the possibility of
performing reliable qualitative analysis based on the signatures of the
analytes; the possibility of performing multianalyte detection based
on the signatures of the analytes; their reliable quantitative assay
of the analytes from any biological fluid like, serum, whole blood,
saliva, and cerebrospinal liquid, their response being independent on
the composition of the matrix of the sample.
Chitosan is a semicrystalline polysaccharide derived from chitin,
one of the most abundant natural polysaccharides.1 The principal
advantages of this polymer are: forms good films and membranes,2
it is antimicrobial and inhibits the growth of a wide variety of fungi,
yeasts, and bacteria, which can be beneficial for use in the field of
biomedicine,3 it is biocompatible, and biodegradable,4 had adhesion
capability, and high mechanical strength.5 This polymer has been used
as modifier in design of several electrochemical sensors6–8 with good
results obtained due to its excellent properties, making it a promising
candidate for stochastic microsensors.
Maltodextrins are complex malto-, oligo-, and polysaccharide
mixtures resulted from hydrolysis of starch, with dextrose equiva-
lent (DE) lower than 20.9 DE represents the percentage of reducing
sugar calculated as glucose on a dry-weight basis, with glucose given
the value 100 or the equivalent of the degree of polymerization of
maltooligosaccharides.10,11 Its structure favorized the stochastic re-
sponse of the sensors, when used for the design of stochastic sensors.
Breast cancer is one of the most prevalent cancers in the world.
The 185 kDa transmembrane ErbB2 protein (Neu/HER2) is overex-
pressed in many breast cancers tumors and is associated with tumor
progression, invasion, metastases and poor prognosis.12,13 It is the
second member of the Epidermal Growth Factor Receptor (EGFR)
tyrosine kinase family.14 Many clinical studies showed that HER-2
is an important factor in prognosis for breast cancer, and also it is
known that patients with HER2-positive have a shorter survival time
than patients with HER-2 negative.15–17 Concentration of HER-2 in
serum between 2 and 15 ng/mL were recorded for healthy patients,
∗ Electrochemical Society Student Member.
∗∗ Electrochemical Society Active Member.
z
E-mail: ralucavanstaden@gmail.com
while concentration of HER-2 in serum higher than 15 ng/mL were
recorded for patients diagnosed with breast cancer.18 Monitoring of
the HER-2 levels in blood can help the efficiency of treatments for the
patients diagnosed with this cancer.19 Current methods used for detec-
tion of HER-2 in serum samples include enzyme-linked immunosor-
bent assay (ELISA),20 but this method is expensive and laborious.18
While immunohistochemistry and ELISA are the chosen techniques
for serum and tissue analysis of HER-2; they are very expensive, and
time consuming, and needs a laborious sample process. Given the
fact that a fast analysis is needed for the patient’s positive for HER-2,
there is a real need to develop minimal invasive methods of analysis,
performed within minutes, in order to save the life of the patients.
Therefore, in this paper, for the first time there were designed
three stochastic microsensors based on diamond paste modified with
chitosan I (n = 371 – 744, chitosan I/DP), chitosan II (n = 868 –
1365, chitosan II/DP), and maltodextrin (DE 4–7) (MD/DP) for fast
screening of whole blood for HER-2.
Experimental
Materials.— Epidermal growth factor receptor 2 (HER-2), natural
monocrystalline diamond powder (particle size 1 μm) (DP), mal-
todextrin (dextrose equivalent 4–7) (MD), chitosan I (n = 371 - 744)
(Deacetylated chitin, KiOmedine-CsU B, Poly (D-glucosamine), and
chitosan II (n = 868 - 1365) (Deacetylated chitin, KiOmedine-CsU
D, Poly (D-glucosamine) were supplied by Sigma - Aldrich. Paraffin
oil, titrisol buffer solution were purchased from Fluka (Buchs,
Switzerland). Deionized water obtained from a Millipore Direct-Q
3 System (Mosheim, France) was used for the preparation of buffer
solution (0.1 mol/L titrisol buffer solution, pH = 7.40). Working
standard HER-2 solutions (0.3 fg/mL to 40 ng/mL were prepared
using serial dilution method.
Samples.— Twelve whole blood samples were obtained from
the University Hospital in Bucharest (Ethics committee approval nr
11/2013) from patients confirmed with breast cancer. Informed con-
sent was obtained from all patients.
Instrumentation.— All measurements were recorded using a
PGSTAT 12 potentiostat/galvanostat (Metrohm) connected to a three-
electrode cell, and linked to a computer via an Eco Chemie (Utretch,
The Netherlands) software version 4.9. Ag/AgCl electrode served as
S3068 ECS Journal of Solid State Science and Technology, 4 (10) S3067-S3070 (2015)

Ag/AgCl
Stochastic
Pt sensor

Figure 1. Set-up of the electrochemical cell used for stochastic mode.

reference electrode and platinum electrode served as auxiliary elec-

trode. A special program developed in our laboratory was used for
automat reading of toff and ton values.

Design of the microsensors.— Solutions of chitosan I, chitosan II

(10−7 mol/L, in HCl: deionized water = 1:4 v/v), and MD (10−3 mol/L
dissolved in water) were used to modify the diamond paste (obtained
from natural diamond powder and paraffin oil). 25 μL solution of
modifier was added to the diamond paste in a ratio of 1:4 (v/w).
The modified paste was placed into a plastic tube with an internal
diameter of 300 μm. Electrical contact between the modified paste
and external circuit was obtained using an Ag wire inserted into the
modified paste. The surface of the microsensors was renewed by
polishing with alumina paper. The microsensors were stored in a dry
place, at room temperature.

Stochastic mode.— All stochastic measurements were performed

at a constant potential of 125 mV, at 25◦ C using an electrochemical
cell filled with standard solutions or whole blood samples (Figure 1).
The toff values (signatures of HER-2, obtained when the current is
dropping to zero value) was identified for HER-2 (Figure 3). The
values of ton were determined and used for obtaining the equation of

Figure 3. Pattern recognition of HER-2 in whole blood samples using the

microsensors based on: a) chitosan I/DP; b) chitosan II/DP; c) MD/DP.

calibration specific for stochastic sensors: 1/ton = A + BxConc.HER-2 ,

where A is the intercept, and B is the sensitivity of the sensor. The
values of 1/ton obtained in the diagram for whole blood sample were
introduced in the equation of calibration in order to obtain the value
of concentration.

Results and Discussion

Response characteristics of stochastic microsensors.— The work-
ing principle of stochastic microsensors is based on channel conduc-
Figure 2. Current development for stochastic sensors. tivity. A potential of 125 mV is applied, and HER-2 is going through
 ECS Journal of Solid State Science and Technology, 4 (10) S3067-S3070 (2015) S3069

Table I. Response characteristics of stochastic microsensors used for pattern recognition of HER-2.

Stochastic microsensors Signature of the Equation of calibration and Linear concentration Sensitivity Limit of determination
based on DP and analyte toff (s) correlation coefficient∗ range (pg mL−1 ) (s−1 /mg mL−1 ) (pg mL−1 )
Chitosan I 2.6 1/ton = 1.23 (±0.05) × 107 × C 3 – 3000 1.23 × 107 3
+ 0.03 (±0.01); r = 0.9995
Chitosan II 2.1 1/ton = 1.47 (±0.02) × 107 × C 30 – 3000 1.47 × 107 30
+ 0.02 (±0.01); r = 0.9997
MD 4.2 1/ton = 8.94 (±0.03) × 105 × C 30 – 30000 8.94 × 105 30
+ 0.05 (±0.01); r = 0.9970

∗ 1/t s−1  and C mg mL−1 .

on

the channel, blocking it in the first stage; in this stage, the value of the
current is dropping to zero; the time measured when the value of the
current is zero is called toff (signature of the analyte) (Figure 2). This
value depends on the size, geometry of the analyte, on its velocity
to unfold (if it is a protein), and on the speed of passing through the
channel. Stage 2 is binding on the channel wall when the electrochem-
ical processes are taken place. Specific for this stage is the value of
ton which is dependent on the concentration of HER-2. Molecules are
getting into the channel/pore in a certain sequence based on their size,
geometry, capacity/speed of unfolding, speed of passing through the
channel/pore, all these parameters being responsible for their signa-
ture (toff value).
The signatures of HER-2 determined using chitosan I/DP, chitosan
II/DP, and MD/DP are shown in Table I; these values were used to
automatically identify the analyte in each diagram obtained after the
analysis of whole blood samples. The ton values were used to determine
the linear concentration range, sensitivity, and limits of determination,
for all stochastic microsensors designed. These microsensors can be
used for analysis of HER-2 in the linear concentration range, between
3 fg/mL and 30 ng/mL. The lower limit of quantification (3 pg/mL)
was obtained when the microsensor based on chitosan I and diamond
paste was used. The highest sensitivity (1.47 × 107 s−1 /mg/mL) was
obtained when the microsensor based on chitosan II and diamond
paste was used.
The microsensors were robust and stable for more than 3 months
when used every day for measurements. RSD (%) values for sensi-
tivity recorded for this period of time was less than 1.00% for every
microsensor.
Selectivity of the sensors was checked versus carcinoembryonic
antigen (CEA), HER1, and CA15-3 (breast tumor antigen) when
different values for toff (signatures) were obtained for the selected
analytes.
Analytical Applications
The analysis of HER-2 was performed for whole blood samples us-
ing the proposed stochastic microsensors. The diagrams were recorded
(examples are given in Figure 3), and HER-2 was identified based on
its signature (Table I), and quantified using the ton values. In Table II
are shown the quantities of HER-2 found in whole blood samples
analysis using stochastic microsensors developed in our laboratory.
The pair-t test performed at 99.00% confidence level shown that there
is no significant difference between the values obtained using the
three sensors (tabulated theoretical value: 4.032) (Table II). Further-
more the results obtained for Bias (%), as well as the results obtained
using the immunohistochemistry (IHC) of the tissue show a good
correlation between the stochastic methods and the standard method.
At the moment, immunohistochemistry is the method used for the
reliable assessment of HER2 in tumoral tissue; therefore there is a
need of biopsy or surgery to extract the tissue sample. This method
is painful and in many cases not recommended. Therefore, the results
obtained for these measurements correlated with the amounts well
known found in whole blood samples, represent a good advancement
in HER2 analysis using a minim invasive analysis.
Recovery tests were performed for the assay of HER-2 in whole
blood samples (Table III). The concentration of HER-2 in whole
blood sample was measured, and after, different amounts of HER-
2 were added to the whole blood sample. For these measurements
were also selected five samples from healthy patients, that did not
contained HER2. The recovery was calculated comparing the value of

Table II. The assay of HER-2 in whole blood samples.

HER-2, ng/mL∗
Sensor based on DP and

Sample no. Chitosan I Chitosan II MD/DP t-test IHC∗∗

1 21.90 ± 0.03 21.18 ± 0.07 21.90 ± 0.01 1.56 ++
2 64.40 ± 0.02 69.60 ± 0.04 63.60 ± 0.03 3.45 +++
3 19.17 ± 0.01 18.70 ± 0.02 19.90 ± 0.02 1.86 +
4 28.00 ± 0.03 22.20 ± 0.02 25.00 ± 0.03 3.20 ++
5 26.40 ± 0.02 25.30 ± 0.02 25.70±0.03 2.20 ++
6 29.50 ± 0.03 28.70 ± 0.02 29.80 ± 0.02 2.32 ++
7 20.30 ± 0.01 20.90 ± 0.01 21.80 ± 0.03 2.06 ++
8 62.94 ± 0.04 62.20 ± 0.02 63.50 ± 0.05 1.97 +++
9 16.70 ± 0.02 15.80 ± 0.02 15.20 ± 0.01 2.07 -
10 39.90 ± 0.02 39.90 ± 0.02 40.75 ± 0.05 1.23 +++
11 23.10 ± 0.01 22.99 ± 0.03 22.10 ± 0.03 2.32 ++
12 45.80 ± 0.03 45.90 ± 0.02 45.60 ± 0.02 1.04 +++
BIAS (%) 1.15 1.23 1.20 - -

∗N = 3.
∗∗ Immunohistochemistry (IHC) of tissue samples (“-” is for concentrations of HER2 in whole blood less than 16 ng/mL, “+” is for concentrations of
HER2 in whole blood between 16 and 20 ng/mL, “++” is for concentrations of HER2 in whole blood between 21 and 30 ng/mL, and “+++” is for
concentrations of HER2 is for concentrations of HER2 in whole blood higher than 31 ng/mL).
S3070 ECS Journal of Solid State Science and Technology, 4 (10) S3067-S3070 (2015)
Table III. Recovery test of HER-2 in whole blood samples.
Mirosensor based on DP and HER-2, Recovery, %∗
Chitosan I 93.73 ± 0.03%
Chitosan II 98.93 ± 0.01%
MD 94.00 ± 0.03%
∗N = 10.
the concentration of analyte added (theoretical value) with the value
of the HER-2 recovered from the whole blood sample. Recoveries
higher than 93.00% were recorded with RSD lower than 0.01%.
The proposed method is far less expensive than ELISA and IHC,
it is faster, and can be easily used for screening of biological fluids
in order to provide to the medical doctor in real time the information
about HER-2.
Conclusions
The stochastic microsensors based on chitosan I/DP, chitosan
II/DP, and MD/DP proved to be reliable screening tools for pattern
recognition of HER-2 in whole blood samples. The most sensitive
microsensor was based on chitosan II and diamond paste. The lowest
limit of determination (3 pg/mL) was obtained for the microsensor
based on chitosan I and diamond paste. The features of the sensors
is their utilization for pattern recognition of HER-2, and screening
tests for fast diagnosis of breast cancer as well as for determining the
efficiency of treatment.
Acknowledgments
This work was supported by UEFISCDI PNII Program Ideas 2011–
2014, Contract nr. 123/05.10.2011. Iuliana Moldoveanu acknowl-
edges the Sectorial Operational Programme Human Resources De-
velopment 2007–2013 of the Ministry of European Funds through the
Financial Agreement POSDRU/159/1.5/S/137390.
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