A Portable Sensing System for Impedance based Detection of
Biotoxin Substances
V. I. Ogurtsov, K. Twomey and J. Pulka
Tyndall National Institute, Lee Maltings, University College Cork, Cork, Ireland
Keywords: Label-free Biosensor, Biotoxin, Immunosensor, Surface Modification, Electrochemical Impedance
Spectroscopy, Portable Instrumentation, Signal Processing.
Abstract: The study describes the development of a portable autonomous biosensing platform for impedance based
detection of biotoxin substances. The platform implements a label-free approach, which is based on detection
of the biosensor interfacial changes due to a bio recognition reaction. The interfacial changes are sensed by
means of Electrochemical Impedance Spectroscopy in a frequency range from 10 Hz to 100 kHz. The platform
comprises of an electrochemical biosensor, portable low-noise mix signal hardware and associated software
incorporating signal processing algorithms for extraction biotarget concentration from the biosensor response.
The biosensor is realized as an on-chip package-free three electrode micro electrochemical cell consisted of
a counter electrode (CE), a reference electrode (RE) and a working electrode (WE) patterned on a single
silicon chip. WE represents an array of 40 um diameter gold disks with 400 m center-to-center distance,
which were undergone of corresponding surface modification for antibody immobilisation. The developed
system was validated by an example of T-2 toxin detection. Performed calibration in the range of 0 – 250 ppm
of T2 toxin concentrations confirmed that the system can provide successful detection of the toxin at the levels
below 25 ppm.
1 INTRODUCTION
Biosensing systems are increasingly used in a range
of different applications including environmental,
clinical, food, agriculture, and security (Bryan et al.,
2013), (Zhang, Du, and Wang, 2015), (Wang, Lu, and
Chen, 2014), (Yong et al., 2015). The conventional
approaches are mainly lab-based and cannot be easily
brought to the point-of-need. Modern microfabricated
biosensors, on the other hand, offer the advantages of
a cost-effective and rapid sample analysis, and
specific and sensitive measurements over the more
traditional methods, which tend to be multi-step (e.g.
ELISA), or to involve sophisticated and expensive
instruments (e.g. HPLC). The ability to apply
semiconductor processing technologies, more
commonly used in the IC industry, in the sensor chip
fabrication (Herzog et al., 2013; Said et al., 2011)
enables large batch production and subsequent
availability of cheap and disposable devices. Typical
microfabricated biosensors incorporate a gold
electrode, or other e.g. platinum, Si
3
N
4
, active layer
upon which different surface chemistries are applied
to form a complete device that is specific and
sensitive to a target of interest. There are different
biosensor technologies Electrochemical, Optical,
Thermal and Piezoelectric. Of these, the
electrochemical biosensors accounted for the largest
share of over 70% of the global biosensors market in
2013, which are expected to maintain their leading
position during the forecast period from 2014 to 2020
(
Transparency Market Research , 2014).
Within the branch of electrochemical sensors,
there are amperometeric, potentiometric,
impedimetric and field-effect transistor (FET)
biosensors (Kafi et al., 2008), (Zhou et al., 2016),
(Ogurtsov, Twomey, and Herzog, 2014). With the
amperometric type biosensors, the changing current
response is monitored with time; for the
potentiometric the voltage is monitored. Both of these
types offer a relatively straight forward measurement
and a rapid analysis method. The impedimetric is
more complex and more sensitive technique. Its
classical implementation is based on the
measurement of an AC current that forms in the
response on the application to the sensor of a
sinusoidal voltage over a set of frequencies.
There is a growing interest in label-free
impedimetric biosensors due to such their advantages
54
Ogurtsov V., Twomey K. and Pulka J.
A Portable Sensing System for Impedance based Detection of Biotoxin Substances.
DOI: 10.5220/0006169200540062
In Proceedings of the 10th Inter national Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2017), pages 54-62
ISBN: 978-989-758-216-5
Copyright
c
2017 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
as simplicity of sample preparation, high sensitivity
and the ability to miniaturise the associated
measurement instrumentation (Bryan et al., 2013),
(Rushworth et al., 2014). A further advantage can be
obtained by using of micro-sized electrodes, which
improve the mass transport (and hence the sensor
sensitivity) owing to the occurrence of radial
diffusion over planar diffusion from macro-size
electrodes resulting in an improvement in the signal-
to-noise ratio and reduction in the
iR drop (Arrigan,
2014)
.
The presented study describes the development
of such portable immunosensing platform capable of
biotoxin detection. The performance of the
biosensing platform was validated by detection of T2-
toxin. It is a trichothecene mycotoxin, which is toxic
to humans and animals. It is naturally occurring
mould byproduct of Fusarium spp. fungus that can be
found in grains such as barley, wheat and oats.
2 EXPERIMENTAL
Description of the Bio Sensing
System
The platform implements a label-free approach,
which is based on detection of the biosensor
interfacial changes due to a bio recognition reaction.
The interfacial changes are sensed by
Electrochemical Impedance Spectroscopy (EIS)
technique (measurement of the biosensor complex
impedance in a wide frequency range) (Lasia, 1999).
The platform structure is shown in Figure 1.
Figure 1: Structure of the biosensing platform.
It comprises of an electrochemical biosensor, low-
noise mix signal portable instrumentation hardware
and associated software incorporating signal
processing algorithms for extraction biotarget
concentration from the biosensor response.
Biosensor Chip
The biosensor is realized as an on-chip package-free
three electrode micro electrochemical cell, which
includes a Pt counter electrode (CE), an Ag/AgCl
reference electrode (RE) and an Au working electrode
(WE) patterned on a single silicon chip as shown in
Figure 2A, Figure 2B and Figure 2C. The WE
represents an array (Figure 2B) consisted of 40 m
diameter recessed gold disks (Figure 2C) arranged in
a hexagonal configuration with 400 m center-to-
center spacing, which were undergone of surface
modification as will be described below. The WE was
placed at the end of the chip to provide its separation
from RE and CE. Such arrangement was
implemented in order to facilitate the bio
functionalization chemistry and prevent both RE and
CE from interference with this process
C B
Figure 2: Photograph of an on-chip package-free three
electrode micro electrochemical cell (A); SEM image of
WE with 40 m microdisk array (B) and SEM image of
recessed gold disk produced by plasma etching of the
silicon nitride isolation layer.
The on-chip micro electrochemical cell was
fabricated at the Central Fabrication Facility at
Tyndall National Institute. The electrodes on the on-
chip microelectrochemical cell were patterned on a Si
substrate of N-Type, <111>-orientation and
fabricated by standard photolithography and lift-off
techniques as described in (Said et al., 2011) and
schematically shown in Figure 3. In brief, firstly, a
silicon oxide layer of 1 μm thickness was thermally
grown on the substrate and alignment marks were
placed on the wafer. Then, the gold working
electrodes, connecting tracks and pads of 150 nm
thick, the platinum counter electrodes of 150 nm
thick and silver reference electrodes of 250 nm thick
were patterned on top of the silicon oxide layer by a
38 mm
WE
RE
CE
10 mm
38 mm
WE
RE
CE
10 mm
A Portable Sensing System for Impedance based Detection of Biotoxin Substances
55
metal lift-off process. In this process 20 nm of Ti was
used to promote the adhesion of the noble metals to
the wafer that in case of Ag was supplemented by 20
nm of Ni. Then, 500 nm of silicon nitride was
deposited on the whole wafer by plasma-enhanced
chemical vapor deposition. The role of the silicon
nitride is to insulate the connecting tracks from the
solution. Openings for the electrodes and the
connecting pads were obtained by plasma etching.
Silver–silver chloride reference electrodes were
prepared with help of chemical oxidation by
immersion of the wafer in a 10 mM FeCl
3
solution for
50 s. The metal was then lifted and a resist layer was
spin coated on top of the wafer to protect the
electrodes during the dicing of the wafer into
individual chips.
Figure 3: Fabrication process flow for the three electrode
on-chip micro electrochemical cell.
Method and Chemicals
All electrochemical experiments were performed in a
Faraday cage, and each measurement used for
obtaining sensor calibration was carried out three
times. EIS measurements were performed by the
described system over a frequency range from 10 Hz
to 100 kHz at an applied potential of DC bias of 0.2
V and an AC amplitude of 10 mV. The frequency
range was defined by hardware specification and
application objective to determine analyte
concentration. The appropriate bias potential was
determined from cyclic voltammetry over a range 0V
to 0.6 V at a scan rate of 100 mV/s. All chemicals
used in this work were purchased from Sigma Aldrich
Ireland Ltd. and utilised as received.
Surface Modification Procedure
The working electrode of the microchemical cell was
undergone of surface modification for attachment of
antibodies capable of receipting the selected biotarget
according to a scheme presented in
Figure 4
.
Figure 4: Surface modification scheme.
This procedure consisted of the following steps:
Electrode Surface Pretreatment. The fabricated
chips were first plasma-cleaned for 10 minutes and
then immersed in HCl:MeOH (1:1, v/v) solution for
15 minutes. They were then sonicated in acetone and
isopropyl alcohol for 5 minutes each; rinsed with
copious amounts of DI water and dry under a stream
of nitrogen.
Silanisation of the WE surfaces was carried out in
3% APTES in MeOH:DI water (19:1) solution for 30
minutes at room temperature. The electrodes were
then rinsed sequentially with MeOH and DI water
before left cure in the oven (dust free) for 15 minutes
at 120°C.
Surface Activation (Cross-Linker Attachment).
Following the curing steps, the silanised electrodes
were immediately immersed in 18 mL of DMF
solution containing 2 mL of 10% pyridine and 0.098
g 1,4-phenylene diisothiocyanate (-PDITC)
(produces 25mM PDITC) for 2 hours. The electrodes
BIODEVICES 2017 - 10th International Conference on Biomedical Electronics and Devices
56
were washed sequentially with DMF and DCE and
dry under N
2
.
Antibody Immobilisation. For this modification,
anti-T2 toxin antibody was diluted in 0.1 M sodium
borate pH 9.3. The working electrodes were
immersed in 100 μL of diluted antibody solution for
2 hours at RT, wrapped in aluminium foil. Then the
electrodes were removed, rinsed with DI water and
dried with N
2
.
Instrumentation
The instrumentation block diagram is shown in
Figure
6
. It represents a two channels EIS system where each
channel contains a signal generator providing in-
phase and quadrature AC signals, transimpedance
amplifier and two (in-phase and quadrature) detection
channels, each consisted of a mixer and a low-pass
filter. These two paths are a sensor channel for the
actual measurement and a reference channel to
compensate for the background signal. The sensor
connected to the reference channel should be not
specific to the target analyte thereby capable of
providing information on the sensor background
signal resulting from varying parameters such as
temperature, pH, nonspecific binding etc. If the
background signal is of negligible value, the
reference channel can be used as the second sensing
channel for another bio target.
A known AC voltage that is generated by the
signal generator is applied across the sensor and the
sensor current is converted to a voltage by the
corresponding transimpedance amplifier. The voltage
is applied across the sensor and the sensor is
amplified and demodulated in the I/Q demodulator
yielding real and imaginary impedance components.
To increase the dynamic range of the module, 2-
bit electronically controlled instrumentation
amplifiers are included in the channel structure. To
facilitate fine module adjustment and calibration,
level shifters on the base of I
2
C controlled DAC are
introduced into the circuit.
The biosensing system hardware is realized
consisted of two modules: Potentiostat and Front-End
Amplifiers (PFEA) and Signal Processing and
Microcontroller (SPM) units.
The PFEA (Figure 5) is designed as a separate
small size unit in order to locate it close to a reservoir
where the biosensor tests the sample solution.
Figure 5: Potentiostat and Front-End Amplifiers module:
photographs of the packaged unit with the sensor chip (A)
and the unit PCB (B).
Figure 6: Instrumentation block diagram.
A
B
A Portable Sensing System for Impedance based Detection of Biotoxin Substances
57
This shortens the length of the connection wires,
which link the biosensor and front-end amplifier,
therefore decreases the leakage current and attenuates
the noise and electromagnetic interference. The
PFEA unit consists of a potentiostat, a temperature
sensor and two transimpedance amplifiers providing
current to voltage transformation of the sensor and
reference (calibration) signals. The potentiosat was
realized on three operational amplifiers OPA2211.
Two identical transimpedance amplifiers were
assembled from OPAMP ADA4647 and
instrumentation amplifier AD8253. The unit was
implemented on a small four-layer PCB of 25 mm x
35 mm dimensions.
The Signal Processing and Microcontroller
(SPM) unit (Figure 7) includes mixing and Analog
Signal Processing (ASP), microcontroller and
peripheral (C) and Power Supply (PS) blocks.
Figure 7: Photograph of Signal Processing and Micro-
controller unit.
The ASP block consists of Input, Switch, Level-
Shifter and four identical Mixer/Low Pass Filter
(LPF) lock-in circuits, which form two identical
analog processing channels. Four-channel 16-bit
digital to analog converters (DAC) incorporated into
the SPM circuit is used to adjust DC levels in the SPM
unit. The block was assembled from AD630 (Mixer),
UAF42 (Low Pass Filter), ADG1419 (Switch),
DAC8574 (DAC with buffered voltage output and
I
2
C compatible two wire serial interface), AD8253
(Instrumental amplifier with 2-bit electronically
controlled gain) and LM4140 (1.024 V voltage
reference).
The C block is based on a high performance 8-
bit ATxmega128A1U microcontroller. It realises
functions of instrumentation control, data acquisition
and communication. It also contains the signal
generator and four 16-bit DACs. The signal generator
is based on AD9854 chip, which is a
DDS synthesizer
capable of parallel generation of precision sine and
cosine signals.
The PS block supplies stabilised low noise
voltages of +8V, -8V, +3.3V and -3.3V to power the
signal generators, microcontroller, ASP and PFEA
system units. The PS block can be powered from a
voltage source in the range of +5V – +12V.
These three blocks formed the main body of the
instrumentation were implemented in three separate 4
layer FR4 PCBs of 51 mm x 90 mm dimensions,
which were integrated in a stack manner taking 55
mm of height as shown in Figure 7.
Signal Processing and Corresponding
Software
The associated with instrumentation software
consists of two highly interrelated parts written for a
host computer and an ATxmega128A1U Atmel
microcontroller. The software ensures
communication between the instrumentation and the
host computer, control of the instrumentation settings
and its operation, implementation of the dedicated
procedure of biosensor impedance spectra
measurement and their signal processing. A smoothed
differential impedance spectrum is used for extraction
an analytical signal that is applied for biosensor
calibration and quantification of the target analyte
concentration. A developed automated procedure for
analytical signal extraction includes the following
steps: smoothing of the initial biosensor impedance
spectrum by Kernel smoothing procedure;
subtraction of a reference background spectrum from
the measured spectra; and finally finding the
maximum of the imaginary component of the
differential impedance spectrum, which is taken as
the analytical signal to be used for biosensor
calibration and calculation of the target concentration
(based on the biosensor calibration and extracted
value of the analytical signal).
The PC software was implemented as user-
friendly multi-tab GUI software where each tab is
associated with the program window contained
means for solutions of assigned tasks. There are three
tabs/windows, namely ‘Protocol’, ‘System
configuration’, and ‘EIS’.
The tab/window ‘Protocol’ allowed for careful
design of frequency sweep measurement protocol for
each frequency point by applying values of frequency
and measurement number with corresponding
adjustment of all electronically controlled hardware
parameters.
BIODEVICES 2017 - 10th International Conference on Biomedical Electronics and Devices
58
Figure 8: Screen short of System configuration PC software tab/window.
The tab/window ‘System configuration’ shown in
Figure 8 provides communication control including
received and transmitted data stream visualisation,
detection and control of communication and stream
errors, data extraction from communication stream,
instrumentation and measurement setting control and
configuration and system operability testing. Tools
for system operability testing include Self-Test
command, which starts Self-test measurement
procedure followed by reporting about the
measurement results in the ‘Test parameters’ plot and
in the ‘Test parameters’ table in ‘Hardware
Configuration’ panel. The program monitors these
parameters before each impedance spectrum
measurements that allows for easy identification of
the hardware and the biosensor operability problem.
3 RESULTS AND DISCUSSION
The tab/window ‘EIS’ is the main instrumentation
software part that is designed to provide all raw
impedance measurements and signal processing
results in different forms as shown in Figure 9. These
include raw sensor impedance measurements
presented by Nyquist (Imaginary impedance part vs.
Real impedance part - ‘Zim vs Zre’) and Bode
(Imaginary impedance part vs. Frequency - ‘Zim vs
F’, Real impedance part vs. Frequency - ‘Zre vs F’
and Phase vs. Frequency - ‘Phase vs F’) plots as well
as the processed Nyquist spectra obtained after
Kernel smoothing and subtraction of the reference
background spectrum from the measured spectra with
analytical signal extraction (see the plot ‘Zim vs Zre
processed’ where the analytical signals extracted are
highlighted by red points on the corresponding
processed impedance spectra) and calculated analyte
concentration in respect to the biosensor calibration
(plot ‘Anal. Signal vs. Concentration’) in both
graphical and table forms.
The experimental results presented in Figure 9
were achieved with T-2 toxin biosensor described
above, which was used for the developed system
validation. Initial and differential raw and smoothed
impedance spectra were obtained for five T2-toxin
concentrations 0, 25, 50, 100 and 250 ppm. In order
A Portable Sensing System for Impedance based Detection of Biotoxin Substances
59
\
Figure 9: Screen short of EIS PC software tab/window.
to provide better distinguishability of the spectra the
measurements were made without repetitions thus
zero measurement errors are shown in the
corresponding table below the “Electrochemical
measurement” panel. As one can see, the initial
impedance spectra were subjected to noise and
application of Kernel’s smoothing allowed for
effective noise suppression. Due to a relatively high
low frequency boundary of the sweep range (10 Hz)
the obtained impedance spectra accounted only for
high and middle frequency parts of a depressed
semicircle response of a microdisc electrode array
associated with semi-infinite radial spherical
diffusion. When toxin concentration increases, real
and imaginary impedance parts both also increase that
reflects a growth of a layer with targeted species
captured by antibodies due the antibody-antigen
binding reaction. If toxin concentration exceeds the
certain level this impedance increase is saturated due
to a depletion of the free antibodies capable of
biorecognition reaction.
As follows from Bode plots the most difference
between spectra corresponding to the different toxin
concentrations is located in a low frequency range
below 250 Hz. Here, the imaginary impedance values
are about one order less than their real counterparts;
they are also more sensitive to variation of T2 toxin
concentration. If frequency increases, both
impedance components decrease but the imaginary
impedance stays relatively constant at the frequencies
below 250 Hz where the influence of the toxin
concentration on the biosensor impedance is the most
noticeable. This frequency behaviour of the
imaginary impedance of the biosensor leads to the
fact that frequency dependence of the impedance
phase represents unimodal smooth function with
maximal values in the range of 56 – 62 degrees
around the frequency of 1 kHz.
The dependence of imaginary impedance against
toxin concentration becomes more apparent if to
study the differential impedance spectra obtained by
subtraction of the spectrum at zero concentration,
BIODEVICES 2017 - 10th International Conference on Biomedical Electronics and Devices
60
which is used as the reference background spectrum,
from the impedance spectra corresponded to nonzero
toxin concentrations. These smoothed differential
impedance spectra are shown in the plot ‘Zim vs Zre
processed’. There are also highlighted by red points
the maximum values of the imaginary impedances on
the corresponding processed impedance spectra.
These values were used as the analytical signals
related to calibration curve of the developed label-
free impedance immunosensor. This curve is shown
in the plot ‘Anal. Signal vs. Concentration’ together
with the analytical signals automatically extracted
from the smoothed differential impedance spectra
presented by the red squares. The calibration curve
represents a nonlinear function that reflects the
competitive nature of the antibody-antigen binding
reaction. Parameters of this calibration function
defined by application of logarithmic regression
analysis are given in Table 1.
Table 1: Calibration parameters of the biosensing system
with T2 toxin biosensor.
Function Slope R
2
Error
y= a·ln(x+b)+c a= 2.412·10
4
0.977 7.144·10
3
Based on these parameters and the extracted values of
analytical signals corresponding T2 concentrations
can be determined as presented in the right table in
the Table panel in Figure 9. As follows from the
calibration curve the developed biosensing portable
system can successfully detect biotoxin at the levels
below 25 ppm.
4 CONCLUSIONS
A portable biosensing platform for impedance based
detection and quantification of biotoxin substances
capable of operation in autonomous mode has been
developed. The platform implements a label-free
approach, which is based on detection of the
biosensor interfacial changes due to a bio recognition
reaction. The interfacial changes are sensed by means
of Electrochemical Impedance Spectroscopy in a
frequency range from 10 Hz to 100 kHz. The platform
comprises of an electrochemical biosensor, portable
low-noise mix signal hardware with embedded
microcontroller and associated software
incorporating hardware and measurement control
together with signal processing algorithms for
extraction biotarget concentration from the biosensor
response. The biosensor is realized as an on-chip
package-free three electrode micro electrochemical
cell consisted of a Pt counter electrode, an Ag/AgCl
reference electrode and an Au working electrode
patterned on a single silicon chip. The WE represents
an array of 40 m diameter gold disks with 400 m
center-to-center distance, which were undergone of
surface modification for antibody immobilisation.
The surface modification was based on APTES –
PDITC procedure. The instrumentation was realised
as a two channel EIS system, which used in-phase and
quadrature demodulation of AC signal for extraction
of the real and imaginary impedance components.
The biosensing system hardware consisted of two
modules: Potentiostat and Front-End Amplifiers
(PFEA) and Signal Processing and Microcontroller
(SPM) units. The corresponding software capable of
autonomous operation contains two interrelated parts
written for the host computer and the embedded
microcontroller. An automated procedure for
analytical signal extraction consists of: smoothing of
the initial biosensor impedance spectrum by Kernel
smoothing procedure; subtraction of a reference
background spectrum from the measured impedance
spectrum; and extraction from differential
impedance spectrum the maximum of the imaginary
component, which is used as an analytical signal for
biosensor calibration and calculation of the target
analyte concentration. The PC software was
implemented as user-friendly multi-tab GUI software
where each tab is associated with the program
window. These are ‘System configuration’,
‘Protocol’ and ‘EIS’ tabs/windows. They provide
communication, instrumentation and measurement
setting control, and system operability testing; allow
for design of impedance measurement protocol for
each frequency sweep point; and present results of
EIS and analyte concentration quantification in
graphical and table forms. The developed biosensing
system was validated with the developed on-chip T2
toxin biosensor. Calibration of the system was
obtained for five toxin concentrations from 0 ppm to
250 ppm. It showed that the developed portable
biosensing system can provide successful detection of
the biotoxin at the levels below 25 ppm.
ACKNOWLEDGEMENTS
Financial support of this work by European
Commission projects FP7-SEC-2011.3.4-2
“HANDHOLD: HANDHeld OLfactory Detector
and H2020-NMP-29-2015 “HISENTS: High level
Integrated SEnsor for NanoToxicity Screening” is
gratefully acknowledged.
A Portable Sensing System for Impedance based Detection of Biotoxin Substances
61
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