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A low-cost miniaturized potentiostat for pointof-care diagnosis

ARTICLE in BIOSENSORS & BIOELECTRONICS JUNE 2014
Impact Factor: 6.45 DOI: 10.1016/j.bios.2014.06.053 Source: PubMed

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Biosensors and Bioelectronics 62 (2014) 249254

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

A low-cost miniaturized potentiostat for point-of-care diagnosis

Andres Felipe Diaz Cruz a, Nicolas Norena a, Ajeet Kaushik b, Shekhar Bhansali a,n
a

BioMEMS Microsystems Laboratory, Electrical and Computer Engineering, Florida International Laboratory, Miami, FL 33174, USA
Center of Personalized Nanomedicine, Institute of Neuroimmune Pharmacology, Department of Immunology, Herbert Wertheim College of Medicine,
Florida International University, Miami, FL 33199, USA

art ic l e i nf o

a b s t r a c t

Article history:
Received 16 March 2014
Received in revised form
4 June 2014
Accepted 16 June 2014
Available online 30 June 2014

This paper presents a novel approach of using a miniaturized potentiostat (M-P) chip (LMP91000)
to perform full range cyclic voltammetry (CV) measurements for the detection of biomarkers. The
LMP91000 evaluation board was recongured to perform three-electrode CV measurements in order to
achieve electrochemical cortisol immunosensing. The microelectrodes for cortisol estimation were
fabricated by immobilizing monoclonal anti-cortisol antibody (Anti-M-Cab) onto self-assembled monolayer (SAM) modied Au microelectrodes. The results obtained using the M-P were compared to those
obtained using a conventional potentiostat. The M-P was successful in measuring cortisol levels in the
range of pM. The outcomes of the studies suggest that M-P can effectively perform biochemical
measurements on three electrode systems, enabling the development of miniature systems for pointof-care (POC) diagnosis.
& 2014 Published by Elsevier B.V.

Keywords:
Miniaturized potentiostat
Point-of-care
Personalized health monitoring/diagnostic
Electrochemical immunosensing
Cortisol

1. Introduction
Portable miniaturized analytical devices for disease detection
at early stages and for monitoring physiological variables at pointof-care (POC) can be useful to personalize health diagnostics for
appropriate effectual treatment (Ahn et al., 2004; Choi et al., 2011;
Gubala et al., 2011; Justino et al., 2013; Kaushik et al., 2014;
Kemmler et al., 2014; Kost, 1995; Kumar et al., 2013; Loncaric et al.,
2012; Luppa et al., 2011; Rusling et al., 2010; Soper et al., 2006;
Tudos et al., 2001; Wang, 2006). POC diagnostic tools are increasingly being developed for quantifying biomarkers and trends
based on time and age. These miniaturized biomedical devices
are also being explored to provide fundamental information that
could form the basis of health informatics and superior treatment
strategies (Ahn et al., 2004; Luppa et al., 2011; Soper et al., 2006).
The existing diagnostic systems are limited to laboratory
facilities and are not suitable for POC due to their high costs, the
necessity of trained personnel, portability, and long waiting to
obtain results (Kaushik et al., 2014). The development of novel
methodologies for adequate diagnosis at early stage for healthcare
monitoring at POC requires a new class of systems. Such systems
must be capable of interacting directly with biological samples in
order to retrieve the desired information regarding the patients
metabolism effortlessly and in real-time (Gubala et al., 2011;
Rusling et al., 2010).
n

Corresponding author. Tel.: 1 305 348 2807.

E-mail address: sbhansa@u.edu (S. Bhansali).

http://dx.doi.org/10.1016/j.bios.2014.06.053
0956-5663/& 2014 Published by Elsevier B.V.

Electrochemical biosensors have recently been employed for

selective, specic and rapid detection of biomarkers within physiological ranges. The sensing performance of such electrochemical biosensors is found to be dependent on the selection of
transducers and immobilizing electro-active matrix e.g. nanostructures (Arya et al., 2012; Kaushik et al., 2014; Solanki et al.,
2011). The introduction of nanomaterials as immobilization
matrices lead to increased sensitivity, linearity, and detection at
pico-molar (pM) level (Arya et al., 2012; Huey et al., 2012; Solanki
et al., 2011). Electrochemical biosensors enable miniaturization
through the use of advanced interdigitated micro-electrodes (IDEs)
congurations that enhance sensing performance and reduce form
factors (Pasha et al., 2014; Vasudev et al., 2013). IDEs have been
used to detect analyte biomarkers in the pM range (Shalev et al.,
2013), and also to enhance the sensitivity of the measurements.
IDE-based electrochemical sensors, having a series of parallel
micro-band electrodes with alternating micro-bands connected
together are useful for rapid reaction kinetics, improved sensitivity, large electrode aspect ratio (w/l) and increased signal-to-noise
ratio (Arya et al., 2010a). These IDEs can be congured as twoelectrode or three-electrode systems, depending on the utilized
transduction technique. For example, in electrochemical impedance sensing, two-electrode based IDE systems attain faster
steady-state current response to enable easier measurements
(Cohen and Kunz, 2000; Iwasaki and Morita, 1995; Morita et al.,
1997; Shalev et al., 2013; Yao and Zhu, 2014). Electrochemical
biosensors integrated in microuidic systems have been explored
for biomarker detection in order to decrease the probability of
human error and the sample volume required. However, such

250

A.F.D. Cruz et al. / Biosensors and Bioelectronics 62 (2014) 249254

systems have limited use at POC detection systems for targets

beyond glucose and estrogen (Kim et al., 2012). The area of
miniaturized electronics integrated with electrochemical biosensors has great signicance for in-eld and on-site diagnostics
(Huey et al., 2012; Maurer et al., 2006; Park et al., 2002; Poh et al.,
2010; Warren et al., 2002; Yao and Zhu, 2014). The junction of
laboratory bio-sensing protocols and miniaturized electronics can
be explored for mass production with great prospects of
commercialization.
To explore a POC electrochemical sensor for a specic target
requires an immobilization matrix compatible to the target analyte, and an electronic setup that provides control on the measurements and records, correlates, stores and displays data (Blanco
et al., 2006; Huang et al., 2007; Steinberg and Lowe, 2004). The
potentiostat is capable of controlling the electrical signals within
the electrodes in two, three or four-electrode systems to allow the
user to observe multiple electrochemical phenomena. Conventional potentiostats are capable of performing several electrochemical measurements such as chronoamperometry, impedance
spectroscopy and CV. However, available potentiostats are costly,
bulky, and not practical for POC applications.
In this paper, we present a low-cost and miniaturized potentiostat (M-P) capable of performing CV for electrochemical immunosensing of cortisol. An open source microcontroller unit, along
with a micro-power electrochemical sensing integrated circuit has
been congured to develop a M-P. This system is capable of
performing three-electrode system based electrochemical measurements (Fig. 1) in which the potential is scanned in both
positive and negative directions at predened sweep rates (mV/s)
for oxidation and reduction reactions (Blanco et al., 2006; Kwakye
and Baeumner, 2007; Steinberg and Lowe, 2004). For cortisol
sensing, an immunosensor is fabricated via immobilizing monoclonal Anti-M-Cab onto SAM modied IDEs (Arya et al., 2010a,b;
Pasha et al., 2014; Vasudev et al., 2013).
Cortisol, a steroid hormone, is an important biomarker for
psychological stress. Thus cortisol detection is gaining prominence
for personalized health monitoring (Kaushik et al., 2014; Singh

et al., 2014). The obtained sensing parameters using M-P are

within the physiological range and are comparable to those
obtained using conventional potentiostats.

2. Materials and methods

2.1. Materials
The LMP91000EVM and the BeagleBone microcontroller were
purchased from Texas Instruments. Dithiobis (succinimidyl propionate) (DTSP) and sodium borohydride (NaBH4) were purchased
from ThermoFisher Scientic. Anti-M-Cab 23304809 was procured from Sigma Aldrich. Phosphate buffered saline (PBS) tablets
and hydrocortisone (cortisol) were purchased from Sigma Aldrich.
All other chemicals were of analytical grade and were used
without further purication. PBS solution (10 mM, pH 7.4) was
prepared by dissolving 1 tablet in 200 mL of deionized water.
Working solutions of hydrocortisone were prepared by dilution in
PBS (10 mM, pH 7.4).
2.2. Electrochemical biosensor fabrication to detect cortisol
The interdigitated electrodes (IDEs) were fabricated on an
oxidized 4 in. silicon wafer using conventional microfabrication
process. First, the electrodes were patterned using UV photolithography. Next, Cr (20 nm) and Au (150 nm) were deposited using an
E-beam evaporator. The microelectrode patterns were nally
obtained on the silicon wafer by a liftoff process.
Phosphate buffer saline (PBS) solution (10 mM, pH 7.4) was
used to prepare the Anti-M-Cab (1 mg/mL) and cortisol solutions.
Prior to functionalization, the electrodes were immersed in freshly
prepared piranha solution to clean the surface. The electrodes
were next immersed in 2 mg/mL solution of DTSP in acetone for
2 h for SAM formation. DTSP was reduced using NaBH4 (10 mg/mL
in DI water). The DTSP/Au electrodes were then rinsed with
acetone and water to remove any unbound DTSP molecules.

Current (A)

Electrochemical Response

Potential (V)

Micro-controller

I2C BUS

SPI BUS

Miniaturized
Potentiostat

Cortisol Sample

Buffer

Anti-M-Cab Cortisol

Fluid Valve
Control

Assay Chamber
SAM Head Group
SAM Functional
Group
IDE/Cr/Si

Waste
Fig. 1. The diagram above presents a high level representation of the cortisol detection device based on a miniaturized potentiostat (M-P). The system uses a DTSP-SAM
based electrochemical cortisol immunosensor, which is integrated to the system through a LTCC microuidic manifold. The electrochemical sensing is performed using the
miniaturized potentiostat, which parameters are congured and controlled by the micro-controller unit.

A.F.D. Cruz et al. / Biosensors and Bioelectronics 62 (2014) 249254

Anti-M-Cab (10 mL) was immobilized on to DTSP/IDE electrode for

2 h followed by carefully washing with PBS (pH 7.4) to remove any
unbound biomolecules. Anti-M-Cab binds covalently (amide bond)
via a facile reaction between an amino group of antibody and a
reactive succinimidyl group of the DTSP SAM surface. Ethanol
amine (10 mL) was immobilized onto Anti-M-Cab/DTSP-SAM/IDE
bio-electrode for 10 min to block unreacted succinimidyl group on
DTSP-SAM. The fabricated bio-electrodes were stored in a refrigerator at 4C when not in use. CV was utilized to characterize the
stepwise fabrication of the bio-electrodes using our designed M-P
and an Autolab Potentiostat/Galvanostat (Eco Chemie, Netherlands). The validation and characterization of our device was
achieved using 5 mL of PBS solution (pH 7.4) containing 5 mM
Fe(CN)63 /4 as a redox probe, in a potential range from  0.6 to
0.6 V at 50 mV/s.
2.3. Design and development of potentiostat
The LMP91000 analog front end can be programmed to perform electrochemical measurements using two-electrode and
three-electrode systems. During the rst attempts of performing
CV measurements through the LMP91000, an external CPU was
used to congure the system through its USB communication
feature. This was accomplished by installing the Sensor AFE
Designer software in a CPU, which allowed the user to interface
with the evaluation board through an external data capture board.
Although most of the parameters were congurable through the
sensor AFE designer software, a CV measurement with a constant
sampling rate was not achievable, since the differential voltage
between the working and reference electrodes could only be
adjusted manually. Also, the utilization of the CPU increased
signicantly the size and the cost of the nal device.
To address these issues, a BeagleBone microcontroller unit was
added to the design due to its adaptability and portability. In this
conguration the LMP91000EVM was powered and controlled by
the microcontroller via its I2C interface. The LMP91000EVM is by
default congured to perform gas-sensing measurements on twoelectrode setups. Therefore, it needed to be recongured to
produce three-lead CV measurements for a portable potentiostat
system. This was achieved by removing the two-wire jumper pin,
which enables the utilization of the working, counter and reference electrodes. Also, the J_MENB jumper was connected to enable
the manual operation mode, which allowed the LMP91000's
registers to be manually set up to perform the measurements
desired (Fig. 2). The LMP91000EVM generates an output voltage
proportional to the current owing through the working electrode.
This voltage is provided by the transimpedance amplier (TIA) at
the output stage, which converts the current in the working
electrode to a voltage in a directly proportional rate (Fig. 3). The
acquisition of the signal response of the potentiostat is sent back
to the microcontroller through the Serial Peripheral Interface (SPI)

2-wire
Lead Gas

2-wire jumper
open

LMP
Pin 2
Pin 3
Pin 19
Pin 20

J-MENB
Manual
J-MENB
SHORT PIN

ADC IC

Pin 1
Pin 5
Pin 3

Fig. 2. The 2-wire jumper short was removed to allow three lead electrochemical
measurements. The J_MENB jumper short was moved to the far left to enable
manual conguration of the LMP 91000 chip. The ADC takes the analog output from
the potentiostat chip and transfers it to the microcontroller unit via SPI.

251

of the ADC161S626 Analog to digital converter, which is embedded

on the LMP91000EVM (Fig. 2).
As previously mentioned, the LMP91000EVM was set up in
manual operation mode, which allows the user to set the function
parameters depending on the type of electrochemical measurement desired. In our case, the TIA Control (TIACN) registers were
congured to assign a value of 35 k and the RLOAD register to
10 . These registers are related with two different parameters,
the TIA control increases or decrease the gain of the transimpedance amplier and the RLOAD register sets the value of the
resistor that bridges the transimpedance amplier and the working electrode. This resistor is used to adjust the value of the
current drawn from the sensor to the TIA during the measurements. Moreover, the reference control register (REFCN) permits
the conguration of the internal zero, bias source, and reference
source. The reference source was xed to be internal, so no
external supply was necessary. The default value for this voltage
is 2.5 V. The internal zero parameter was assigned to be 67% of the
reference voltage, which set the output voltage of the board to be
1.67 V when there is no electrochemical activity in the sensor
connected to the three-electrode system. The BIAS_SIGN register
was preset to switch automatically from negative to positive and
from positive to negative in order to obtain a full range CV. Finally,
the BIAS register was controlled by a routine that changed the
value of the differential voltage between the working and the
reference electrode according to the values assigned to the REFCN
parameters. By programming the LMP91000EVM with the specications previously mentioned, we were able to perform CV with
variable sampling rates in the potential range from  0.6 V to
0.6 V. As previously mentioned, the LMP91000EVM outputs a
voltage proportional to the cell current. The value of this current
can be found by plugging the output voltage of the LMP91000EVM
in the transimpedance amplier transfer function equation, taking
into consideration the parameters set for the CV measurement
using the equation Iout = (Vout VINT _ Z )/TIAgain .
2.4. System testing and validation
Once the operational parameters of the potentiostat circuit
were enabled through the microcontroller unit to achieve CV, the
device was tested to validate its functionality. It was observed that
the device effectively performed full range CV measurements on
three-electrode set-ups. The data obtained was stored in the
internal memory of the microcontroller, where it was analyzed
and correlated to a certain analyte's concentration level. The
device was designed in such way that the data obtained was also
exported via a Secure Shell (SSH) as a text le to be plotted in a
host computer using a Matlab routine (Fig. 4).
For validation, CV studies of IDEs were performed in PBS (pH
7.4) containing 5 mM Fe(II)/Fe(III) redox moieties using a conventional laboratory potentiostat and the M-P presented in this
document (Fig. 5). For consistency, the scan rate (50 mV/s) and
the detection limits ( 0.6 r Vwe  re r0.6) were congured to be
the same in both experiments. The resulting plots were compared,
and it was found that both CV curves exhibit well-dened
oxidation and reduction peaks of the redox moieties with identical
magnitude of current response (5% variation in magnitude of
current response). M-P based results of IDE's electrochemical
response studies were found to be repeatable and reproducible
with 2% current variation (data not shown). It was also observed
that in several tests the cell current was enough to drive the
output of the transimpedance amplier to the biasing voltage of
the potentiostat circuit, which caused clapping in the peaks of the
plots obtained. In order to avoid such saturation in the output of
the potentiostat circuit, the transimpedance gain was set to 35 k,

252

A.F.D. Cruz et al. / Biosensors and Bioelectronics 62 (2014) 249254

VDO

VREF

SCL

LMP91000
3- Lead
Electrochemical
Cell
CE

CE

A1 -

VREF
DIVIDER

VARIABLE
BIAS

I2C INTERFACE
AND
CONTROL
REGISTERS

SDA

CONTROLLER

MENB

RE

WE

RE

TEMP SENSOR

DGNO

R LOAD
WE

VOUT

+
TLA

REFCN
REF_SOURCE Internal
INT_ZERO 67%
BIAS_SIGN +/ - sweep

TIA 35 K

RLOAD 10

RTIA
C1

C2

AGNO

Fig. 3. LMP91000 Schematic. The parameters highlighted show the modications made to the conguration of the LMP91000 in order to achieve cyclic voltammetry
measurements on three electrode systems. The I2C interface of the LMP91000 was used to enable the communication with the micro-controller unit.

Three-Electrode
System

Matlab program
coded to plot multiple
measurement to be
analyzed

Full range CV
Measurement

Measurement date
stores in
microcontroller SD
card

Through SSH (secure

shell) the data is sent
to a computer as a
text file

BeagleBones &
LMP 9100EVM

Resulting
Plots

Matlab program
coded to plot the CV
curves
Computer

Fig. 4. The block diagram above shows the process implemented for the testing and validation of the system designed. Through the Beaglebone microcontroller, the
LMP91000EVM was congured to perform cyclic voltammetry measurements on three-electrode systems. The data obtained is stored in the SD card of the Beaglebone, it is
also transferred via SSH to a host computer where it can be plotted for further analysis.

which was sufcient to perform all the measurements required in

the development of our device.
2.5. Electrochemical performance, validation, and cortisol sensing
studies
To optimize the stepwise fabrication of the electrochemical
immunosensor, M-P based CV studies of (a) DTSP-SAM/IDE, (b)
Anti-M-Cab/DTSP-SAM/IDE and (c) EA/Anti-M-Cab/DTSP-SAM/IDE
in 5 mL PBS (pH 7.4) containing 5 mM Fe(II)/Fe(III) at 50 mV/s in a
potential range from  0.6 to 0.6 V are shown in Fig. 5B. The
electrochemical response of DTSP-SAM/IDE (curve b) is lower than
that of IDE (curve a) due to inhibited electron transport caused by
SAM formation via thiol bonding between DTSP-SAM and Au of
IDEs. The immobilization of Anti-M-Cab due to covalent binding
between Anti-M-Cab and DTSP via amide bond formation onto
DTSP-SAM/IDE further decreases the electrochemical response
current of Anti-M-Cab/DTSP-SAM/IDE immunoelectrode (curve
c). At the nal stage of blocking of non-binding sites of immunoelectrode, the magnitude of electrochemical current was observed
lower than that of Anti-M-Cab/DTSP-SAM/IDE immunoelectrode
(curve d). This is due to the non-conducting nature of EA, which
reveals successful immobilization blocks of the non-binding sites
on the immunoelectrode. Consequently, there is a reduction of

electron transport from electrolyte to IDEs. The obtained results

related to the fabrication of the immunosensor are similar to
those obtained in our previous studies and successfully validated
the performance of our developed M-P for electrochemical
measurements.
The M-P was used to measure electrochemical sensing
response studies of EA/Anti-M-Cab/DTSP-SAM/IDE immunosensor
as a function cortisol concentration (10 pM to 500 nM) in 5 mL PBS
(pH 7.4) containing 5 mM [Fe(CN)6]3  /4  with a potential range of
 0.6 to 0.6 V at a scan rate of 50 mV/s. 30 minutes were used as
the incubation time for optimum binding of each cortisol concentration (5 mL) on the sensor surface followed by washing with PBS
to remove unbound molecules prior to each measurement in
triplet set. It was observed that the magnitude of the electrochemical response decreases upon adding cortisol due to the
formation of insulating immuno-complex between Anti-M-Cab
and cortisol, which further hinders the electron transport from
electrolyte to IDEs (S1). A linear relation was observed between
the magnitude of the current response and logarithm of cortisol
concentration which obeys a linear equation [I response current (A)
1.95  10  5 (A) 1.24  10  6 log[cortisol concentration (M)] A;
R0.989]. EA/Anti-M-Cab/DTSP-SAM/IDE immunosensor exhibited a linearity range of 10 pM to 500 nM, a lowest detection
concentration of 1 pM, a sensitivity of 1.24 mA/(M) with the

A.F.D. Cruz et al. / Biosensors and Bioelectronics 62 (2014) 249254

high afnity between Anti-M-Cab and cortisol. The sensing parameters such as selectivity and stability of EA/Anti-M-Cab/DTSPSAM/IDE have been discussed in our previous report (Pasha et al.,
2014; Vasudev et al., 2013). In brief, CV studies related to the
selectivity of EA/Anti-M-Cab/DTSP-SAM/IDE immunoelectrode
with respect to interferents such as PSA (100 pM), NSE (100 pM),
EGFR (100 pM), and BSA cortisol (100 pM) with respect to
cortisol (100 pM). Our developed cortisol immunosensor exhibits
signicant electrochemical change when adding cortisol (  15%)
and minimum change on mentioned interferents (12%).

-5

1.5x10

a = CV curve of IDE (Autolab)

-5

1.0x10

b = CV curve of IDE (M-P)

Current(A)

-6

5.0x10

0.0
-6

-5.0x10

-5

3. Conclusion

-1.0x10

-5

-1.5x10

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

Potential(V)
-5

1.0x10

a = IDE-Au electrode
b = SAM-DTSP/IDE
c = Anti-M-Cab/DSTP-SAM/IDE
d = EA/Anti M-Cab/SAM-DTSP/IDE

a
c
d

-6

5.0x10

Current (A)

253

0.0

We have customized a miniaturized LMP91000EVM potentiostat as a transducer to develop a portable healthcare monitoring
device. The operational voltage and scan rate was optimized for CV
measurements using a Fe(II)/Fe(III) redox probe. An electrochemical immunosensor based on Anti-M-Cab immobilized over SAM
modied IDEs was successfully interfaced with our M-P to detect
cortisol within the physiological range. The miniaturized electrochemical sensing system demonstrates the system capability to
detect cortisol at 1 pM with a linearity of 10 pM to 500 nM and
with a sensitivity of 1.24 mM. The obtained results are comparable
to conventional electrochemical analyzers and suggest that this
miniaturized, low cost and portable biosensor prototype can be
used to detect biomarkers at POC.

-6

-5.0x10

Acknowledgments

-5

-1.0x10

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

Potential (V)

This research work was partially supported by NSF-NERC

(1160483). Authors acknowledge AMERI@FIU for research facilities. Authors also thank to Dr. Abhay Vasudev (Engineer@Intel) for
scientic discussion and help in work progress.

-5

1.95x10

Appendix A. Supplementary information

Lowest detection limit = 1 pM
Linearity range = 10 pM - 500 nM
Sensitvity = 1.24 A/M
Regression Coefficient = 0.989
S.D. = 0.44A

-5

Current (A)

1.80x10

-5

1.65x10

Supplementary data associated with this article can be found in

the online version at http://dx.doi.org/10.1016/j.bios.2014.06.053.

-5

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1.50x10
1.35x10

-5

1.20x10

-5

1.05x10

E-11

E-10

E-09

E-08

E-07

5E-6

Log [Cortisol Concentration (M)]

Fig. 5. (A) Comparison of CV studies obtained of Au-IDE using Autolab (a) and M-P
(b) in 5 mL PBS (pH 7.4) containing 5 mM [Fe(CV)6]3  /4  redox moieties at 50 mV/
s. The obtained curves were identical and reveal that the system design matches
the performance of a conventional potentiostat. (B) CV studies obtained of
(a) IDE-Au electrode, (b) DTSP-SAM modied IDE (c), Anti-M-Cab/DTSP-SAM/IDE
electrode and (d) EA/Anti-M-Cab/DTSP-SAM/IDE immunoelectrode in 5 mL PBS (pH
7.4) containing 5 mM [Fe(CV)6]3  /4  as redox probe at 50 mV/s using M-P,
(C) Linear calibration curve plotted between the magnitude of response current
and cortisol concentration (10 pM to 500 nM) obtained using M-P.

regression coefcient of 0.989 and standard deviation of 0.44 A

using M-B potentiostat. The association contact was estimated as
5  1012 L mol  1 using LineweaverBurke-like plot demonstrates

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