sensors

Article
Competitive USB-Powered Hand-Held Potentiostat
for POC Applications: An HRP Detection Case
Yaiza Montes-Cebrián 1, * , Albert Álvarez-Carulla 1 , Gisela Ruiz-Vega 2 ,
Jordi Colomer-Farrarons 1 , Manel Puig-Vidal 1 , Eva Baldrich 2,3 and Pere Ll. Miribel-Català 1
1 Department of Electronics and Biomedical Engineering, Faculty of Physics, Universitat de Barcelona,
08028 Barcelona, Spain; [email protected] (A.Á.-C.); [email protected] (J.C.-F.);
[email protected] (M.P.-V.); [email protected] (P.L.M.-C.)
2 Diagnostic Nanotools Group, Cibbim-Nanomedicine, Vall Hebron Research Institute (VHIR), Universitat
Autònoma de Barcelona, 08035 Barcelona, Spain; [email protected] (G.R.-V.); [email protected] (E.B.)
3 CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 28029 Madrid, Spain
* Correspondence: [email protected]; Tel.: +34-93-4020876




Received: 10 November 2019; Accepted: 3 December 2019; Published: 6 December 2019


Abstract: Considerable efforts are made to develop Point-of-Care (POC) diagnostic tests. POC
devices have the potential to match or surpass conventional systems regarding time, accuracy, and
cost, and they are significantly easier to operate by or close to the patient. This strongly depends
on the availability of miniaturized measurement equipment able to provide a fast and sensitive
response. This paper presents a low-cost, portable, miniaturized USB-powered potentiostat for
electrochemical analysis, which has been designed, fabricated, characterized, and tested against
three forms of high-cost commercial equipment. The portable platform has a final size of 10.5 × 5.8
× 2.5 cm, a weight of 41 g, and an approximate manufacturing cost of $85 USD. It includes three
main components: the power module which generates a stable voltage and a negative supply, the
front-end module that comprises a dual-supply potentiostat, and the back-end module, composed
of a microcontroller unit and a LabVIEW-based graphic user interface, granting plug-and-play
and easy-to-use operation on any computer. The performance of this prototype was evaluated by
detecting chronoamperometrically horseradish peroxidase (HRP), the enzymatic label most widely
used in electrochemical biosensors. As will be shown, the miniaturized platform detected HRP at
concentrations ranging from 0.01 ng·mL−1 to 1 µg·mL−1 , with results comparable to those obtained
with the three commercial electrochemical systems.

Keywords: horseradish peroxidase (HRP) chronoamperometry; electrochemical biosensor;

reconfigurable potentiostat; portable; low-cost electronics; USB-powered

1. Introduction
Traditionally, diagnostic tests are performed at central laboratories equipped with automated
bench-top analyzers that provide highly reproducible and quantitative diagnostic results. Consequently,
patients must often wait for long periods before receiving their test results. This circumstance is
most common in developing countries and rural areas, where the lack of access to basic diagnostic
equipment and trained personnel is an additional challenge [1]. This issue has resulted in an interest
to develop Point-of-Care (POC) testing devices in recent years [2]. POC systems are diagnostic
instruments that provide rapid results geographically near the patient, even when handled by
untrained personnel. Rigorous requirements are set for POC diagnostic systems following the World
Health Organization (WHO) ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid
and Robust, Equipment-free and Delivered to end-users) [3]. According to this standard, POC

Sensors 2019, 19, 5388; doi:10.3390/s19245388 www.mdpi.com/journal/sensors

Sensors 2019, 19, 5388 2 of 13

platforms must deliver quick results for early-stage disease detection to enable rapid intervention and
improve patient quality of life. The provided results must be accurate, reproducible, and optimally
quantitative, and they must be comparable to those obtained by bench-top analyzers at central
laboratories. Furthermore, these POC devices must be inexpensive, handheld, or at least portable and
easy-to-use, making them usable by non-professional personnel.
Many portable POCs are based on electrochemical detection [4–6]. Nowadays, glucose monitoring
POC devices (glucometers) are the most widespread miniaturized test systems. Of the main reasons,
glucose sensors are inexpensive, easy to produce, small, and easy-to-use [7]. Most glucometers are
based on potentiostats, measurement equipment that allow for the studying of oxidation-reduction
(redox) reactions taking place in a test solution or at the electrode surface. In this context, the redox
activity generates a current proportional to the concentration of the electrochemically active molecules
that are being monitored. These systems are suitable in a wide range of applications, such as medical
and health care monitoring [8], environmental measurements [9], or construction and characterization
of portable biosensors [10], among others. There are different approaches to implement such solutions
depending on the requirements of each situation [2], where the budget constraints are one of the
main considerations. For instance, conventional bench-top potentiostats designed for research can
perform a wide variety of electrochemical analysis. However, they are typically complex, expensive,
and tend to be bulky, making them unsuitable for POC applications. In contrast, some research groups
have developed miniaturized potentiostat-based single-chip platforms [11,12]. These devices are very
small, low-powered, and customizable for specific applications, although the fabrication costs are too
elevated for POC implementation.
The use of commercial off-the-shield (COTS) integrated circuits (IC) is an affordable way to
miniaturize instrumentation at minimum cost. Some examples based on potentiostats constructed with
COTS components can be found in the state-of-the-art for a wide range of applications [13–16]. For
instance, a portable system was developed based on a LMP91000EVM potentiostat (Texas Instruments;
Dallas, TX, USA) and a Raspberry Pi 2 Model B microcontroller, which accomplished amperometric
detection of progesterone in undiluted saliva making use of disposable immunosensors [17]. A similar
COTS-based potentiostat was proposed in [18], which included a wireless module to transmit the
measurements to a remote database. The effectiveness of the proposal was assessed by measuring
ascorbic acid and comparing the results with those provided by a commercial potentiostat. Another
example able to transmit data to a PC or via Bluetooth communication is the portable electrochemical
amperometric analyzer proposed in [19]. In this study, the performance and reliability of the platform
were validated using an indium tin oxide glass electrode. The low-cost miniaturized potentiostat
reported in [20] was also based on an LMP91000 evaluation board, this time assembled to a BeagleBone
development board. The system performed cyclic voltammetry (CV) measurements to achieve
electrochemical cortisol immunosensing, obtaining a limit of detection of 1 pM of cortisol with a
sensitivity of 1.24 µM. In a different approach, Muñoz-Martinez et al. [21] described a system capable
of performing electrochemical sensing over system-on-a-chip platforms. To validate the system, they
performed diverse electrochemical experiments and a comparison between the COTS system and a
commercial potentiostat. Alternatively, smartphones have been exploited as a resource for powering
the system, data processing, and Big-Data management [22,23]. One of the most recent advances in
the field is the employment of self-powered platforms based on the use of a fuel cell (FC) acting as
a power source. In this scenario, the same FC may even act simultaneously as a power source and
as a sensor [24]. These solutions use smartphone resources like an audio earphone port [25] or NFC
functionality [26].
The main features required for portable electrochemical instrumentation systems are the following:
to have a small size, low power consumption, high precision measurements and low fabrication, and
maintenance costs. Taking into account these considerations, we have designed a miniaturized, robust,
and customizable USB-based system for amperometric detection. The presented system, dubbed
AmpStat, is composed of three parts: (a) a connector that houses the disposable amperometric sensor,
Sensors 2019, 19, 5388 3 of 13

(b) an embedded electronic system for measurement acquisition, and (c) AmpView, custom software
Sensors
which2019, 19, x FOR
displays andPEER REVIEW
stores the results. The size of the miniaturized platform is 10.5 × 5.8 × 2.5 3 ofcm
15
and it weighs 41 g. The full-custom circuit, which contains the power module and the front-end
module
module, and the front-end
operates module,
a low-voltage operates
condition a low-voltage
of 3.6 condition up
V and only consumes of 3.6 V and
to 235 µA.only consumes
Moreover, up
a single
to 235 µA. has
prototype Moreover, a single prototype
an approximate manufacturinghas ancost
approximate
of $85 USD. manufacturing cost of $85 USD.
Operation
Operation of the proposed system was initially evaluated by
of the proposed system was initially evaluated detecting amperometrically
by detecting amperometrically
horseradish peroxidase (HRP). HRP has a particular commercial
horseradish peroxidase (HRP). HRP has a particular commercial and medical interest in and medical interest in the
the molecular
molecular biology,biotechnology,
biology, medicine, medicine, biotechnology,
and diagnosticand diagnostic
industry fields, industry
as well asfields, as well as broad
broad applicability in life
applicability in life sciences [27]. For instance, HRP is very common
sciences [27]. For instance, HRP is very common in biomedical applications, in which in biomedical applications,
it is used into
which
catalyzeit is used to catalyze
hydrogels [28]. Onhydrogels [28]. On
the other hand, HRPtheand
other hand,variety
a wide HRP and a wide variety
of peroxidase of peroxidase
enzyme mimetics
enzyme mimetics
are extensively are extensively
employed as electrodeemployed
modifiers,as bioreceptors,
electrode modifiers,
and labelsbioreceptors, and labelsand
to produce enzymatic to
produce enzymatic and sandwich electrochemical
sandwich electrochemical biosensors [29–31]. biosensors [29–31].
As
As will
will be
be shown,
shown, thethe USB-powered
USB-powered prototype
prototype developed
developed here here provided
provided current
current measurement
measurement
from 5 nA to 11 µA in real-time and could be adjusted to register currents
from 5 nA to 11 µA in real-time and could be adjusted to register currents up to 3 mA. This up to 3 mA. This allowed
allowed
detecting HRP at concentrations ranging from 0.01 ng·mL −1 to 1 µg·mL−1
−1 −1 using screen-printed carbon
detecting HRP at concentrations ranging from 0.01 ng·mL to 1 µg·mL using screen-printed carbon
electrodes
electrodes (SPCE)
(SPCE) and
and aa ready-to-use
ready-to-use commercial
commercial substrate
substrate solution,
solution, with
with results
results comparable
comparable to to those
those
obtained with three-commercial potentiostats.
obtained with three-commercial potentiostats.

2. Materials
Materials and
and Methods
Methods

Portable Potentiostat
2.1. Portable Potentiostat
The architecture of the POC system is divided into three parts (Figure 1): (a) (a) aa Front-End
Front-End Module
Module
(FEM), which drives the sensor,
sensor, measures
measures the
the current
currentprovided
providedby bythe
thesensor
sensor(I(ISENSE
SENSE)) and translates

the current into voltage (VOUT

OUT),),(b)
(b)aaPower
PowerModule
Module(PM)(PM) that
that generates
generates aa stable
stable voltage
voltage and virtual
ground, creating a negative supply that allows measuring negative signa, and c) a Back-End Module
(BEM), comprised of a microcontroller unit (MCU),(MCU), which
which processes
processes the
the V OUT,, translates
VOUT translates itit into
into a
current and sends the result via USB to the computer, and a graphic user interface (GUI), which
displays the data on the computer. The block diagram of the system is depicted in Figure 1.

Block diagram
Figure 1. Block diagram of
of the
the miniaturized
miniaturized portable potentiostat.
Sensors 2019, 19, 5388 4 of 13

2.1.1. Front-End Module (FEM): Signal Acquisition Module Description

This module performs two tasks: (a) drives the sensor electrodes to the desired voltage (VIN ) and
(b) measures the current provided by the sensor (ISENSE ), translating it into voltage (VOUT ) through a
Transimpedance Amplifier (TIA) to be read by the MCU.
For VIN generation, we use an adjustable voltage reference based on the AD5321 IC (Analog
Devices; Norwood, Massachusetts, USA). This is a 12-bit voltage output Digital-to-Analog Converter
(DAC), which has a typical consumption of 120 µA at 3 V and 0.05 µA in power-down mode. The
DAC is controlled by the MCU through a 2-wire serial interface, compatible with the I2C protocol.
The designed potentiostat is based on a three-electrode topology. Two operational amplifiers
(A1 and A2) polarize the sensor and track VIN to the reference electrode (RE), whose low-input-bias
current avoids voltage distortion. An operational amplifier in transimpedance configuration (A3)
performs the current readout and translates the current registered between the counter (CE) and the
working electrodes (WE) into a voltage signal (VOUT ) by means of a sensing resistor (RTIA ). The system
includes two gain resistors (RTIA1 and RTIA2 ) to measure a wide current range. Two switches (SW1
and SW2 ) connect the suitable resistor, depending on the current range to measure. In this way, if the
system detects that the measurement is out of range, it automatically changes the RTIA to adjust the
measurement circuit to the current range. Two capacitors, CTIA1 and CTIA2 , are connected in parallel
with the gain resistors. Both passive components act as a filter, avoiding noise in the corresponding
measurement range. Finally, a unity gain buffer amplifier (A4), located between the TIA and the MCU,
isolates both parts in order to avoid errors in measurement.
As stated before, the potentiostat circuit translates ISENSE into VOUT through RTIA . Both parameters
are related according to Equation (1),

VOUT = −RTIA · ISENSE (1)

in which VOUT has a negative value. Accordingly, a dual-supply operational amplifier (positive and
negative operation voltage) is required to perform the electrochemical measurement. The operational
amplifier used here is a LPV521 (Texas Instruments, Dallas, TX, USA). This nano-power amplifier
operates from 1.6 V up to 5.5 V with a typical current consumption of 345 nA. It has a typical
offset voltage of 0.1 mV at 1.8 V and a typical bias current of 0.01 pA at 1.8 V. A low-resistance and
low-power consumption switch (ADG702; Analog Devices; Norwood, MA, USA) is used to change the
current range, which has a typical current consumption of 1 nA and a typical on-resistance of 40 Ω.
Moreover, all the resistors used in the system have a 1% tolerance to assure a minimum uncertainty in
the measurement.

2.1.2. Back-End Module (BEM): Processing and Display Module Description

The Back-end module is composed of a MCU and a GUI. The MCU used is the MSP430FR5969
LaunchPad development kit. It is a low-cost evaluation module for rapid prototyping, which is
based on the MSP430FR5969 microcontroller and includes on-board emulation for programming and
debugging. In addition, it is simplified by a 20-pin header, which allows for quick access to General
Purpose Input/Output ports (GPIOs), peripherals for communication, Analog-to-Digital Converters
(ADC), timers, etc. The MCU controls the DAC, which generates VIN signal through an I2 C protocol
and acquires the VOUT from the FEM by means of the ADC. It also activates the suitable switch, which
sets the best RTIA value assuring the maximum measurement range. Furthermore, it processes the
obtained data, translating it into current and sending it to the computer via USB port.
The full-custom GUI, AmpVIEW, is developed using LabVIEW (National Instruments; Austin,
TX, USA), a development software for visual language programming. AmpVIEW controls the system,
including the measurement time and applied voltage and, at the same time, it registers and displays
the chronoamperometry measurement in real-time. Figure 2 summarizes the procedure followed to
perform measurements and a picture of the GUI. Once the program has been turned on, the user must
Sensors 2019, 19, 5388 5 of 13

Sensors
set the2019, 19, x FOR PEER
pretreatment andREVIEW
measurement parameters (time and sensor’s polarization voltage) and5must
of 15

press the “Start Measurement” button. An error message appears on the display when the device has
the device connected, the MCU configures the GPIOs and the ADCs ports, it controls the DAC
not been connected to the computer and the process ends. In the case of having the device connected,
through the I2C protocol and it starts the chronoamperometry. While the FEM drives the sensor, the
the MCU configures the GPIOs and the ADCs ports, it controls the DAC through the I2 C protocol
MCU acquires the VOUT signal and it sends the result via USB to the GUI, which displays the data in
and it starts the chronoamperometry. While the FEM drives the sensor, the MCU acquires the VOUT
real-time. When the measurement is finished, the user can save the data in a file pressing the “Export
signal and it sends the result via USB to the GUI, which displays the data in real-time. When the
Data” button, which generates a csv or xlsx file with the chronoamperometry data. In addition, the
measurement is finished, the user can save the data in a file pressing the “Export Data” button, which
measurement can be aborted at any moment by pressing the “Stop Measurement” button.
generates a csv or xlsx file with the chronoamperometry data. In addition, the measurement can be
aborted at any moment by pressing the “Stop Measurement” button.

Figure 2. (a)
Figure (a)Diagram
Diagramflow
flowofof
thethe
designed program.
designed (b) Chronoamperometry
program. curvecurve
(b) Chronoamperometry measured with
measured
AmpVIEW.
with AmpVIEW.

2.1.3. Power Module

2.1.3. Power Module (PM):
(PM): Power
Power Supply
Supply Module
Module Description
Description
The
The full-custom
full-custom circuit
circuit board
board is is connected
connected toto the
the MCU
MCU board,
board, which
which operates
operates with
with the
the power
power
provided by the computer USB port. The MCU board provides 3.6 V (VMCU) to
provided by the computer USB port. The MCU board provides 3.6 V (VMCU) to the full-custom the full-custom circuit
that contains the FEM and the PM. In order to avoid variations in the power supply,
circuit that contains the FEM and the PM. In order to avoid variations in the power supply, the V MCUthe Vsent
is MCU
to the AP3330 Low Dropout linear Regulator (LDO), which provides an output with
is sent to the AP3330 Low Dropout linear Regulator (LDO), which provides an output with a stable a stable and
regulated voltage
and regulated of 3.6of
voltage V 3.6
(VPM V).(VAs stated in the previous section, it is necessary to have dual supply
PM). As stated in the previous section, it is necessary to have dual
operational amplifiers to measure
supply operational amplifiers to measurethe negative voltage obtained
the negative at the potentiostat’s
voltage obtained output (Voutput
at the potentiostat’s OUT ).

(VOUT).
To generate the negative voltage supply, we created a virtual ground with a low quiescent
current LDO (ADP125 IC; Analog Devices; Norwood, Massachusetts, USA). This provides an output
voltage of 3 V that is set as a virtual ground, by creating a non-symmetrical power supply of 0.6 V
Sensors 2019, 19, 5388 6 of 13

To generate the negative voltage supply, we created a virtual ground with a low quiescent current
LDO (ADP125 IC; Analog Devices; Norwood, Massachusetts, USA). This provides an output voltage
of 3 V that is set as a virtual ground, by creating a non-symmetrical power supply of 0.6 V (+V) and −3
V (−V), allowing a wide negative measurement range. The ADP125 has a quiescent current of 45 µA
with no-load and a maximum quiescent current of 210 µA in case of maximum load (500 mA).

2.2. Electrochemical Measurements

2.2.1. Electrochemical HRP Detection Procedure

The chronoamperometric detection of HRP (Ref. P6782, Sigma Aldrich, San Luis, MO, USA) was
performed in parallel using the full-custom AmpStat potentiostat and three commercial potentiostats.
Disposable screen-printed carbon electrodes (SPCE; Ref. DRP-110; Dropsens; Llanera, Spain) were
employed for this purpose, each one featuring a 4-mm carbon working electrode, a carbon counter
electrode, and a silver pseudo-reference electrode. Before they were used, SPCE were rinsed with
ethanol 70% and water, dried under airflow and characterized by CV in 0.1 M KCl, 1 mM K4 Fe(CN)6 .
For detection, HRP was diluted serially in miliQ water, obtaining eight final HRP concentrations
(0.01, 1, 10, 25, 50, 100, 500, and 1000 ng·mL−1 ) and a negative control without HRP. The measurement
equipment was turned on to register current at 0.00 V vs. the Ag pseudo-reference. Then, the SPCE
was plugged and 45 µL of ready-to-use 3,30 ,5,50 -Tetramethylbenzidine (TMB) Liquid Substrate System
(Sigma Aldrich, Ref. T0440, which contains a mixture of TMB and H2 O2 in a buffer of undisclosed
composition) were pipetted onto the electrodes. Next, 5 µL of HRP were added and the enzymatic
reaction proceeded while the current was registered for 300 s more.

2.2.2. Data Analysis

Data shown in the graphs correspond to the averages of no less than three independent replicates,
each one obtained using a new SPCE, and the error bars show their standard deviation (SD). The assay
limits of detection (LOQ) and quantification (LOQ) were calculated using the average of the blanks
plus 3 and 10 times their SD, respectively. The variability was estimated in terms of the variation
coefficient (%CV = (SD/mean) × 100). The sensitivity corresponded to the slope of the linear range of
the assay and the signal-to-noise ratio (SNR) was the signal registered for each HRP concentration
divided by the averaged signal of the blanks.

3. Results and Discussion

In this work, we report the design, production, and analytical evaluation of a USB-powered
portable potentiostat. The AmpStat prototype was designed to detect amperometrically the activity
of HRP, an enzyme widely used for the detection of electrochemical biosensors, although it could be
easily adapted for other applications.

3.1. Test and Calibration of the Electronic Platform

Figure 3 shows the final device composed by the full-custom Printed Circuit Board (PCB) connected
to the MCU board (Figure 3a) and the control AmpVIEW software (Figure 3b). The whole system
measures 10.5 × 5.8 × 2.5 cm and it weighs 41 g. The PCB, which contains the FEM and the PM, is
connected to the MCU board through a 20-pin header. Since the FEM circuit measures low currents, we
implemented a guard ring used to protect high-impedance circuit nodes from surface leakage currents.
It is a copper ring driven by a low-impedance source to the same voltage as the high impedance node.
We designed and simulated the electronic circuit with Multisim program (National Instruments;
Austin, TX, USA), and we made the PCB layout using Ultiboard software (National Instruments;
Austin, TX, USA). The MCU was programmed with Code Composer Studio (Texas Instruments; Dallas,
TX, USA), an integrated development environment (IDE) to develop applications for Texas Instruments
embedded processors.
Sensors 2019, 19, 5388 7 of 13
Sensors 2019, 19, x FOR PEER REVIEW 8 of 15

Figure 3.
Figure 3. Picture
Pictureof
ofthe
theprototype
prototype developed.
developed. (a)(a) AmpStat
AmpStat potentiostat
potentiostat composed
composed byfull-custom
by the the full-custom
PCB
PCB (copper
(copper board)board)
and theand
MCU the MCU
board board
(red (red
board). (b)board).
AmpVIEW (b) software
AmpVIEW softwareanregistering
registering amperometry an
amperometry
in real time. in real time.

We designed
The electronics and simulated the and
characterization electronic circuit with
a preliminary Multisim
study towardsprogram (National
the analytical Instruments;
validation of the
Austin, TX, USA), and we made the PCB layout using Ultiboard software
platform were carried out with a Source Measurement Unit (SMU) B2962A (Keysight Technology; (National Instruments;
Austin,
Santa TX,CA,
Rosa, USA).
USA) TheandMCU was programmed
an oscilloscope MSO-X 3034Awith Code Composer
(Agilent Studio
Technologies; (Texas
Santa Instruments;
Clara, CA, USA).
Dallas,
This TX, USA),
allowed an integrated
analyzing development
the AmpStat performanceenvironment
and deviation (IDE)by to develop the
measuring applications for Texas
ISENSE current. For
Instruments
this purpose,embedded
the SMU was processors.
connected to the electronics platform and was programmed to apply a
rampThe electronics
of current fromcharacterization
0 µA to 11.2 µAand a preliminary
(maximum currentstudy
thattowards
the system the analytical
can measure) validation
while Iof the
SENSE
platform
was were carried
measured. The maximumout withdeviation
a Sourcebetween
Measurement Unit measured
the current (SMU) B2962A and the(Keysight
current Technology;
applied was
Santa
of Rosa,
4.26%. TheCA, USA) and
AmpStat an oscilloscope
potentiostat, composed MSO-X
by the 3034A (Agilent
PCB and MCUTechnologies;
board, consumes Santa6.45
Clara,
mA CA,at 5
USA).
V, This the
although allowed
PCB, analyzing the AmpStat
which contains the FEM performance
and PM, onlyand deviation
consumes 235 by
µA,measuring
and can workthe Iwith
SENSE

current. For
voltages this
of 3.6 purpose,
V. The the SMU
difference was consumptions
between connected to the electronics
is due to the MCU platform
board,and wascontains
which programmedmany
to apply a ramp
functionalities thatofincrease
currentprototype
from 0 µA to 11.2 µA (maximum
consumption. The developed current
systemthatisthe
ablesystem can measure)
to measure currents
while
up ISENSEµA
to 11.2 was
withmeasured.
resolution The maximum
0.13 deviation
nA. However, it canbetween the current
be configured measured
to measure and up
currents thetocurrent
3 mA.
applied was of
Furthermore, 4.26%.
the system The AmpStat
can polarizepotentiostat, composedtoby
the sensor electrodes the PCB
voltages upand MCU
to 3.6 V. board, consumes
6.45 mA at 5 V, although
Compared the PCB,AmpStat
to other works, which contains the FEMcurrent
had a broader and PM, only and
range consumes 235 µA,
resolution, and still
while can
work with voltages
maintaining comparableof 3.6 operation
V. The difference
voltage between consumptions
[32,33]. The measurement is due to the MCU
deviation board, which
of AmpStAT was
contains
lower thanmany functionalities
the proposed that
by [34], increase
which was prototype
the only workconsumption.
we could find The in developed
which this system is able
parameter wasto
measure currents
discussed. Although upthe
to 11.2 µA withreported
potentiostat resolution 0.13consumes
in [11] nA. However, it can itbeisconfigured
less power, to measure
a fully-integrated chip
currents up
designed in to 3 mA. Furthermore,
a 0.35-µm the system can
bulk-CMOS technology andpolarize the sensor
can measure a more electrodes to voltages
limited current range.up to 3.6
V.
Compared to other works, AmpStat had a broader current range and resolution, while still
maintaining comparable operation voltage [32,33]. The measurement deviation of AmpStAT was
lower than the proposed by [34], which was the only work we could find in which this parameter
 The equipment was tested by detecting HRP concentrations ranging from 0.01 ng·mL−1 to 1000
ng·mL−1, as well as a negative control without HRP. HPR was detected by monitoring its activity
using a commercial ready-to-use substrate solution that contained TMB and H2O2. In this system,
HRP catalyzed the reduction of H2O2 coupled to the oxidation of TMB. The resulting oxidized TMB
was
Sensors then
2019, 19, reduced
5388 at the surface of a SPCE, which registered a reduction current that was proportional 8 of 13
to the amount of HRP present in solution (insert in Figure 4a). The AmpStat prototype was able to
apply voltages up to 3.6 V between the WE and the RE. However, sensor characterization revealed
3.2. System
that theVerification
best potentialby for
HRP thisElectrochemical
application wasDetection
0.00 V vs. the Ag pseudo-reference, and these were the
measurement conditions used here.
The equipment was tested by detecting HRP concentrations ranging from 0.01 ng·mL−1 to 1000
−1
Figure 4 shows an example of chronoamperometric detection for 500 ng·mL−1 of HRP, and the
ng·mL , as well
procedure as a negative
followed control
to perform without
each HRP.
detection. HPRwe
First, was detectedthe
connected by AmpStat
monitoring its activity
to the computer,using
a commercial
executed the ready-to-use substrate
AmpView program, solution
and pressedthat contained
the “Start” buttonTMB and Hthe
to record 2 Ocurrent
2 . In this system,
signal HRP
(Figure
catalyzed
4b (1)).the
Thereduction of H2 Oshowing
software started 2 coupledtheto the oxidation of TMB.
chronoamperometry The resulting
in real-time oxidizeddisplay.
on the computer TMB was
thenThen,
reduced at the surface
we plugged the SPCE of atoSPCE, which registered
the potentiostat (Figure 4ba (2))
reduction current
and pipetted 45 that
µL ofwas
TMB proportional
onto the
to the amount of
electrodes, HRPproduced
which present in solutioncurrent
a transient (insertfluctuation
in Figure 4a). The4bAmpStat
(Figure (3)). We prototype
then addedwas 5 µLable
of to
apply HRP (Figureup
voltages 4b to
(4)),
3.6andV allowed
betweenthe enzymatic
the WE andreaction
the RE.toHowever,
take place sensor
while the current was registered
characterization revealed
for 300
that the bests.potential
Finally, when theapplication
for this measurement ended,
was 0.00 or the the
V vs. “Stop”
Ag button was pressed,and
pseudo-reference, datathese
was saved
were the
in csv or xlsx format.
measurement conditions used here.

Figure
Figure 4. (a)4. Example
(a) Example
of aofchronoamperometry
a chronoamperometryregistered
registered for
for 500 ng·mL−1−1
500 ng·mL ofof
HRP.
HRP.(b)(b)
The procedure
The procedure
followed for current measurement: (1) First, the AmpStat potentiostat was connected viatoUSB
followed for current measurement: (1) First, the AmpStat potentiostat was connected via USB the to
computer, the AmpView software executed, and the “Start” button clicked on;
the computer, the AmpView software executed, and the “Start” button clicked on; (2) the SPCE was(2) the SPCE was
plugged to the connector, and (3) 45 µL of TMB were added; then, to begin the enzymatic reaction,
plugged to the connector, and (3) 45 µL of TMB were added; then, to begin the enzymatic reaction, (4) 5
(4) 5 µL of HRP were pipetted on the electrodes, and the current was registered during 300 s; finally,
µL of HRP were pipetted on the electrodes, and the current was registered during 300 s; finally, the
the “Stop” button was pressed and the data saved.
“Stop” button was pressed and the data saved.

Figure 4 shows an example of chronoamperometric detection for 500 ng·mL−1 of HRP, and the
procedure followed to perform each detection. First, we connected the AmpStat to the computer, executed
the AmpView program, and pressed the “Start” button to record the current signal (Figure 4b(1)). The
software started showing the chronoamperometry in real-time on the computer display. Then, we plugged
the SPCE to the potentiostat (Figure 4b(2)) and pipetted 45 µL of TMB onto the electrodes, which produced
a transient current fluctuation (Figure 4b(3)). We then added 5 µL of HRP (Figure 4b(4)), and allowed the
enzymatic reaction to take place while the current was registered for 300 s. Finally, when the measurement
ended, or the “Stop” button was pressed, data was saved in csv or xlsx format.
Figure 5a shows a histogram summarizing the currents registered over time for increasing
HRP concentrations and employing alternatively the customized AmpStat and the three commercial
potentiostats used in parallel as the reference standards. As can be seen, the equipment generated
Sensors 2019, 19, 5388 9 of 13

comparable results. In general, the enzymatic reaction was fast, and the highest currents were registered
over the first 100 s for HRP concentrations above 25 ng·mL−1 . In contrast, detection of lower HRP
concentrations improved for longer measurement times. Additionally, the fourth equipment displayed
the highest reproducibility at 300 s, when current stabilization had been reached. In all cases, the
currents registered ranged 20–50 nA for the blanks, increased proportionally to HRP concentration
up to 100 ng·mL−1 , and increased to a lesser extent or plateaued for higher HRP contents when the
reaction was substrate-limited (Figure 5b).
Sensors 2019, 19, x FOR PEER REVIEW 11 of 15

(a) Currents
Figure 5. Figure recorded
5. (a) Currents by the
recorded by four potentiostats
the four potentiostatsused forHRP
used for HRPconcentrations
concentrations ranging
ranging from 0from 0
to 1000 ng·mL −1ng·mL
to 1000 afters,100
after−1100 200s,s200
and s and
300300
s ofs of reactionwith
reaction with the
thesubstrate. (b)(b)
substrate. Currents registered
Currents after after
registered
300 s of reaction
300 s of reaction with the with the substrate
substrate (n =(n3).= 3).
Among the three commercial equipment that were studied, the potentiostat-3 was the one that
measured the lowest currents for all HRP concentrations, but also presented the smallest data
Sensors 2019, 19, 5388 10 of 13

Among
Sensors 2019, 19, the
x FORthree
PEERcommercial
REVIEW equipment that were studied, the potentiostat-3 was the one
12 of 15
that measured the lowest currents for all HRP concentrations, but also presented the smallest data
dispersion.
dispersion. On On the the other
other hand,
hand, the
the potentiostat-2
potentiostat-2 registered
registered the
the highest
highest currents
currents and
and the
the most
most stable
stable
and
and reproducible
reproduciblebackground
backgroundnoise
noisein
inthe
theblanks
blanks(see
(seethe
the currents
currentsregistered
registeredover
overtime
timeforforthe
the blanks
blanks
in
in Figure
Figure 5a).
5a). Interestingly,
Interestingly,the
theAmpStat
AmpStatportable
portable system
system developed
developed here
here measured
measured similar
similar currents
currents
and
and comparable
comparable result result dispersion
dispersion than
than the
the potentiostat-2
potentiostat-2 (%CV ranging of 4.7–21% in both cases).
Nevertheless,
Nevertheless,the theAmpStat
AmpStat displayed
displayedmore variable
more blank
variable measures.
blank This This
measures. negatively affected
negatively the assay
affected the
SNR,
assaywhich was calculated
SNR, which by using
was calculated by the blank
using the values (Figure(Figure
blank values 6). 6).

Figure6.6.SNR
Figure SNRof
ofthe
thefour
fourpotentiostats
potentiostatsfor
forHRP
HRPconcentrations
concentrationsranging
rangingfrom
from0.1
0.1toto1000
1000ng mL−1−1.
ngmL

Table
Table11summarizes
summarizesthe thefigures
figuresof ofmerit
meritcalculated
calculatedforfor the
the HRP
HRP detection
detection assay,
assay,carried
carriedout
out with
with
the four electrochemical systems. The four potentiostats displayed comparable
the four electrochemical systems. The four potentiostats displayed comparable LOD and LOQ LOD and LOQ in terms
in
of minimal
terms current measurable
of minimal and quantifiable,
current measurable respectively.respectively.
and quantifiable, However, inHowever,
terms of HRP concentration,
in terms of HRP
both LOD and LOQ
concentration, both were
LODhigher
and LOQ(andwere
thus worse)
higher if HRP
(and wasworse)
thus measured using
if HRP themeasured
was potentiostat-3.
usingThethe
customized AmpStat potentiostat designed here exhibited an LOD of 0.83 ng·mL −1 and an LOQ of 1.52
potentiostat-3. The customized AmpStat potentiostat designed here exhibited an LOD of 0.83 ng·mL−1
ng·mL −1 of HRP, which were values only slightly higher than those provided by the potentiostat-1
and an LOQ of 1.52 ng·mL−1 of HRP, which were values only slightly higher than those provided by
and
the potentiostat-1 and(0.52–0.56
the potentiostat-2 ng·mL−1 and
the potentiostat-2 1.16–1.61
(0.52–0.56 ng·mL
ng·mL
−1 , respectively). Additionally, assay
−1 and 1.16–1.61 ng·mL−1, respectively).
sensitivity, calculated as the slope of the corresponding assay linear range (spanningassay
from 0.01 ng·mL −1
Additionally, assay sensitivity, calculated as the slope of the corresponding linear range
to 50 ng·mLfrom−1 ), was comparable for 50
all three
(spanning 0.01 ng·mL−1 to ng·mLequipment. These parameters
−1), was comparable for all were
threereasonably
equipment. accurate,
These
taking into account that the AmpStat is a homemade prototype with an approximate
parameters were reasonably accurate, taking into account that the AmpStat is a homemade prototype manufacturing
cost
withofan$85 USD.
approximate manufacturing cost of $85 USD.

Table 1. Comparison of the figures of merit of the HRP detection assay, carried out with the four
Table 1. Comparison of the figures of merit of the HRP detection assay, carried out with the four
tested potentiostats.
tested potentiostats.
AmpStat Potentiostat-1 Potentiostat-2 Poteniostat-3
AmpStat Potentiostat-1 Potentiostat-2 Poteniostat-3
LOD
LOD
Current (µA) 0.05 0.05 0.04 0.04
Current (µA) −1 ) 0.05 0.05 0.04 0.04
Concentration (ng·mL−1 0.83 0.52 0.56 1.27
Concentration (ng·mL ) 0.83 0.52 0.56 1.27
LOQ
LOQ
Current (µA) 0.07 0.08 0.07 0.06
Current (µA)
Concentration (ng·mL−1 ) 0.07
1.52 0.08
1.16 0.07
1.61 0.06
2.89
Concentration (ng·mL−1) 1.52 1.16 1.61 2.89
Sensitivity
0.0328 0.0326 0.0305 0.0219
(ng·mL−1·µA−1)
Weight (g) 41 1600 480 5433
10.5 × 5.8 × 22.2 × 20.5 × 13.2 × 10.0 × 360.7 × 233.7 ×
Dimensions (cm)
2.5 7.5 3.6 116.9
Cost (USD) 85 11,013 4087 16,000
Sensors 2019, 19, 5388 11 of 13

Table 1. Cont.

AmpStat Potentiostat-1 Potentiostat-2 Poteniostat-3

Sensitivity
0.0328 0.0326 0.0305 0.0219
(ng·mL−1 ·µA−1 )
Weight (g) 41 1600 480 5433
Dimensions (cm) 10.5 × 5.8 × 2.5 22.2 × 20.5 × 7.5 13.2 × 10.0 × 3.6 360.7 × 233.7 × 116.9
Cost (USD) 85 11,013 4087 16,000

4. Conclusions
In this paper, we demonstrated experimentally the performance of AmpStat, a full-custom low-cost
potentiostat. We described in detail the design, production, and preliminary analytical evaluation
of this portable high-performance prototype, which is entirely powered and controlled by USB and
includes a user-friendly interface that makes it plug-and-play and easy-to-use.
The potentiostat is composed of three blocks: the PM, which generates a stable voltage and a
virtual ground, the potentiostat-based FEM, and the BEM, which includes an MCU and the GUI.
Furthermore, we developed a full-custom software, called AmpVIEW, which controls the system,
presents the chronoamperometry in real-time on a computer display, and allows saving the data in
different file formats. The low-cost platform consumes 6.45 mA at 3.6 V. However, the full-custom
designed PCB, which contains the instrumentation, only consumes 235 µA. Furthermore, the developed
potentiostat can drive the sensor electrodes to voltages up to 3.6 V and measure currents up to 11.2 µA,
although it can be adjusted to measure a maximum current of 3 mA.
We confirmed the efficacy of the AmpStat prototype by detecting HRP in a concentration range
from 0.01 ng·mL−1 to 1 µg·mL−1 . As has been shown, the results obtained with the AmpStat were
comparable to those obtained using three commercial electrochemical systems that were significantly
more expensive. Our equipment displayed LOD of 0.83 ng·mL−1 , LOQ of 1.52 ng·mL−1 , and sensitivity
of 0.0305 µA·mL·ng−1 .
Furthermore, this platform could be additionally optimized in the future to produce a tailored
compact system. For instance, size reduction could be achieved by creating a full-custom board
with an integrated microcontroller unit. The software could also be upgraded to perform current
averaging within defined measurement time ranges and/or interpolation in preloaded calibration plots.
Also, the system could integrate additional functionalities based on mobile resources. For example,
the incorporation of a Bluetooth module to connect the system to a smartphone and a rechargeable
battery would facilitate the employment in POC testing, resulting in a more compact and flexible
electrochemical detection solution.

Author Contributions: Y.M.-C. developed, implemented and tested the Point-of-Care (POC) electronic device
and A.Á.-C. developed the acquisition software. Y.M.-C. and G.R.-V. did the experimental study and analyzed
and interpreted the acquired data. Y.M.-C. wrote the manuscript and approved the final version of the manuscript.
Besides, P.L.M.-C., E.B., M.P.-V. and J.C.-F. supervised the development of the device. P.L.M.-C., J.C.-F., M.P.-V.,
G.R.-V. and A.Á.-C. have discussed the resultant data, contributed in the manuscript and approved the final
version for its publication.
Funding: This research was funded by the Spanish Ministry of Economy, Agencia Estatal de Investigación, Fondo
de Investigaciones Sanitarias of Instituto de Salud Carlos III (ISCIII) and Fondo Europeo de Desarrollo Regional
(AEI/FEDER, UE), through projects TEC2016-78284-C3-3-R and DTS17/00145. EB is funded by a Miguel Servet II
contract from ISCIII-FEDER (CPII18/00025). GR is supported by a VHIR predoctoral fellowship funded by Amics
del VHIR. Diagnostic Nanotools is a Consolidated Group supported by the Secretaria d’Universitats i Recerca of
Generalitat de Catalunya (Grant 2017 SGR 240).
Conflicts of Interest: The authors declare no conflict of interest.
Sensors 2019, 19, 5388 12 of 13

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