Journal Pone 0263738

PLOS ONE
RESEARCH ARTICLE
TBISTAT: An open-source, wireless portable,

electrochemical impedance spectroscopy
capable potentiostat for the point-of-care
detection of S100B in plasma samples
Francisco Burgos-Flórez1,2☯, Alexander Rodrı́guez1☯, Eliana Cervera1,
Valtencir Zucolotto3, Marco Sanjuán2, Pedro J. Villalba ID1*
1 Department of Medicine, Biotechnology Research Group, Universidad del Norte, Barranquilla, Colombia,
2 Department of Mechanical Engineering, Rational Use of Energy and Preservation of the Environment
a1111111111 Group (UREMA), Universidad del Norte, Barranquilla, Colombia, 3 GNano–Nanomedicine and
a1111111111 Nanotoxicology Group, São Carlos Institute of Physics, University of São Paulo, São Carlos, São Paulo,
a1111111111 Brazil
a1111111111
a1111111111 ☯ These authors contributed equally to this work.
* [email protected]
Abstract
OPEN ACCESS
Citation: Burgos-Flórez F, Rodrı́guez A, Cervera E, Point-of-Care (POC) testing for biomarker detection demands techniques that are easy to
Zucolotto V, Sanjuán M, Villalba PJ (2022) use, readily available, low-cost, and with rapid response times. This paper describes the
TBISTAT: An open-source, wireless portable, development of a fully open-source, modular, wireless, battery-powered, smartphone-con-
electrochemical impedance spectroscopy capable
trolled, low-cost potentiostat capable of conducting electrochemical impedance spectros-
potentiostat for the point-of-care detection of
S100B in plasma samples. PLoS ONE 17(2): copy for the electrochemical detection of the S100B protein captured in an ANTI-S100B
e0263738. https://doi.org/10.1371/journal. functionalized thin-film gold interdigitated electrode platform to support traumatic brain injury
pone.0263738 diagnosis and treatment. EIS results from the developed potentiostat were validated with a
Editor: Kalisadhan Mukherjee, Pandit Deendayal commercial benchtop potentiostat by comparing impedance magnitude and phase values
Petroleum University, India, INDIA along the EIS frequency range. In addition, an experimental design was performed for
Received: August 24, 2021 detecting S100B in spiked human plasma samples with S100B concentrations of clinical util-
Accepted: January 25, 2022 ity, and a calibration curve was found for quantifying S100B detection. No statistically signifi-
cant differences were found between EIS results from the developed potentiostat and the
Published: February 7, 2022
commercial potentiostat. Statistically significant differences in the changes in charge trans-
Copyright: © 2022 Burgos-Flórez et al. This is an
fer resistance signal between each tested S100B concentration (p < 0.05) were found, with
open access article distributed under the terms of
the Creative Commons Attribution License, which a limit of detection of 35.73 pg/mL. The modularity of the proposed potentiostat allows easier
permits unrestricted use, distribution, and component changes according to the application demands in power, frequency excitation
reproduction in any medium, provided the original ranges, wireless communication protocol, signal amplification and transduction, precision,
author and source are credited.
and sampling frequency of ADC, among others, when compared to state-of-the-art open-
Data Availability Statement: All relevant data are source EIS potentiostats. In addition, the use of minimal, easy acquirable open-source hard-
within the paper and its Supporting Information
files.
ware and software, high-level filtering, accurate ADC, Fast Fourier Transform with low spec-
tral leakage, wireless communication, and the simple user interface provides a framework
Funding: This work was funded by Departamento
Administrativo de Ciencia, Tecnologı́a e Innovación
for facilitating EIS analysis and developing new affordable instrumentation for POC biosen-
(COLCIENCIAS) grant number 757–2016 (FB) and sors integrated systems.
by Gobernación del Atlántico call number 809–
2018 (AR). The funders had no role in study
PLOS ONE | https://doi.org/10.1371/journal.pone.0263738 February 7, 2022 1 / 25

PLOS ONE TBISTAT: Portable potentiostat for the detection of S100B
design, data collection and analysis, decision to Introduction

publish, or preparation of the manuscript.
Traumatic brain injury (TBI) is a brain dysfunction produced by an external force, usually due
Competing interests: The authors have declared
to a sudden movement or blow to the head. Delays in the clinical identification of neurological
that no competing interests exist.
impairment during the acute phase of TBI lead to higher mortality among patients [1]. This
delay could often be related to the subjective and qualitative nature of the Glasgow Coma Scale
(GCS), a technique routinely used as a strategy to classify the TBI severity [2]. In addition to
the GCS, other invasive and non-invasive neuromonitoring techniques help to establish crite-
ria for medical decisions. However, they require expertise and advanced medical skills and
demand high costs for healthcare systems, limiting their availability in many resource-con-
strained environments, as in low-middle income countries, where the clinical examination is,
in many cases, the only accessible tool for neuromonitoring [3].
S100B, a calcium-binding dimeric protein (MW: 21 KDa) expressed in astrocytes and
found in very low levels under physiological conditions in serum/plasma, has been previously
suggested as a potential biomarker for TBI [4, 5]. Following a TBI, S100B is released from dam-
aged nerve cells into the bloodstream by passing through the blood-brain barrier (BBB), which
could be disrupted after head primary injury [6]. The clinical significance of S100B depends
on the type and severity of the brain damage [7]. Various cutoff values of S100B have been pro-
posed for identifying brain injury [8]. A cutoff level of 100pg/mL [9] has been used in the mild
TBI to discard the presence of intracranial hemorrhages in the CT scans, and values closer to
30 pg/mL have been reported as an indicator of BBB permeability even with no associated
symptoms [10]. Likewise, patients with moderate to severe TBI could display higher serum/
plasmatic S100B levels in the order of ng/mL, which correlates to intracranial hypertension,
neurological worsening, and poor response to treatment [7]. In the clinical context, the mea-
surement of biomarkers such as S100B demands a technique that is easy to use, readily avail-
able, low-cost, and with a rapid response time, ideally at the Point-of-Care (POC).
The advances in material science, nano-fabrication, molecular biology, and immunology
have supported the continuous generation of new POC electrochemical biosensors capable of
detecting a myriad of biological substances of interest in medicine [11–15]. Among electro-
chemical detection techniques, electrochemical impedance spectroscopy (EIS) has been one of
the most widely used for impedance measurements due to its minor system interference, fast
response, and accurate, reliable results [16]. EIS has been effectively used for affinity biosensors
[17–21] since it can monitor events such as antigen-antibody binding (Ag-Ab) that occur on
the surface of the electrodes, where small changes in impedance are proportional to the con-
centration of the measured antigen.
To fulfill the objectives of POC technology, the development of portable, precise, minimal
sample preparation requirements, high-quality and cost-effective instrumentation, and tech-
niques that allow the detection of the analytes such as S100B are essential [22]. Researchers in
need of performing precise analytical EIS tests for biosensing applications often employ a bench-
top potentiostat, a highly-priced (more than USD 10,000), heavy, patented, commercial device.
Current electronics technology has enabled simplifying the large, heavy, and expensive benchtop
potentiostats. Several companies, such as Gamry, PalmSens, Ivium, and Metrohm, have devel-
oped portable variants of their benchtops with similar specifications but with prices still above
USD 1,000. As explained by Dryden et al. [23], commercial potentiostats function as black boxes,
as they give limited information about their hardware and software technical specifications,
which can make the development of new measurement techniques and integration with other
instruments challenging for researchers, educators, and for further product integration [24]. In
addition, current potentiostat prices still prohibit their widespread use, specifically in resource-
limited settings. Still, most efforts in the literature have been devoted to biosensor

Table 1. Feature comparison of literature reviewed potentiostats with the proposed device.
Reference EIS 2–3 Electrode Android Independent Wireless Windows/ Linux Fully documented open-source
Systems integration Impedance circuit Integration hardware and software
Punter-Villagrasa ✓ ✓ × × × ✓ ×
(2014) [28]
Zhang (2016) [27] ✓ ✓ ✓ × ✓ × ×
Pruna (2017) [26] ✓ × × × × ✓ ×
Ainla (2018) UWED × ✓ × × ✓ × ×
[24]
Jiang (2019) [12] ✓ ✓ × ✓ × ✓ ×
Jenkins (2019) ✓ ✓ ✓ × ✓ × ✓
ABE-Stat [22]
Proposed device ✓ ✓ ✓ ✓ ✓ × ✓
✓Feature is present on the potentiostat. × Feature is not present on the potentiostat.
https://doi.org/10.1371/journal.pone.0263738.t001
miniaturization, increased sensibility, and reproducibility, whereas commercial potentiostats

have remained expensive, requiring time-consuming tasks for adequate configuration [25].
Various research groups have embraced the task of developing low-cost EIS-capable portable
potentiostats which can be available to the general public for their replication and adaptation to
their own needs (Table 1). To our knowledge, potentiostats made by Jenkins et al. [22], Jiang et al.
[12], Pruna et al. [26], and Zhang et al. [27] are the only ones whose functionality allows EIS.
Even though considerable details have been given for the above prototypes, only Jenkin’s et al.
[22] ABE-Stat provides wireless smartphone-controlled electrochemical detection and fully docu-
mented open-source hardware and software. A portable potentiostat with wireless connectivity to
smartphones would facilitate electrochemical analysis at the POC and in emergencies at remote
locations, where access to a computer or wired connection to a device is unlikely, such as the case
of S100B detection soon after TBI occurs. In addition, technology developed in an open-source
format allows researchers to quickly adopt the device’s design and develop specific-purpose
potentiostats without the need to design and test the electronics from scratch [24].
It should be noted that the only current truly open-source potentiostat capable of perform-
ing EIS is the one developed by Jenkins et al. [22]. However, as the authors indicate, there are
still drawbacks in the precision and generation of reliable results during electrochemical
impedance measurements. First, the presence of discontinuities at characteristic frequencies (2
Hz, 60 Hz, and 2 kHz) is likely due to the way ABE-stat calculates impedance and instabilities
associated with the control amplifier during small-signal measurements. Authors also describe
the need for a more robust microcontroller unit (MCU) that provides a higher number of gen-
eral-purpose input-output ports (GPIOs) and a less noise-prone Bluetooth module (BM), ide-
ally a Bluetooth low energy one (BLE). In addition, the use of the AD5933 as impedance
analyzer with a 1024-points single-frequency 16 MHz clock-dependent Discrete Fourier
Transform (DFT) causes higher noise and lower precision in EIS analyses, given its nonlinear
nature, and prevents accurate impedance determination at frequencies below 1 kHz. Further-
more, higher errors due to spectral leakage are found if evaluated frequencies do not corre-
spond to integer numbers of cycles over the sampled period. The authors also express the
inability of the device to generate coherent results for nonlinear non-resistive loads, which is
critical when making Nyquist plots of electrochemical systems, considering their capacitive
nature. Therefore, the definition of circular regions of the said diagram is significantly affected,
as greater inaccuracies in charge transfer resistance (RCT) values might show up. RCT
depends on the change in the capacitance of the bio-functionalized surface of the working

electrode, mainly given by the number of antigen-antibody bonds formed after the sample is
applied. Therefore, increasing the device’s precision for its use in affinity biosensors such as
the one employed in this work is necessary.
For the above reasons, this paper describes the development of a fully open-source modular,
low-cost, portable potentiostat capable of performing EIS for the electrochemical detection
and quantification of the S100B protein biomarker captured in a two-electrode platform,
referred to from now on as TBISTAT. The overall design approach is inspired by the compo-
nent selection, layout, and firmware design of Ainla [24] and Jenkins [22]. However, the design
has been made modular to allow easy component changes according to the application
demands in power, frequency excitation ranges, wireless communication protocol, signal
amplification and transduction, precision, and sampling frequency of analog digital conver-
sion (ADC), among others. TBISTAT features an independent impedance analyzer circuit,
using the AD5933 as only a signal generator with a programmable external clock, and can be
interfaced with both classic Bluetooth and BLE modules. In addition, the true 12-bit inbuilt
ADC of its MCU and active hardware and digital filter reduces current variance during ADC.
TBISTAT is also controlled by an Android smartphone application that performs a 1000-point
real Fast Fourier Transform (FFT) of a non-integer number of cycles over sample periods to
achieve low-spectral leakage transformation for logarithmic frequency points.
The TBISTAT functionality is assessed and validated by conducting EIS on three experi-
mental systems: An AUTOLAB dummy cell circuit composed of a 100 O resistor in series with
the parallel circuit of a 1μF ceramic capacitor and a 1 KO resistor; Bare thin-film gold interdig-
itated electrodes (AUIDEs) drop casted with 10uL of 10mM K3[Fe(CN)6] in 0.2M KCl solu-
tion; ANTI-S100B functionalized thin-film AUIDEs exposed to 316 pg/ml of S100B spiked
human plasma samples, and drop casted with 10uL of 10mM K3[Fe(CN)6] in 0.2M KCl as
support solution. EIS results from the developed potentiostat were validated with an Autolab/
M204 benchtop potentiostat by comparing impedance magnitude and phase values along the
EIS frequency range and through a T-test that compared means of changes in capacitance val-
ues (ΔC) using single frequency analysis (SFA). In addition, an experimental design was per-
formed for detecting S100B in spiked human plasma samples with S100B concentrations of
clinical utility (31pg/mL, 100 pg/mL, and 316pg/mL) using the change in charge transfer resis-
tance (ΔRCT) as the response variable. A one-way Welch ANOVA was applied to check for
differences between treatment groups (S100B concentrations), followed by Games-Howell as a
post-hoc test. A regression model (calibration curve) was developed together with a lack of fit
test to determine model adequacy to the response variable.
The use of minimal, modular, easy acquirable open-source hardware and software, together
with high-level filtering, low-cost, accurate ADC, smartphone-executed FFT, adequate analog/
digital ground/power planes, wireless communication, and a simple user interface, makes the
TBISTAT a framework for facilitating EIS analysis for POC applications such as S100B detec-
tion and provides new opportunities for the development of affordable diagnostics, sensors,
and wearable devices. To our knowledge, this is the first portable potentiostat entirely built for
measuring S100B concentration on plasma samples using EIS. The open-source nature of this
work encourages users to optimize this design for their purposes and needs.
Materials and methods

Ethics statement
The study was conducted according to the guidelines of the Declaration of Helsinki, and
approved by the Ethics Committee of UNIVERSIDAD DEL NORTE, Barranquilla-Colombia
(act number 167, approved on 25-01-2018).

Electrodes and reagents

AUIDEs with 180 pairs of interdigitated gold electrodes (5/5 μm, electrode/gap) were obtained
from Micrux Technologies, Spain. Bovine Serum albumin (BSA), 1-ethyl-3-(3-dimethylami-
nopropyl) carbodiimide (EDC), phosphate-buffered saline (PBS), 30% Hydrogen Peroxide,
30% Ammonium Hydroxide, and potassium ferrocyanide (III) powder <10um, 99% (702587)
were obtained from Sigma-Aldrich (US). Cysteamine >98.0% (30070) was purchased from
Sigma-Aldrich (Brazil). Potassium chloride (P217500) ACS 99.0 a 100% was obtained from
Fisher (US). All reagents used were of analytical grade. Deionized water (DW) (MiliQ1) was
employed to prepare all solutions. Recombinant Anti-S100B antibody [EP1576Y]—Astrocyte
Marker and Recombinant Human S100B protein (ab55570) were acquired from Abcam (US).
Biosensor construction
AUIDEs biosensor construction comprised three consecutive processes: cleaning, self-assem-
bled monolayer (SAM) formation, and surface functionalization (Fig 1). AUIDEs were first
thoroughly cleaned following RCA-1 cleaning protocol by immersion in a 5:1:1 DW, 27%
NH4OH, 30% H2O2 solution for one minute, and then generously rinsed with DW and dried
using N₂. An EIS essay was performed in potassium ferrocyanide to assess the cleaning effi-
ciency of AUIDEs working surface, which were again rinsed with DW and dried using N₂. For
the SAM formation, ten microliters of 0.5M cysteamine in PBS were drop-casted on the
AUIDEs WE surface and left covered at room temperature (RT) for one hour on a rotary
machine to improve Au-cysteamine molecular interactions. AUIDEs were then rinsed with
DW and dried using N₂. Subsequently, a solution of 0.5 M EDC in 10mM PBS (pH 7.4) with
anti-S100B at 50ug/ml was prepared and vortex mixed every 15 minutes during two hours at
RT for anti-S100B carboxyl group activation. The zero-length cross-linking functionalization
of anti-S100B on cysteamine-modified AUIDEs was performed by drop-casting ten microliters
of the EDC-anti-S100B solution on the AUIDE WEs surface, which were then left covered at
RT for 12 hours on a rotary machine for anti-S100B conjugation to the Au-cysteamine SAM.
Electrodes were then carefully rinsed with DW and dried using N₂. An EIS scan was then per-
formed with potassium ferrocyanide to characterize the electrochemical behavior of the func-
tionalized WEs. AUIDEs WEs were then blocked with 10 microliters of 0.5% BSA in 10 mM
PBS and left covered at RT overnight on a rotary machine. Electrodes were then thoroughly
Fig 1. Graphical representation of the covalent immobilization of anti-S100B onto AUIDEs using a cysteamine/EDC approach.
https://doi.org/10.1371/journal.pone.0263738.g001

rinsed with DW and dried using N₂ at RT, and an EIS was done in potassium ferrocyanide to
set a single reference RCT baseline for each AUIDE. Small reagent concentrations were used
in this work to reduce manufacturing costs. In addition, to avoid undesirable fixation of
reagents to the recipient walls, low retention tips and tubes were used to dilute, aliquot, and
drop-casting antibodies and crosslinker molecules during functionalization and further S100B
tests.
Preparation of spiked human plasma samples

Human whole blood was obtained from a venipuncture of a healthy donor with previous writ-
ten consent under the Universidad del Norte (Barranquilla, Colombia) ethics committee No.
167. Whole blood, collected to Gel & EDTA K2 tubes (Improvacuter1 722350202), was then
centrifuged for 30min at 10,000g to remove proteins with molecular weight (MW) over 30
KDa. Human plasma was extracted by pipetting and aliquoting in low retention tubes. Each
aliquot was spiked with corresponding amounts of the S100B protein to obtain aliquots of 31,
100 and 316 pg/mL and, after resuspended, stored at -20˚C in low retention tubes. Each spiked
human plasma sample was thawed 15 min before the test and resuspended to be drop-casted
on the AUIDE WE surface.
High-level representation of potentiostat system

Fig 2 shows a general representation of the system. The proposed design features a lithium-ion
polymer battery (LiPo) with a boost circuit that powers a Teensy LC MCU and an HC06 Blue-
tooth 2.0+EDR (B2.0) module. The MCU powers a 3.3 V digital-analog front-end potentiostat
circuit (PC), which interfaces with an Android application running on a smartphone (or a tab-
let) using the BM connected to the MCU. The smartphone provides a user interface to wire-
lessly connect with the MCU via Bluetooth, calibrate the device impedance measurements,
perform EIS on the electrochemical two-electrode biosensor platform, and display the imped-
ance measurements in a real-time Nyquist plot as EIS experimental runs take place on the bio-
sensor platform. During an EIS measurement, the PC excites the electrochemical cell, which in
turn generates an electrochemical output current (EOC) which is transduced and amplified
using a transimpedance amplifier (TIA) and acquired with the Teensy LC ADC. The Teensy
LC MCU sends digitalized acquisitions to the smartphone app via Bluetooth, and a real-time
FFT of the incoming data is performed in the Android application, which results in amplitude
and phase data for each frequency point in the EIS frequency spectrum. The Nyquist plot is
constructed in real-time as each FFT is done during the excitation of the electrochemical bio-
sensor system. The S100B concentration is obtained by calculating the ΔRCT from the EIS
using a regression model which relates ΔRCT to the S100B concentration. Once EIS is com-
pleted, the Android application shows the value of S100B concentration and information
regarding possible brain injury related to the found S100B concentration.
Hardware design
LiPo battery boost, charger, protection circuit module. A 3.7 V, 1000 mAh lithium-ion
polymer battery (LiPo) was used as a power supply for the BM and MCU. The PC is powered
by a 3.3 V low-dropout regulator (LDO) inherent to the MCU. Considering that the MCU and
BM require a 5 V voltage supply and that LiPo batteries are rechargeable and have a significant
risk of igniting if a short circuit happens, a boost, USB charger, short-circuit protection circuit
was employed to fulfill these purposes. Additional details are found in S1 File.
Wireless communication with Bluetooth module. The TBISTAT communicates with a
smartphone or a tablet using either the wireless BLE protocol or B2.0. A host program in the

Fig 2. General representation of the system components.

smartphone receives the input parameters of the experiment from the user, communicates the
experimental protocol to the TBISTAT, receives the raw data (ADC) of the EIS measurement,
and visualizes it for the user in a real-time Nyquist plot. The modules HC06 and HM-10 were
used for performing wireless communication between TBISTAT and a smartphone.
Communication between BMs and MCU is done using the universal asynchronous
receiver/transmitter (UART) interface, which is a block of circuitry responsible for imple-
menting serial communication using two channels, one for transmission (Tx) and one for
reception (Rx). Both modules permit serial transfer as high as 115200 Bauds. Additional details
about the BMs technical specifications are found in S1 File.
MCU. A Teensy LC MCU was employed as the control unit for the developed potentio-
stat. This MCU features an ARM Cortex-M0+ processor at 48 MHz, 62K Flash, 8K RAM,
12-bit analog input & output, hardware Serial, 400 KHz I2C, USB, and a total of 27 Input-out-
put (IO) pins. Teensy LC flash memory programming is achieved by the Teensy Loader appli-
cation, which is compatible with the popular open-source Arduino IDE. Hence, code can be
written and compiled in Arduino and automatically uploaded using the Teensy loader applica-
tion, requiring only a USB connection between the MCU and a computer.

Fig 3. Potentiostat circuit with TIA circuit. Analog switches (MAX4641 and ADG704) are controlled by Teensy LC (not shown) and isolate the
electrochemical cell. The TIA gain is controlled by one of four resistors selected by the ADG704 switch.
Teensy LC processor speed and 12-bit ADC allows interrupt-driven ADC with sampling
rates as high as 200 KHz, greatly surpassing the sampling criterion defined in the Nyquist–
Shannon sampling theorem for a 10 KHz signal [29]. In addition, the presence of multiple Dig-
ital IOs and fast UART allows controlling multiple analog switches and fast serial transfer. The
8K RAM allows allocation of up to 2000 unsigned 16-bits integers for each EIS frequency mea-
surement point for sufficient FFT frequency resolution [30]. I2C communication is employed
for controlling the SI5351 clock generator IC and the direct digital synthesizer (DDS) AD5933
IC. The former capabilities are not fully met by Ainla et al. RFDUINO MCU [24] or Jenkins
et al. ESP12S MCU [22] without including additional ICs (like peripheral ADC IC or addition
I/O ports for ESP12S, or MCU change for higher processor speeds (RFDUINO)) which
increase manufacturing costs and device size. The circuit schematics and design description of
the MCU module board are found in S1 File.
Digital-analog front-end potentiostat circuit. Fig 3 shows an overall circuit schematic of
the PC. The digital-analog front-end circuit comprises three modules: The AC excitation sig-
nal design module made up of the clock generator IC, the DDS, and the signal conditioning
circuit; The PC module made up of analog switch ICs, TIA circuit, and signal filtering circuit;
and the mixed-signal grounding module.
The generation of an AC signal from DC voltage sources relies on the adequate use of a
DDS IC. Similar to previous studies [22, 27], The TBISTAT employs the AD5933 network ana-
lyzer IC, which consists of a 27-bit direct digital synthesis (DDS) sine excitation voltage

generator, a digital-to-analog converter (DAC), and a programmable gain amplifier (PGA)

which determines the AC signal amplitude in four possible ranges [31].
The AD5933 can produce 1KHz-100KHz AC signals without any external components. For
the excitation signal frequency to go below 1 kHz, the clock that drives the AD5933 must be
scaled down. Similar to the implementation made by Zhang et al. [27], this work uses the
S5351A IC as a clock generator connected to the external clock pin on the AD5933, which
allows an excitation bandwidth increase between 1 Hz - 100Khz.
During EIS, electrochemical systems are commonly perturbed with AC excitation voltages
of about 5 to 10 mVp to preserve the linear behavior of the EOC [32]. Potentials as small as 2
mVp and as large as 20 mVp have been employed [33]. The AD5933 can produce AC signals
with four preprogrammed AC amplitudes and DC-bias voltages which, if not corrected, could
induce polarization across the electrochemical cell under test and significantly alter the imped-
ance response of the system by inducing a DC component in the EOC [31]. Therefore, a high
pass filter/amplitude reduction circuit was designed to cancel the DC component and achieve
a 10 mVp, 3.3/2 VDC, biased excitation signal. This circuit induces a DC bias of 3.3/2 V to pro-
vide a reference DC voltage via a simple voltage divider circuit, also known as virtual ground
voltage (VGV). This feature allows the AC signal component to swing around the middle of
the 0–3.3V range for a unipolar power supply (0 to 3.3V). An operational amplifier (OAMP)
was used as a buffer of the filtered signal and for low output impedance (less than 1 O). In this
way, using the above circuit, only one OAMP is needed to attain filtering, amplitude reduction,
VGV, and low output impedance of the excitation signal. To isolate high-frequency harmonics
from wireless communication and the AD5933 MCLK itself, a 100 KHz two-pole SKLPF was
implemented in a second OAMP circuit.
Two high-speed, low-voltage analog switches (MAX4641 IC) are employed and controlled
by the EIS digital control signal coming from the MCU for isolating the electrochemical cell
counter, reference, and working electrodes. During an EIS measurement, EIS output is set
high, which closes the MAX4641 analog switches, and the AC excitation signal perturbs the
electrochemical cell. Even though the AD5933 IC is capable of performing transduction of the
EOC through an embedded TIA and executing a DFT itself for the obtention of phase and
magnitude values of the analyzed impedance, previous works have noted the presence of sig-
nificant errors arising from DC and AC spectral leakage given by the limitations of the
AD5933 impedance analyzer implementation [31, 34–37]. Among them, discontinuities in the
test phasor, DC and AC leakage, and overall inability to measure impedance at low-frequency
signals (less than 2 KHz) for nonlinear non-resistive loads have been reported, which are cru-
cial since they usually correspond to circular portions of Nyquist plots in many electrochemi-
cal systems. AD5933 performs a single-point DFT, meaning that the analysis or correlation
frequency in its core is always at the same frequency as the current output excitation fre-
quency. If the input signal period over the 1024-point sample interval is an integer, there will
be a smooth transition from the end of one period to the beginning of the next one. However,
if this is not met, there will not be a smooth transition between beginning and end, and spec-
tral leakage will ensue. As explained by Matsiev et al. [37], the errors reported in the literature
are erroneously attributed to spectral leakage and could, instead, be the result of the disconti-
nuity in the test phasor, which induces DC and AC leakage. The conventional calibration pro-
duces a single multiplicative gain factor that leads to substantial errors and undue
disappointment in the device performance [36], especially at the lower end of the operating
frequency range, which could explain the unreliability of Jenkins et al. ABE-Stat at frequencies
below 2 KHz [22].
For the above reasons, this work discards using the AD5933 impedance analyzer circuit
and proposes that the EOC is transduced by an electrometer grade amplifier (LMP7721 IC),

whose output voltage amplitude is controlled by one of four resistors selected by the analog
multiplexer ADG704 IC. Similar to the high-pass filter circuit used in the AC excitation signal
design module, a 3.3/2 V VGV is fixed on the positive input of the TIA via a simple voltage
divider circuit. This circuit ensures low DC bias between the input excitation signal and output
voltage of the TIA, which is a sufficient condition for EIS of the ANTI-S100B functionalized
AUIDEs to detect S100B, as seen in Rodriguez et al. [38]. The TIA voltage signal passes
through a fourth-order 10 KHz SKLPF for high-frequency noise filtering using an LMP7702
OAMP IC. The cutoff frequency is determined following the EIS measurement specifications
established by Rodrı́guez et al. [38] for S100B detection, and the ADC conversion is performed
using the MCU ADC. Additional details about the PC design are found in S1 File.
Software design
An Android application was developed to interface with the MCU firmware over either B2.0
or BLE protocols. The used libraries have GNU General Public License v3, which permits
unrestricted use for software development.
Using the firmware developed for RFDUINO by Ainla et al. [24] as a reference, the Teensy
LC main loop is equipped to listen, interpret, and execute the commands sent by the smart-
phone in the string format. The MCU serial module receives serial commands in the above
format from the Android application for setting AD5933 external clock frequency, number of
FFT points to acquire, TIA gain resistance, DDS word for AC excitation, sampling frequency,
and initializing EIS scans.
The user interface of the Android application comprises only two buttons (connect and
start), a checkbox for calibration, and a text input space for selecting calibration resistance
value. The user initially presses the connect button to establish a Bluetooth connection
between the smartphone and the BM connected to the MCU. When the start button is pressed,
the smartphone configures MCU for EIS and starts measuring each frequency point. If the Cal-
ibration checkbox is checked before pressing start, the user must input a number correspond-
ing to the calibration resistance to be used. Then, the smartphone performs calibration of
system phase and magnitude for each EIS frequency for the defined calibration resistance.
When data coming from BM is received, an FFT is performed. If the user is performing cali-
bration, the magnitude and phase are stored on an excel file. If the user is performing a con-
ventional measurement to detect the S100B biomarker, a Nyquist plot is displayed in real-time
as FFT on each EOC received data is performed. Once EIS is finished, ΔRCT of the EIS is cal-
culated for finding S100B concentration, which is shown together with TBI diagnosis support
information. Additional details of the software implementation of the EIS measurements are
discussed in S2 File, while source code for MCU firmware and Android Studio application
files can be found in S3 File.
S100B regression model and TBI support information

There is no easy way to obtain RCT from a Nyquist plot since it is difficult to define an electro-
chemical model that accurately fits the impedance data. Only proprietary software for this pur-
pose is found in the literature, which would restrict TBISTAT use. Hence, a simple linear
regression model of the lowest three frequency points of the Nyquist plot is performed to
obtain an approximate RCT at y = 0. The calibration model of ΔRCT vs S100B using AUIDEs
is used for determining sample S100B concentration. The value of the S100B concentration is
then shown together with TBI classification and treatment support information for the health
care professionals, which is based on previously reported results about the clinical utility of
S100B for diagnosis and treatment of TBI [4, 5].

Prototype fabrication
Circuit schematics and printed circuit board (PCB) designs were made in the free web-based
electronic design automation (EDA) suite EasyEDA. The PCB designs can be found in S1 File.
PCB fabrication was done by JLCPCB (China) upon sending the Gerber files of each designed
PCB. Gerber files and schematics can be found in S4 File.
A surface mount technology (SMT) screen printing stencil was also ordered from JLCPCB
for each PCB. Circuit components were acquired from Digikey (USA), LCSC (China), and
AliExpress (China). PCB passive components assembly was performed via manual soldering,
while ICs were soldered by screen printing liquid solder using the provided SMT stencils, fol-
lowed by heating with a temperature-controlled hot air gun of a VIVO HOME 892D solder
rework station (USA). The bill of materials (BOM) for each PCB is found in S4 File.
System calibration and impedance measurement

Calibration of each TBISTAT is done on the assumption that system phase and magnitude are
properties that depend on the accuracy of the VGV used for EIS (� 1.65 V for 3.3 V DC sup-
ply), the frequency response of the PC for the excitation signal frequency range, and the intrin-
sic variability of the functionality of the passive and active components that constitute the PC.
In order to find system impedance, one must measure the magnitude and phase of the EOC
produced by a two-terminal cell with a known resistor value since it does not alter the system
phase as it is a non-capacitive or inductive load. Using the FFT, TBISTAT estimates a signal
magnitude and phase of the frequency bin corresponding to each AC excitation signal fre-
quency. This magnitude is stored and related to the resistor value and the measured phase.
Care must be taken to ensure that the signal magnitude acquired for the defined resistor is
unsaturated (less than 1.6 AC voltage amplitude with respect to reference VGV) and shows a
high signal-to-noise ratio (at least 50 mV of AC voltage amplitude with respect to VGV). Cali-
bration must be done using as many resistor values as possible to achieve high precision
throughout the impedance detection range for all available settings [22]. In this work, 103.5
Ω,141.9 Ω, 238 Ω, 393 Ω, 601 Ω, 1183 Ω, 1360 Ω, 1830 Ω, 2960 Ω, 5530 Ω, 11330 Ω, 12980 Ω,
15600 Ω, 18200 Ω, 24300 Ω, and 33200 Ω resistors were used for calibration. Resistors of 1183
Ω and 11330 Ω are used for both the upper and lower limits of impedance measurement
ranges of the 10 KΩ and 100 KΩ TIA resistances, respectively. Hence, a total of 18 resistors
were employed for calibration. Therefore, calibration of the impedance measurements (system
magnitude and phase) was done for a total of 18 different settings (one possible AC amplitude
peak of 10 mVp, three different TIA gain resistances for measuring impedances between 100
O and 35 OK, and six different resistors for each TIA gain resistance). A calibration file exam-
ple for the mentioned resistor values is found in S5 File.
Once calibration has been performed, the user can press start on the Android Application,
which will trigger EIS measurements and real-time Nyquist plot construction for the electro-
chemical cell under test. Additional details about TBISTAT calibration and EIS measurements
can be found in S1 File.
Electrical characterization
Power requirements. Power consumption was estimated by measuring current draw
from a fully charged (3.7 V) 1000 mAh LiPo battery using a Fluke 117 handheld multimeter
(USA) for both standby and active EIS measurements of a 100 Ω resistor connected between
the working and counter/reference electrodes in a two-electrode configuration.
Electrical noise. TBISTAT noise was evaluated by performing continuous EIS scans on
500O, 5 KO, and 15 KO resistors in a two-electrode configuration perturbed with 10 Hz, 100

Hz, 1000 Hz, and 10000 Hz 10 mV amplitude AC excitation voltages. EOC noise was estimated
as the root mean squared error (RMSE) values for 100 observations recorded using the 10 KO,
100 KO and 1 MO TIA resistors, with the appropriate sampling frequency for each defined sig-
nal input frequency to decrease spectral leakage during FFT, and using the 12-bits default
ADC resolution of the Teensy LC. RMS current noise is given by:
FFT magnitude:VSS
IRMS ½nA� ¼ ð1Þ
ADCres:TIAR
Where VSS is the 3.3 VDC voltage source powering the PC, ADCres corresponds to the
ADC resolution equal to 4096, and TIAR refers to the value in Ohms of the TIA resistance (10
KΩ, 100 KΩ or 1 MΩ).
System validation
TBISTAT was employed for performing EIS on three experimental systems: An AUTOLAB
dummy cell circuit composed of a 100 Ω resistor in series with the parallel circuit of a 1μF
ceramic capacitor and a 1 KΩ resistor; bare thin-film AUIDEs drop casted with 10uL of 10mM
K3[Fe(CN)6] in 0.2M KCl solution; ANTI-S100B functionalized thin-film AUIDEs exposed to
316 pg/ml of S100B spiked human plasma samples, and drop casted with 10uL of 10mM K3
[Fe(CN)6] in 0.2M KCl as support solution.
Validation was done using two methods: Visual comparison of impedance magnitude and
phase responses along the EIS frequency range obtained by TBISAT and an Autolab/M204
benchtop potentiostat/galvanostat (Metrohm1) equipped with an Autolab1 FRA32 module
controlled by the NOVA 2.11 software, and a T-test statistical comparison between ΔC results
at f = 31.6 Hz obtained by TBISAT and the benchtop potentiostat. Capacitance was obtained
from EIS scans following the method described by Rodriguez et al. [38]. The statistical analysis
was done using Statgraphics Centurion 18. The assumptions of normality, homoscedasticity,
and independence of residuals were assessed to establish the statistical validity of the T-test. All
statistical tests were considered significant with a p-value lower than 0.05.
S100B detection using TBISTAT

EIS measurements were performed using the TBISTAT to quantify the S100B protein. A sin-
gle-factor experimental design with four replicates was made to assess the effect of S100B con-
centration on the RCT obtained from the EIS of each AUIDEs biosensor platform. The
biomarker S100B was tested in three levels set in a logarithmic scale, using concentrations with
clinical utility: 31pg/mL (log₁₀ = 1.5), 100 pg/mL (log₁₀ = 2), and 316pg/mL (log₁₀ = 2.5). The
ΔRCT was selected as the response variable, and it was defined as the difference between the
RCT obtained from EIS for S100B testing (tRCT) and the basal RCT (bRCT) obtained from
EIS on anti-S100B/BSA functionalized working surface.
Ten microliters of the spiked human plasma samples were drop casted on the WEs surface
of AUIDEs and left to dry at RT. WEs were rinsed with DW after 15 minutes of antigen-anti-
body binding and left covered at RT. AUIDEs were then connected to the TBISTAT for the
electrochemical measurements and tested in a frequency range of 1 to 10,000Hz with a 10
mVp analog excitation signal. Electrochemical measurements were carried out using 10uL of
10mM K3[Fe(CN)6] in 0.2M KCl as a support solution. Variation of RCT was recorded to
evaluate changes in impedance after 15 minutes of antigen-antibody binding. Typical semicir-
cular behavior in the range corresponding to high frequencies associated with the electrode
redox probe was observed.

A statistical analysis was done using Statgraphics Centurion 19. Initially, the assumptions of
normality, homoscedasticity, and independence of residuals were assessed to establish statisti-
cal validity graphically and analytically. A one-way Welch ANOVA was applied to check for
differences between treatment groups (S100B concentrations), followed by Games-Howell as a
post-hoc test. A regression model (calibration curve) was developed together with a lack of fit
test to determine model adequacy to the response variable. Model suitability was established
considering global model significance, coefficients significance, and analysis of residuals struc-
ture. All statistical tests were considered significant with a p-value lower than 0.05.
The limit of detection (LOD) was determined by Eq 2, as follows:
3:3 SD
LOD ¼ ð2Þ
m
where SD is the average standard deviation of the response, and m is the calibration sensitivity,
determined by the slope of the calibration curve.
Results
Fabrication
Fig 4 shows the fabricated hardware modules connected for EIS measurements and the devel-
oped Android application running on an Android 7.1 OnePlus 5T smartphone. The hardware
occupies 72 cm2 and 216 cm3 when extended on a planar surface, as seen in Fig 4. Significant
reductions in device volume can be achieved by positioning each PCB on top of each other.
Power requirements
Table 2 shows the current draw of TBISTAT for continuous use in standby mode and during
EIS using a 1000 mAh LiPo battery as energy source. Battery life can last up to 11 hours of con-
tinuous EIS scans with a 100 O resistor as measured impedance. Higher duration can be
achieved using a 2000 mAh LiPo battery, which should last for at least 22 hours of continuous
EIS measurements.
Electrical noise
TBISTAT current noise stayed between 0.3 nA and 130 nA for the range of frequencies and
impedances used (Fig 5). The noise was higher in low frequency-low impedance EIS scans and
remained below 10 nA at higher frequencies and higher TIA resistors values. TBISTAT noise
levels were comparable to those reported by Jenkins et al. [22], even though teensy LC has 8
bits less nominal ADC resolution than the 24-bit ADC used by the former. The noise values
presented on TBISTAT could be explained by the use of hardware (Sallen-Key) and software
filters (five-point median filter) and a highly precise impedance measurement device with low
spectral leakage given by accurate frequency resolution definition and a 1000-point FFT.
System validation and performance

Dummy cell experiment. TBISTAT EIS magnitude and phase values were highly similar
to those obtained with the reference instrument (Fig 6). However, as expected, higher discrep-
ancies were found at frequencies lower than 10 Hz, which could be explained by higher signal
distortion and gain loss due to the high pass filter conditioning AD5933 AC signal generator.
Bare AUIDEs drop casted with 10uL of 10mM K3[Fe(CN)6] in 0.2M KCl solution. EIS
scans phase values in the potassium ferricyanide solution using TBISTAT showed higher dif-
ferences (circa 2˚) to those found with the reference instrument (Fig 7). Magnitude values

Fig 4. Components of TBISTAT connected for EIS measurement on an AUIDE cell. LiPo battery and boost circuit (bottom left) powers the MCU through
two cables (red 5 V, black ground). The BM is connected to the MCU (top left) using four-pin male-female headers. The MCU is connected to the PC through
ten cables (two for 3.3 V and ground (red and black), two for I2C communication (SDA purple, SCL white), one for ADC (yellow), and five for Analog switches
and TIA resistance multiplexer (blue)). A card reader (top right) is connected to the electrochemical cell female header connector (bottom right of PC). The
smartphone is shown running the TBISTAT application.
Table 2. Current draw of TBISTAT for standby and operation modes.

Condition Current Battery duration
Standby mode� 60 mA 17
��
EIS (1Hz to 10KHz) 90 mA 11
�
AD5933 not powered, BM powered, but no scans being made.
��
Continuous EIS scans.

Fig 5. Current noise of TBISTAT for the 10–10000 Hz bandwidth and 10 KΩ-1MΩ TIA gain resistors.
were reasonably close to the reference instrument (less than 2% differences). Higher variations
in phase values were found in lower frequencies (less than 10 Hz), which resulted in marked
differences with the reference instrument. Nevertheless, the TBISTAT employed calibration
strategy successfully prevented discontinuities in impedance measurements when the TIA
resistor was increased.
Fig 6. EIS magnitude and phase plots for TBISTAT and reference instrument for dummy cell excitation.

Fig 7. EIS magnitude and phase plots for TBISTAT and reference instrument for measurements in 10mM K3[Fe(CN)6] in 0.2M KCl solution.
ANTI-S100B functionalized thin-film AUIDEs exposed to 316 pg/ml of S100B spiked

human plasma samples. EIS scans with TBISTAT resulted in similar phase and magnitude
values to those obtained with the reference instrument (Fig 8). Higher impedance at lower fre-
quencies showed higher variations with respect to the reference instrument, which resulted in
marked differences in real and complex values in the obtained Nyquist plots (Fig 9). The use of
a five-point median filter allowed higher precision than expected in the 1–10 Hz frequency
range, which was thought to sustain altered phase/magnitude values due to the high amplifier
gain and low-current scenario given by the high magnitude impedance. The programmable
external clock of the AD5933 prevented excitation signal distortion at frequencies below 1
KHz, and the designed impedance analyzer system maintained sufficient precision in the mea-
surements (less than 2.5% difference in magnitude and phase value vs. reference instrument),
which together accounted for a sufficiently accurate EIS capable portable potentiostat. A video
showing TBISTAT operation during S100B electrochemical detection using AUIDEs can be
found in the S6 File.
Fig 8. EIS magnitude and phase plots for TBISTAT and reference instrument in ANTI-S100B functionalized thin-film AUIDEs exposed to 316 pg/ml of
S100B spiked human plasma samples.

Fig 9. Nyquist plot comparisons of TBISTAT and reference instrument in detecting S100B in an ANTI-S100B functionalized thin-film AUIDEs exposed
to 316 pg/ml of S100B spiked human plasma sample.
A T-test was carried out between the capacitance results obtained with TBISTAT and the
reference instrument (Table 3). Both standardized skewness and kurtosis values were within
the (-2,2) range, so samples could be considered to come from normal distributions. No signif-
icant differences were found in the sample variances using Levene’s test, and no structure was
found in the residual plot. Considering that the T-test p-value was higher than 0.05, the null
hypothesis could not be rejected. Thus, there was no statistically significant difference between
Table 3. Statistical analysis of single-frequency analysis experiment.

Relative change in capacitance (%) of Au/Cys/Ab/BSA/316 pg/ml
AUIDEs
Experimental run BENCHTOP TBISTAT
1 49.17264105 45.39904809
2 56.02429244 54.42103458
3 51.60599865 48.04601753
4 55.83877576 56.6168629
5 48.88026492 54.80168404
Mean 52.30439456 51.85692943
Standard deviation 52.93074526 53.1485057
Skewness 0.221046 -0.581177
Kurtosis -1.38102 -0.991419
Test P-Value
Levene’s 1.81495 0.2148
T-test H0: μ1 = μ2, α = 0.05 0.167647 0.871021

Fig 10. Nyquist plots of AUIDEs experimental results in spiked human plasma samples for the quantification of S100B in the 31–316 pg/mL range.
the means of the two samples. Additional information regarding the results of the statistical
analysis for potentiostat validation can be found in S7 File.
S100B detection using TBISTAT

EIS measurements of S100B were performed in spiked human plasma samples to evaluate a
possible future application of these biosensors in medical diagnosis. The results obtained from
the EIS of the biosensor in the presence of 10mM K₃[Fe(CN)₆] redox probe for the quantifica-
tion of S100B were assessed, exhibiting non-homogeneity of variance. A normal distribution
for the response variable (ΔRCT) was observed and residual independence. The analysis of var-
iance (Welch-ANOVA) and the post hoc analysis showed statistically significant differences in
the ΔRCT signal between each tested concentration (p < 0.05). Information regarding experi-
mental runs and statistical analysis are found in S7 File.
Fig 10 shows the EIS spectra obtained for the quantification of S100B in the range of detec-
tion. The basal signal corresponds to the AUIDE/Cys/anti-S100B/BSA without plasma addi-
tion, while negative control refers to plasma without S100B. A proportional increment was
observed in the ΔRCT with the successive increments of the S100B concentration. The
response of electrodes (ΔRCT) was consistent and showed a maximum relative standard devia-
tion (RSD) of 19.15%, indicating good reproducibility of the S100B detection.
The response of the platform in the linear detection range between 31 and 316 pg/mL was
modeled by the regression equation y = 1789.73+ 54.9336 � x (n = 4), where x is the concentra-
tion of S100B (pg/mL) in real scale and y the change in RCT (ΔRCT) measured in Ohms (Fig
11). Each point on the calibration curve represents each independent measurement, and the
error bar represents the standard error of the mean. The model was found suitable for the
experimental results since its coefficients were significant, no structure was found on its

Fig 11. Calibration curve for S100B in spiked human plasma using AUIDEs. y = ΔRCT; x = [S100B] (pg/mL).
residuals, and the lack of fit test was not significant. The LOD obtained for AUIDEs was 35.73
pg/mL. The statistical test results are found in S7 File.
Brain injury support information

TBISTAT takes ΔRCT values from the EIS scan for finding S100B concentration in AUIDEs
(Fig 12). Brain injury support information is shown as a message that suggests the presence or
absence of a TBI depending on the measured S100B concentration in the blood sample.
Discussion
This work has developed an open-source EIS-capable portable potentiostat controlled by a
wirelessly interfaced Android application that detects and quantifies the concentration of the
S100B TBI biomarker from plasma samples in clinically relevant conditions. Previous work
from Jenkins et al. [22], Ainla et al. [24], and Pruna et al. [26] has laid a solid foundation in the
understanding of the intricacies and possibilities in terms of building a portable, wireless
potentiostat prototype that is affordable and easy to adapt to a wide range of applications con-
cerned with the detection of an analyte of interest.

Fig 12. Brain injury support information after the EIS test.
Apart from Jenkins ABE-Stat, to our knowledge, the TBISTAT is the only other portable,
wireless, open-source potentiostat capable of conducting EIS measurements. The TBISTAT
circuit components and PCB fabrication can be acquired for less than 80 USD for one unit,
comparable to the 105 USD cost of Jenkins ABE-Stat. Circuit components suppliers and pack-
aging have been carefully chosen to allow fast acquisition and easy PCB assembly using stan-
dard soldering techniques with a hot iron and a soldering rework station. In addition, the
circuit schematics, Gerber files, BOMs, operation videos, calibration files, MCU firmware, and
Android application project have been made freely available for download without demanding
any usage permits. Software and hardware development interfaces such as Arduino IDE,
Android Studio, and EasyEDA allow rapid setup, circumventing the need for costly licenses
and allowing complete access to every aspect of the prototype design and fabrication.
Even though the TBISTAT was designed to detect S100B, several improvements of previous
works have been attained to provide more robust and precise impedance measurements.
While the TBISTAT was not designed to be a general-purpose potentiostat, configured to per-
form voltammetric assays and user-defined DC-biased EIS scans as Jenkins et al. ABE-Stat, its
performance has been increased in the 1-10KHz, 100–35 KO ranges when compared to the
former. The Teensy LC MCU employed in the TBISTAT possesses a higher number of GPIOs
than Jenkins ABE-Stat ESP8266. In addition, the Teensy LC true 12-bit ADC allows interrupt-
driven ADC with sampling rates as high as 200 KHz, thus avoiding the use of leakage-prone
AD5933 single-point DFT [37]. Jenkins et al. employed a 24-point DFT for excitation frequen-
cies below 60 HZ using a DAC software-programmed AC excitation signal, a variation which
was thought to result in significant discontinuities in impedance measurements at 2 Hz, 60
Hz, and 2 kHz using ABE-Stat, especially when the current signal was not a perfect sinusoid.
The TBISTAT improves the design of Jenkin’s impedance analyzer by utilizing its own imped-
ance analyzer circuit and a customized 1000-points FFT programmed in the Android applica-
tion. The accurate sampling frequency definition in the Android application of the TBISTAT

allows a fully customizable FFT frequency resolution for logarithmically spaced AC excitation
signal frequency points, significantly reducing FFT spectral leakage and avoiding the use of
windowing techniques before or after FFT. Furthermore, impedance measurement disconti-
nuities when the TIA resistor changes as impedance increases are reduced using the same cali-
bration resistors at the frequency range limits. In addition, the presence of a fourth-pole Sallen
key LPF reduces the amplitude and duration of the perturbation, which in turn decreases the
overall impact of this event on the electrochemical equilibrium of the AUIDE cell.
S100B detection using TBISTAT and AUIDEs biosensor platform exhibited an acceptable
global performance in terms of stability and reproducibility. Given the higher steric hindrance
due to the anti-S100B and S100B protein interaction as S100B concentration increased and the
electrostatic repulsive forces between the S100B and negatively charged redox species in sup-
port solution (10mM K3[Fe(CN)6] in 0.2M KCl) [39], a proportional increment was consis-
tently observed in the RCT to the successive increments in S100B concentration.
The use of AUIDEs with portable potentiostat such as TBISTAT offers various advantages
to facilitate further industrial development and commercialization of POCT devices. First, a
simple cleaning protocol (RCA-1) before functionalization is feasible for AUIDEs, avoiding
expensive reagents and considerably reducing the time required. Secondly, AUIDEs possess a
small planar detection area, which allows smaller cell volumes, thus reducing the quantity of
antibody solution needed for biosensor functionalization without a negative impact on the
biosensor performance. In third place, AUIDEs electrodes do not require a reference electrode
for analyte detection during impedance measurements, making the further development of a
POC system easier, with fewer operational amplifiers needed in the instrument.
Even though other studies report a wider range of detection and lower LODs for S100B
[40–42], this work demonstrates an effective detection of S100B in a clinically relevant range
since plasmatic S100B concentrations lower than a cutoff of 100pg/mL [9] ruled out the pres-
ence of bleeding in the CT, while levels below 30pg/mL ruled out BBB disruption [10]. Fur-
thermore, many of the sensors found in the literature exhibit limitations for a fast analysis due
to the requirement of sandwich-type immunoassays and fluorescent labels. Label-free and sim-
ple functionalization chemistry are valuable features of our biosensor, which provides a prom-
ising alternative for the fast analysis of biomarkers, even for very small sample volumes.
One simple but important contribution of TBISTAT is the methods it uses to quantify
S100B concentration from EIS scans in the AUIDEs biosensor platform. After EIS is done,
S100B concentration is calculated using the RCT obtained as the intercept at y = 0 of a three-
point regression model from the lower frequency EIS scan, which is then used as input for cal-
culating ΔRCT and S100B with the regression model. In this way, no electrochemical circle fit
is needed, and the S100B concentration estimate defines TBI support information for patient
diagnosis and treatment.
Limitations
Various limitations should be described for users interested in using the proposed prototype
for their specific purposes. Even though S100B detection using AUIDEs did not require chang-
ing DC bias above zero volts, other applications might require such adjustments. A fully pro-
grammable DC bias voltage potentiostat using the Teensy LC 12-bits DAC together with a
fourth-pole Sallen-Key LPF such as the one designed by Ainla et al. [24] for the UWED is cur-
rently being developed.
The TBISTAT can only be used in the 1Hz-10KHz frequency range to measure impedances
between 100Ω and 35KΩ. Replacing the Teensy LC 48 MHz cortex-M0+ with a 96 MHz cor-
tex-M4 Teensy 3.2 could increase sampling frequency and allow the ADC of higher frequency

signals without incurring any PCB changes. In addition, a 100 KHz Sallen-key LPF could
replace the 10KHz already found on TBISTAT. Higher impedances can be measured by using
the 10 MΩ TIA resistor already found on TBISTAT.
The TBISTAT employs a simple voltage divider circuit for VGV and uses 0.1% tolerance
resistors to achieve a low (less than 5 mV) DC offset between reference and working electrodes.
This value reduces the usable AC voltage span for ADC to less than 500mV, as it induces DC cur-
rents in the TIA in the lower impedance ranges of each TIA gain resistor setting which change
VGV to less than 3.3/2 V. In addition, this circuit is dependent on device voltage supply, which
can have slight variations following LiPo battery discharge. The addition of a 3.3 V high precision
analog voltage reference using an ISL60002 IC and a 3.3 V Rail splitter for analog reference such
as TL2426 IC [22] could be used as an alternative to the VGV circuits employed in the proposed
design to allow a more precise reference voltage for zero DC bias EIS measurements.
Further studies are needed to identify nuisance factors affecting the electrochemical mea-
surements using TBISTAT, such as temperature, humidity, electrical noise, and tests in real
settings beyond the laboratory. Experiments should be carried out at clinical facilities with TBI
patient’s blood for both the biosensor platform and the developed potentiostat at a clinically
relevant range of concentrations of S100B. Moreover, the inability to measure S100B directly
in plasma samples without the need for additional redox probes should be circumvented in
future iterations of the developed biosensor platform so that electrochemical detection can be
made in a non-faradaic manner. More detailed studies are now underway to implement non-
faradaic electrochemical measurements using AUIDEs since the use of a redox solution could
represent a limit for scaling the technology to a marketable stage.
Finally, machine learning algorithms for EIS analysis could support TBI diagnosis and
treatment by using supervised learning in EIS Nyquist plot analysis. This route could pose an
alternate solution to employing an electrochemical circle fit algorithm to find RCT value and
eventually S100B concentration and its correlation to TBI outcomes.
Conclusions
We have described the development and validation of a portable, wireless, open-source poten-
tiostat capable of performing EIS on AUIDEs to detect and quantify S100B in plasma at clini-
cally relevant concentrations. The TBISTAT occupies 216 cm3, weighs 120 g, and has an
approximate manufacturing cost of 80 USD. Its design is built upon the potentiostats made by
Jenkins et al. [22] and Ainla et al. [24], improving accuracy in phase and magnitude measure-
ments and reducing discontinuities in the overall impedance calculations along the 1-10KHz
frequency excitation range and between 100O and 35KO. Source code for MCU firmware and
Android application, Gerber files, schematics, and device operation video of TBISTAT have
been made freely available for download to promote its use, enhancement, and employment in
applications in either medical, animal, food or agroindustry. Furthermore, the modularity of
the design allows easier component changes according to the application demands in power,
frequency excitation ranges, wireless communication protocol, signal amplification and trans-
duction, precision, and sampling frequency of ADC, among others. In addition, the use of
minimal, easy acquirable open-source hardware and software, together with high-level filter-
ing, low-cost, accurate ADC, wireless communication, and the simple user interface, provides
a framework for facilitating EIS analysis for similar POC applications such as the one pre-
sented in this work and for developing affordable diagnostics and POC biosensors integrated
systems. Improvements to the prototype, such as adding a high precision analog voltage refer-
ence and a 3.3 V Rail splitter for analog reference, could increase the device capabilities and
range of possible applications to meet user-specific demands.

Supporting information
S1 File. Hardware considerations. Additional details concerning hardware design, circuit
schematics and PCB design.
(DOCX)
S2 File. Software design. Additional details concerning software design.
(DOCX)
S3 File. MCU firmware and Android application files.
(RAR)
S4 File. BOMs, circuit schematics and Gerber files.
(RAR)
S5 File. TBISTAT calibration file.
(XLS)
S6 File. Video file.
(MP4)
S7 File. Statistical tests and figures.
(DOCX)
S8 File. Configuration values for AC excitation signal and ADC.
(XLSX)
Author Contributions
Conceptualization: Francisco Burgos-Flórez, Alexander Rodrı́guez, Valtencir Zucolotto,
Pedro J. Villalba.
Data curation: Francisco Burgos-Flórez, Alexander Rodrı́guez.
Formal analysis: Francisco Burgos-Flórez, Alexander Rodrı́guez.
Funding acquisition: Valtencir Zucolotto, Marco Sanjuán, Pedro J. Villalba.
Investigation: Francisco Burgos-Flórez, Alexander Rodrı́guez.
Methodology: Francisco Burgos-Flórez, Alexander Rodrı́guez, Marco Sanjuán, Pedro J.
Villalba.
Project administration: Valtencir Zucolotto, Marco Sanjuán, Pedro J. Villalba.
Resources: Francisco Burgos-Flórez, Alexander Rodrı́guez, Valtencir Zucolotto, Marco San-
juán, Pedro J. Villalba.
Software: Francisco Burgos-Flórez, Alexander Rodrı́guez.
Supervision: Valtencir Zucolotto, Marco Sanjuán, Pedro J. Villalba.
Validation: Francisco Burgos-Flórez, Alexander Rodrı́guez, Eliana Cervera.
Visualization: Francisco Burgos-Flórez, Alexander Rodrı́guez, Eliana Cervera.
Writing – original draft: Francisco Burgos-Flórez, Alexander Rodrı́guez, Eliana Cervera.
Writing – review & editing: Francisco Burgos-Flórez, Alexander Rodrı́guez, Eliana Cervera,
Valtencir Zucolotto, Marco Sanjuán, Pedro J. Villalba.

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PLOS ONE

TBISTAT: An open-source, wireless portable,

PLOS ONE | https://doi.org/10.1371/journal.pone.0263738 February 7, 2022 1 / 25

design, data collection and analysis, decision to Introduction

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✓Feature is present on the potentiostat. × Feature is not present on the potentiostat.

miniaturization, increased sensibility, and reproducibility, whereas commercial potentiostats

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Materials and methods

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Electrodes and reagents

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Preparation of spiked human plasma samples

High-level representation of potentiostat system

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Fig 2. General representation of the system components.

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generator, a digital-to-analog converter (DAC), and a programmable gain amplifier (PGA)

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S100B regression model and TBI support information

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System calibration and impedance measurement

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S100B detection using TBISTAT

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System validation and performance

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Table 2. Current draw of TBISTAT for standby and operation modes.

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ANTI-S100B functionalized thin-film AUIDEs exposed to 316 pg/ml of S100B spiked

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Table 3. Statistical analysis of single-frequency analysis experiment.

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S100B detection using TBISTAT

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Brain injury support information

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