Chemosensors 11 00244

chemosensors
Review
Electrochemical Sweat Sensors
Emanuel Bilbao 1 , Octavio Garate 1 , Theo Rodríguez Campos 1,2 , Mariano Roberti 3 , Mijal Mass 3 ,
Alex Lozano 1,3 , Gloria Longinotti 1 , Leandro Monsalve 1,4 and Gabriel Ybarra 1, *
1 Functional Nanomaterials Group, INTI-Micro and Nanotechnologies,

National Institute of Industrial Technology, Av. Gral. Paz 5445, San Martín B1650WAB, Argentina;
ebilbao@inti.gob.ar (E.B.); ogarate@inti.gob.ar (O.G.); trodriguez@inti.gob.ar (T.R.C.);
alozano@inti.gob.ar (A.L.); mlonginotti@inti.gob.ar (G.L.); monsalve@inti.gob.ar (L.M.)
2 Institute for Industrial Quality (INCALIN) (INTI—UNSAM), Av. Gral. Paz 5445,
San Martín B1650WAB, Argentina
3 Printed Electronics & Rapid Prototyping Group, INTI-Micro and Nanotechnologies,
National Institute of Industrial Technology, Av. Gral. Paz 5445, San Martín B1650WAB, Argentina;
froberti@inti.gob.ar (M.R.); mmass@inti.gob.ar (M.M.)
4 CONICET—INTI—UNSAM, Av. Gral. Paz 5445, San Martín B1650WAB, Argentina
* Correspondence: gybarra@inti.gob.ar
Abstract: Sweat analysis by means of minimally invasive wearable sensors is considered a potentially
disruptive method for assessing clinical parameters, with exciting applications in early medical
diagnostics and high-performance sports. Electrochemical sensors and biosensors are especially
attractive because of the possibility of the electronic integration of wearable devices. In this article, we
review several aspects regarding the potentialities and present limitations of electrochemical sweat
(bio)sensors, including: the main target analytes and their relationships with clinical conditions; most
usual electrochemical techniques of transduction used according to the nature of the target analytes;
issues connected to the collection of representative sweat samples; aspects regarding the associated,
miniaturized electronic instrumentation used for signal processing and communication; and signal
processing by machine learning.
Citation: Bilbao, E.; Garate, O.; Keywords: diagnostics; electrochemical biosensors; potentiometric sensors; sweat analysis
Rodríguez Campos, T.; Roberti, M.;
Mass, M.; Lozano, A.; Longinotti, G.;
Monsalve, L.; Ybarra, G.
Electrochemical Sweat Sensors.
1. Introduction
Chemosensors 2023, 11, 244.
1.1. Electrochemical Biosensing of Sweat Composition as a Diagnostic Tool
https://doi.org/10.3390/
chemosensors11040244 Eccrine sweat is an aqueous biofluid excreted by millions of glands distributed all
over the human body. These glands have an important thermoregulator role and excretory
Academic Editor: Gabriela Broncová
functions aiming to eliminate excess wastes and toxicants [1]. Eccrine sweat is mostly
composed of electrolytes and metabolites, but it also contains vitamins, hormones, peptides,
Received: 20 January 2023 and macromolecules in lower concentrations [2]. The composition of sweat can be related to
Revised: 1 April 2023 the physiological human body status and clinical conditions (Table 1). For instance, chloride
Accepted: 3 April 2023 sweat concentration measurement is the gold standard for cystic fibrosis detection [2],
Published: 14 April 2023 glucose level monitoring is crucial for healthy control of diabetic patients, and sweat lactate
level measurements could be a good candidate for sport physiology assessment [3].
The compounds of interest present in sweat can be determined using electrochemical
sensors and biosensors. The detection approach used in each case is greatly determined by
Copyright: © 2023 by the authors.
the nature of the analyte, which we can roughly classify into three kinds: electrolytes, small
Licensee MDPI, Basel, Switzerland.
organic molecules, and macromolecules and intermediate-size (bio)molecules (proteins,
This article is an open access article
steroid hormones, and so on). For instance, ion-sensitive electrodes are the technique of
distributed under the terms and
conditions of the Creative Commons
choice for the selective determination of ions in concentrations higher than 1 mM. Ions in
Attribution (CC BY) license (https://
low concentration (heavy metals such as Zn, Cd, Pb, Cu and Hg, present at trace levels)
creativecommons.org/licenses/by/ have also been detected, albeit by employing more sensitive voltammetric techniques,
4.0/). such as square wave anodic stripping voltammetry [4]. Similarly, the standard means
Chemosensors 2023, 11, 244. https://doi.org/10.3390/chemosensors11040244 https://www.mdpi.com/journal/chemosensors

Chemosensors 2023, 11, 244 2 of 46
of electrochemical determination of the concentrations of small organic molecules (e.g.,

glucose, lactate, ethanol, etc.) is amperometric enzymatic biosensing. Redox enzymes are
used to specifically detect the substrate (analyte), and its catalytic activity (reaction rate)
is converted into an electronic current proportional to the analyte concentration. Finally,
affinity biosensors are the preferred choice in cases of intermediate-size (bio)molecules
and macromolecules. The occurrence of antigen-antibody events is electrochemically
detected (usually by means of a voltammetric technique or by electrochemical impedance
spectroscopy, EIS) on the surface of a sensing electrode, where the biorecognition element
(either the antigen or the antibody) has been immobilized.
Table 1. Key analytes in sweat and the related detection methods. Adapted with permission from
Refs. [5], Copyright 2021, and [6], Copyright 2023.
Concentration Disease
Target Analyte Sensing Modality Ref.
in Sweat Correlation
Dehydration, hyponatremia,
Na+ 10–100 mM [7,8]
electrolyte imbalances
Cl− 10–100 mM Dehydration, cystic fibrosis [7,9]
Ion-selective
Ions K+ 1–18.5 mM Hypokalaemia, muscle cramps [7]
potentiometry
Myeloma, cirrhosis, renal failure,
Ca2+ 0.41–12.4 mM [9]
acid–base balance disorder
Pathogenesis of skin diseases,
pH 3–8 [10]
wound healing
Shift from aerobic to anaerobic
NH4 + 0.1–1 mM [10]
metabolic conditions
Glucose 10–200 µM Diabetes [7]
Cystic fibrosis, stress ischaemia,
Lactate 5–20 mM Amperometric [11]
Metabolites lactic acidosis
enzymatic biosensors
Alcoholism, hepatitis B, diabetes,
Ethanol 2–30 mM [12]
drunk driving
Uric acid 2–10 mM Hyperuricemia, gout, kidney disease [13]
Tumours, cancer, kidney disease,
Ascorbic acid 10–50 µM [13]
thrombosis, stones
Voltammetry,
electrochemical
Hormones Cortisol 22–390 nM Stress [14]
impedance
spectroscopy
Square wave
Peptides voltammetry,
Macromolecules Proteins (antibodies, - electrochemical
antigens, enzymes) impedance
spectroscopy
Especially attractive is the possibility of minimally invasive analysis provided by

wearable electrochemical sweat sensors. Wearable devices (or simply wearables) are
autonomous, non-invasive, and on-body devices aiming to collect and analyse data. They
are usually incorporated into smartwatches, glasses, earbuds, skin stickers or tattoos,
patches, and jewellery. Health wearables perform specific healthcare functions, such as
the measurement of physiological parameters. These wearables incorporate mechanical,
chemical, and biochemical sensors to monitor physical parameters and key analytes or
biomarkers in biofluids. In all instances, smart sensors, artificial intelligence (AI), big
data, and internet of things (IoT) technologies may be implied to acquire, process, and
wirelessly transmit health condition information to the user or healthcare provider, using
this powerful tool for disease management, diagnosis, prediction, and follow-up [15].
1.2. Key Health-Related Examples of Sweat Biochemical Analysis

Cystic fibrosis (CF) is a genetic disorder characterized by the production of thick
and sticky mucus due to elevated concentrations of chloride in extracellular liquid that
affects the lungs, pancreas, and other organs. Sweat testing is consider the gold standard
for CF diagnosis. Sweat is collected, and its level of chloride concentration is measured.
Levels higher than 60 mM are consistent with the diagnosis of CF. According to Cystic
Fibrosis Foundation guidelines, sweat must be quantitatively analysed for chloride by
electrochemical methods or by automated analysers using ion-selective electrodes [16].
Glucose monitoring systems can be classified as invasive, minimally invasive, and
non-invasive. The standard finger-prick test to determine the level of glucose in blood
and the interstitial fluid glucose sensor, which are commercially available and highly
diffused, can be considered invasive and minimally invasive, respectively. Even though
blood testing is considered the gold standard, there is a trend toward migrating to less
invasive and painless techniques for analysing alternative biofluids, such as saliva, tears,
and sweat. Sweat-based sensors are considered the least invasive method to assess glucose
levels. Moyer et al. [17] reported that there is a statically significant correlation between
glucose concentrations in blood and in sweat, with glucose present in sweat representing
about 1–2% of the glucose in blood. However, Moyer and most of the other authors
affirmed that the greatest technological challenge of this alternative technique is to acquire
a representative and interferent-free sweat sample to obtain accurate measurements.
Rapid detection and precise monitoring of therapeutic and non-legal drugs are es-
sential for the healthcare system due to the current high level of substance abuse. Some
researchers work on the development of innovative systems for drug monitoring based
on sweat sensors. Xue et al. [18] developed a microfluidic system based on a competitive
ELISA technique to detect drug-related metabolites in sweat. They reported a rapid system
to quantify, in around 16 min, methadone, methamphetamine, amphetamine, and THC
using approx. 4 µL of sweat. Moreover, Tai et al. [19] implemented a methylxanthine
drug-monitoring wearable platform equipped with an electrochemical differential pulse
voltammetry sensing module with sensitivity of 110 nA µM−1 . In addition, Teymourian
et al. [20] included in their review the detection in sweat of heroin and its metabolites,
codeine, fentanyl, methadone, cocaine and its metabolites, ethanol, and amphetamine-type
stimulants, in the order of nanograms to a few micrograms.
Sport technologies were introduced to help users to reach their desired fitness level or
maximum potential. Sports and wellness sensors, which are mostly wearable, gather infor-
mation about the physiological wearers’ status, their performance, and the environmental
conditions [21]. Sweat sensors are relevant in sports applications because the monitoring of
sweat composition variations along training routines could provide enough information
to assess athletes’ status and personalize their training. Parrilla et al. [22] developed a
potentiometric ion patch based on multiple-wall carbon nanotubes (MWCNTs) electrodes
to monitor pH, Cl− , K+ , and Na+ in sweat. Researchers have paid special attention to lactate
because, first, its sweat concentration is higher than its blood concentration [3] and, second,
because there is a possible correlation between its concentration and exercise intensity [23].
For instance, Zhang et al. [24] developed a wearable enzymatic lactate biosensor mounted
on eyeglasses. This device presented a linear response up to 25 mM in a phosphate-buffered
solution with a pH of 7.
2. Sampling Techniques
2.1. Traditional Methods of Sampling Sweat
In traditional analysis under laboratory conditions, sampling or collection is also a
crucial step in sweat analysis due to its great impact on the accuracy of the determination
of electrolyte and metabolite concentrations [25]. In all instances, the procedures require
trained operators and must satisfy biosecurity requirements to avoid producing injuries to
the user and contaminating the sample.
Traditional sampling methods include systemic and local methods. The systemic
method is the whole-body wash-down, and it is considered the gold standard for whole
body loss of Na+ and other electrolytes [25]. As its name suggests, it consists of washing
with deionized water the whole body, the user’s clothes, and the equipment before and
after an exercise routine in a closed system under controlled conditions. The sample
obtained is a interferent-free biofluid, and the normal evaporative process is maintained

undisturbed [25].
Local methods include patches, polymer bags and films, scraping/ripping/gathering,
and macroducts. Patches are a low-cost, flexible, in-body, and disposable alternative
to collect local sweat in a hydrophilic porous structure [25]. Polymer bags and films
involve a simple and disposable method to collect sweat. They usually consist of an
adhesive rubber that covers the skin and a polymeric bag to accumulate the biofluid.
In the scraping/dripping/gathering method, sweat is collected in capillary tubes, test
tubes, or beakers. Even though it is a simple and inexpensive method, there are many
sources of potential error, especially due to evaporation and contamination. Macroducts
are commercial disposable products mostly used in CF diagnosis that consist of a spiral
plastic tube to collect sweat. The collecting procedure takes at least 30 min.
2.2. Sampling Methods for Wearable Systems: Sweat Generation and Collection
Wearable sensors for continuous or intermittent monitoring of biomolecules from
body fluids are highly desired as integrated platforms in the human body [26]. Some of the
desired properties of the new wearable sensors are stretchability, ultra-thinness, biocompat-
ibility, biodegradability, and self-healing [27]. For example, since a sweat collecting system
is in direct contact with the skin, it should be fabricated from a smooth, non-irritating, and
anti-allergic material so that it does not generate inflammation on the skin after wearing
the sensors.
Wearable devices have evolved gradually with a mix of multiplexed biosensing,
microfluidic sampling, and transport systems integrated with flexible materials and body
attachments for improved wearability and simplicity [28]. These wearables hold promise
and are capable of greater understanding of the correlations between analyte concentrations
within the blood or non-invasive biofluids and feedback to the patient, which is significantly
important in timely diagnosis, treatment, and control of medical conditions [28].
Sweat sensors offers an innovative and non-invasive alternative for physiological
monitoring. With adequately processed data by AI and big data techniques, they could
be a powerful tool to guide clinical decision-making. However, even though the field
of electrochemical sensors and biosensors is mature, measuring sweat samples presents
several challenges, especially regarding wearable sweat sensors. For instance, the lifetime
of wearable electrochemical biosensors is significantly dependent on the long-term stability
of functional materials modified on the flexible substrate, the effect of pH changes in
sweat on the sensing device, and signal fluctuations caused by the bending of sensors.
For all applications, researchers and developers agree that the main drawback of sweat
sensors is obtaining a sufficient sample of sweat in a short period time that represents
the physiological status of the human body without interferents and contaminants [3].
Therefore, sampling is a key point for the development of sweat sensors.
The extraction of a representative sample involves two processes: sweat generation,
followed by efficient collection.
With respect to sweat generation, sweat glands can be stimulated by steam or dry heat
exposition (thermal stimulation) and exercise. Thus, in the case of sweat analysis of athletes,
sweating is spontaneously produced by physical exercise. However, for health-related
sweat analysis, patients are usually at rest, and sweating must be artificially and locally
generated. One of the most commonly used pharmacological techniques to stimulate
sweating is iontophoresis [1]. Iontophoresis is an established process used to induce
ion/molecular flow by applying a mild electric current across the skin, and it is widely
used in clinics for diagnostic and therapeutic purposes [2]. Iontophoretic transdermal drug
delivery is used to distribute agonist drugs (e.g., pilocarpine, carbachol, or lidocaine) from
an electrode across the skin (Figure 1). However, in some cases, the iontophoretic drug may
interfere with the electrochemical determination. Thus, a protective layer of agarose on top
of the electrode has been implemented to avoid drug-related interference [29].
may interfere with the electrochemical determination. Thus, a protect
se on top of the electrode has been implemented to avoid drug-related
Figure 1. Schematic diagram of an iontophoretic-sensing tattoo device, containing the iontophoretic

e 1. Schematic diagramelectrodes
of an (IEs;iontophoretic-sensing
anode and cathode) and three sensingtattoo device,
electrodes (working, containing
reference, and counter the
electrodes: WE, RE, and CE, respectively). Reprinted with permission from [12]. Copyright (2016),
odes (IEs; anode and cathode) and
American Chemical three sensing electrodes (working, reference
Society.
odes: WE, RE, and CE, respectively). Reprinted
The acquisition of representative sampleswith permission
requires from
sweat to be efficiently [12].
collected and Cop
ican Chemical Society. transported to the sensing electrode. Several approaches and materials have been employed
to achieve this end, involving the use of functional absorbent materials, superhydropho-
bic/superhydrophilic surfaces, sweat guidance, and epidermal microfluidic systems.
First, absorbent materials, such as nonwoven fabrics, papers, and hydrogels, accu-
The acquisition of representative samples requires sweat to be efficien
mulate sweat in their pores and efficiently guide it to the sensor surface. Furthermore,
these materials can be functionalized with selective components. For instance, Li et al. [30]
ransported to the sensing electrode. Several approaches and materials ha
developed a sensing platform for real-time sweat analysis combining paper, hydrophobic
wax, conducting electrodes, and active materials (MXene/methylene blue). As result, a
d to achieve this end, involving the use of functional absorbent materia
sweat-based glucose and lactate sensor with sensitivity 2.4 nA µM−1 was developed.
Second, surface wettability can be modified to collect, accumulate, and directionally
hobic/superhydrophilic surfaces, sweat guidance, and epidermal micro
transport sweat samples from superhydrophobic to superhydrophilic zones. For instance,
He et al. [31] developed a band aid that combines superhydrophobic and superhydrophilic
microarrays with colorimetric biosensors for sweat assessment. In addition, Dai et al. [32]
modified a Janus fabric surface, obtaining an array of hydrophilic pores to unidirectionally
First, absorbent materials, such as nonwoven fabrics, papers, and hydr
pump accumulated sweat.
Third, researchers have developed more sophisticated and innovative methods to drive
te sweat in their pores and efficiently guide it to the sensor surface. F
biofluid to the sensor system through microchannels in polymeric substrates, for example,
polydimethylsiloxane (PDMS). Sweat flows through the channels thanks to capillary action
materials can be functionalized with selective components. For instance,
and the pressure of the hydraulic pumping action of sweat glands [25].
Finally, microfluidic systems are a trend in wearable development. Researchers
oped a sensing platform for real-time sweat analysis combining paper, h
have combined sensors, microfluidics, and electronics into on-body devices. However,
Hoovels et al. [25] reported that it is not trivial to find wearable devices that properly
conducting electrodes, and active materials (MXene/methylene blue).
function on a microfluidic basis to analyse sweat. Cao et al. proposed a three-dimensional
t-based glucose and lactate sensor with sensitivity 2.4 nA μM was deve
paper-based microfluidic electrochemical integrated device for real-time monitoring −1
sweat metabolites and electrolytes (Figure 2). The collector had five stacked layers: a sweat
of
Second, surface wettability can be modified to collect, accumulate, and d

collector, a vertical channel, a transverse channel, an electrode layer, and a sweat evaporator.
Beyond these components, the capillary effect of paper helps the sweat to flow into the
port sweat samples from superhydrophobic to superhydrophilic zones. F
paper-based devices, contributing to sweat collection.
al. [31] developed a band aid that combines superhydrophobic and s

c microarrays with colorimetric biosensors for sweat assessment. In add
2] modified a Janus fabric surface, obtaining an array of hydrophilic po
microfluidic electrochemical integrated device for real-time monitoring of sweat metabo-
lites and electrolytes (Figure 2). The collector had five stacked layers: a sweat collector, a
vertical channel, a transverse channel, an electrode layer, and a sweat evaporator. Beyond
these
Chemosensors 2023, components,
11, 244 the capillary effect of paper helps the sweat to flow into the paper-6 of 46
based devices, contributing to sweat collection.
Figure 2. Schematic diagram of the three-dimensional

Figure 2. Schematic paper-based microfluidic
diagram of the three-dimensional electrochemical
paper-based microfluidic in-
electrochemical
tegrated device. (A)integrated
The layered structure
device. of thestructure
(A) The layered device.of3D-PMED includes includes
the device. 3D-PMED the sweatthe collector,
sweat collector,
vertical channel,
vertical channel, transverse channel,transverse channel,
electrode electrode
layer, layer,evaporator.
and sweat and sweat evaporator.
The yellowThe yellow
areas areas
or theor the
white areas on the device were hydrophobic areas formed by wax screen-printing or hydrophilicareas
white areas on the device were hydrophobic areas formed by wax screen-printing or hydrophilic
areas of filter paper, of filter paper, respectively. A three-electrode electrochemical sensor created by screen-printing was
respectively. A three-electrode electrochemical sensor created by screen-print-
affixed on the electrode layer of the device using double-sided adhesive tape. (B) The schematic dia-
ing was affixed on the electrode layer of the device using double-sided adhesive tape. (B) The sche-
gram of the 3D-PMED applied on the skin of a human. A 3D-flow channel was formed by folding the
matic diagram of the 3D-PMED applied on the skin of a human. A 3D-flow channel was formed by
device with the tiered structure to help the sweat to flow from the skin into the device and thus refresh
folding the device with the tiered structure to help the sweat to flow from the skin into the device
the sweat under the electrodes. Reproduced with permission from Ref. [33], Copyright 2019, RSC.
and thus refresh the sweat under the electrodes. Reproduced with permission from Ref. [33], Cop-
yright 2019, RSC. Sweat collection remains an important topic in wearable sensors, as the fluid flow
under the patch is important for real-time, continuous analysis of sweat and long-term
Sweat comfort
collection of the an
remains skin. Additionally,
important topic sweat accumulation
in wearable under as
sensors, sensors will cause
the fluid flowskin
discomfort [18].
under the patch is important for real-time, continuous analysis of sweat and long-term
comfort of the skin. Additionally,
3. Sensing sweat
Electrolytes accumulation
in Sweat under sensors
with Potentiometric will cause
Ion-Selective skin dis-
Electrodes
comfort [18]. As previously detailed, electrolytes are among the most targeted biomarkers within
sweat analytics due to their roles in hydration, osmotic balance, and muscle stimulation.
Within
3. Sensing Electrolytes in theSweat
proposed with sensing techniques, amperometry,
Potentiometric Ion-Selective potentiometry,
Electrodesand conductom-
etry stand out as both suitable and relevant candidates. However, potentiometry is the
As previouslymost
detailed,
commonly electrolytes
used techniqueare among the most targeted
for the determination biomarkers
of electrolytes within
with electrochemical
sweat analytics due to their roles in hydration, osmotic balance, and muscle stimulation.
sweat sensors.
Potentiometry
Within the proposed sensing is a technique
techniques, that allows for
amperometry, electrolyte quantification
potentiometry, according to the
and conductome-
Nernst equation, even at relatively low concentrations of sweat, based on the use of a dual
try stand out as both suitable and relevant candidates. However, potentiometry is the
probe sensor composed of an ion-selective electrode (ISE) and a reference electrode (RE).
most commonly used technique
Recent advancesfor thefield
in the determination
of flexible andof electrolytes
printable withnanomaterial
electronics, electrochemical synthesis,
sweat sensors. and chemical functionalization have laid a solid foundation to overcome the limitations of
Potentiometryproof-of-concept
is a technique approaches
that allowsand develop sensors asquantification
for electrolyte skin-mounted systems throughtodrop
according
casting (DC), screen printing (SP), or ink-jet printing (IJP) [34,35].
the Nernst equation, even at relatively low concentrations of sweat, based on the use of a
Drop casting is a simple film-forming technique proposed by many researchers to
dual probe sensor develop
composed of an
a diverse ion-selective
array electrode
of electrochemical sensors(ISE) andthea need
without reference electrode
for specific equipment.
(RE). Recent advances in the field of flexible and printable electronics, nanomaterial
Regarding REs, this accessible methodology is used to chemically modify the surfaces syn- of
commercial
thesis, and chemical screen printed
functionalization or solid-state
have laid a solid electrodes throughtoa simple
foundation overcomeprocesstheoflimi-
casting a
tations of proof-of-concept approaches and develop sensors as skin-mounted systems in a
solution containing the desired material over the desired electrode surface, resulting
layer after solvent evaporation [36].
through drop casting (DC), screen printing (SP), or ink-jet printing (IJP) [34,35].
On the other hand, inkjet printing and screen printing have been reported as the most
Drop casting promising
is a simple film-forming
approaches technique
for simple, proposed production
fast, and inexpensive by many researchers to
of sensing platforms.
develop a diverse array of electrochemical sensors without the need for specific equip-
Their cutting edge technologies can be attributed to the miniaturization of electrochemical
sensors,
ment. Regarding REs, thisreducing
accessiblethe sample volume required
methodology is usedto as
tolittle as a few microliters,
chemically modify the allowing
sur- for
the size reduction of the diagnostic platform into which they are integrated. Furthermore,
faces of commercial screen printed or solid-state electrodes through a simple process of
casting a solution containing the desired material over the desired electrode surface, re-
sulting in a layer after solvent evaporation [36].
, x FOR PEER REVIEW 8 of 47
3.1.1. Reference Electrodes: Structural Overview

the as-obtained screen-printed (SPEs) or inkjet-printed electrodes (IJPEs) can be further
As stated previously,
and easily the REs constructed
functionalized throughusing DC are based
physicochemical on a layered
procedures to confer modifica-
qualities such as
tion of commercialspecificity,
SC or solid-state
selectivity, electrodes
and stability.as base templates
Alongside (further
the previously details
mentioned can be SP
advantages,
and IJP mass production capabilities encompass the totality of
found in Section 3.1.3). As a result, the following structural overview addresses, for sim- their potentialities, justifying
SP and IJP as suitable candidates complying with the ongoing demands for rapid and
plicity’s sake, the three approaches (DC, SP, and IJP) considering the RE as a bottom-up
accurate in-situ analyses through portable devices. Adapting conventional ISE and RE
layered structure (Figure
to these3b,c).
techniques represents a hindrance, for example, the constant attempts by the
The first reported Ag/AgCl
scientific community REs presented
to develop a robustupandto low-cost
three distinct
alternativefunctional layers glass
to the conventional
silver/silver chloride (Ag/AgCl) gel RE [37].
[46,47]. The first layer is conductive and is responsible for allowing efficient electron trans-
Considering the functional importance of both ISE and RE, the following section
fer while closing the electrical circuit. The second layer, referred as to the ion-to-electron
addresses structural, performative, and suitability characteristics, allowing, at the end, for
layer, is the one thatthecontains Ag/AgCl
presentation and establishes
of a general a reference
conclusion regarding potential,
the state of ISE andas RE
explained
development
above (Equations (1) andandthe(2)). In addition
pending challengestothat
thishave
basic
yettwo-layered
to be overcome. structure, an additional
layer (the dielectric layer) is required for printed reference electrodes (PREs) to control the
3.1. Reference Electrode
functional area of each layer exposed to the measurement solution and to protect the elec-
The Ag/AgCl electrode is almost universally used as a reference electrode in electro-
trical connections pads.
chemical sweat sensors. There are several reasons for this choice: biocompatibility of the
Unfortunately,materials,
there iscompatibility
no component in its
with the structure
sweat’s pH, andtocommercially
offset the dilution-induced
available inks for SP and
decrease in [Cl−] withIJP.time, which
Therefore, weresults
considerin subsequent
exclusively potentialRE
the Ag/AgCl fluctuations,
in this section.as indicated
by the Nernst equation. AsThese
illustrated in Figure 3a, the
characteristics areconventional
those of a RE is structurally composed
pseudo-reference of a glass
electrode,
vessel containing a silver wire coated with silver chloride, immersed in a KCl solution with
limiting its field applications since environmental and human samples contain either pro-
a constant concentration. Potassium chloride is generally used as the electrolyte because
teins or anions thatKaffect
+ and Clthe stability
− present of their
similar high electrochemical potential
mobilities, which improve the considerably.
overall transient response
of the RE.
Figure 3. (a) Illustration of the components present in a traditional reference electrode. (b,c) Stratified
Figure 3. (a) Illustration of the components present in a traditional reference electrode. (b,c) Strati-
view presenting structural differences between a printed pseudo-reference electrode (b) and a printed
fied view presenting structural differences between a printed pseudo-reference electrode (b) and a
reference electrode (c).
printed reference electrode (c).
From an operational standpoint, classical Ag/AgCl RE features a reversible electron
Considering transfer reaction involving
all the limitations of thea proposed
slightly soluble salt (AgCl) using
approaches and a metallic electrode (Ag):
pseudo-reference
electrodes, the next step in RE development was aimed at printing or casting any neces- (1)
AgCl (s) + e−
Ag (s) + Cl− (aq)
sary additional layers to improve reproducibility, repeatability, lifetime, and chloride ion
As a product
concentration susceptibility, withofitsthisperformance
reaction, if the chloride
resemblingconcentration
that ofisakept constant, an
traditional unequiv-
elec-
ocal and stable electrode potential is defined following the Nernst equation:
trode glass Ag/AgCl gel RE. Concerning this development, Andrade et al. [48] provided
an alternative for RE development that would E = Esolve
0
− 2.3the limitations
RT/zF log [Cl− ] of pseudo-reference (2)
electrodes. In their work, the authors presented a glassy carbon (GC) drop casted RE, con-
where E0 is the electrode standard half-cell potential (V), R is the molar gas constant
taining a layer of Ag/AgCl alongside a membrane cocktail of polyvinyl butyral (PVB) and
(J K−1 mol−1 ), T is the absolute temperature (K), z is the number of involved electrons
NaCl. The RE showed a stable potential
transferred in the over aF wide
half-reaction, is the range
Faradayofconstant
concentrations
(C mol−1 ),for
andseveral
[Cl− ] is the
chemical species, no significant drift, and a considerable lifetime of four months.
The reason why this electrode presented similar performance compared with a con-
ventional double-junction reference electrode can be interpreted from the standalone
functionality of its components. First, the Ag/AgCl layer governs, in the absence of any
concentration of chloride ions (M). Under this thermodynamic correlation, a conventional

Ag/AgCl electrode displays, at a temperature of 25 ◦ C, a [Cl− ] dependent Nernstian
response of −59.16 mV/pCl. In addition, conditions of KCl oversaturation (between 3.5 M
and 4 M) ensure a stable half-cell potential in a broad spectrum of solutions, a desirable
quality for any RE.
Recent publications have indicated that the RE is an essential component in any
potentiometric device since the response of an ISE is meaningless without a reference
against which to compare it [38–45]. Additionally, replicating both the structural and the
functional characteristics of a conventional RE in portable or wearable ISEs is not a trifling
matter since DC, SP, and IJP are the methodologies selected in recent times to develop REs
that meet the technical requirements of this type of device.
3.1.1. Reference Electrodes: Structural Overview

As stated previously, the REs constructed using DC are based on a layered modification
of commercial SC or solid-state electrodes as base templates (further details can be found
in Section 3.1.3). As a result, the following structural overview addresses, for simplicity’s
sake, the three approaches (DC, SP, and IJP) considering the RE as a bottom-up layered
structure (Figure 3b,c).
The first reported Ag/AgCl REs presented up to three distinct functional layers [46,47].
The first layer is conductive and is responsible for allowing efficient electron transfer while
closing the electrical circuit. The second layer, referred as to the ion-to-electron layer, is
the one that contains Ag/AgCl and establishes a reference potential, as explained above
(Equations (1) and (2)). In addition to this basic two-layered structure, an additional
layer (the dielectric layer) is required for printed reference electrodes (PREs) to control
the functional area of each layer exposed to the measurement solution and to protect the
electrical connections pads.
Unfortunately, there is no component in its structure to offset the dilution-induced
decrease in [Cl− ] with time, which results in subsequent potential fluctuations, as indicated
by the Nernst equation. These characteristics are those of a pseudo-reference electrode,
limiting its field applications since environmental and human samples contain either
proteins or anions that affect the stability of their electrochemical potential considerably.
Considering all the limitations of the proposed approaches using pseudo-reference
electrodes, the next step in RE development was aimed at printing or casting any necessary
additional layers to improve reproducibility, repeatability, lifetime, and chloride ion concen-
tration susceptibility, with its performance resembling that of a traditional electrode glass
Ag/AgCl gel RE. Concerning this development, Andrade et al. [48] provided an alternative
for RE development that would solve the limitations of pseudo-reference electrodes. In
their work, the authors presented a glassy carbon (GC) drop casted RE, containing a layer
of Ag/AgCl alongside a membrane cocktail of polyvinyl butyral (PVB) and NaCl. The RE
showed a stable potential over a wide range of concentrations for several chemical species,
no significant drift, and a considerable lifetime of four months.
The reason why this electrode presented similar performance compared with a conven-
tional double-junction reference electrode can be interpreted from the standalone function-
ality of its components. First, the Ag/AgCl layer governs, in the absence of any secondary
redox couples, the ion-to-electron transduction process. Second, an excess of chloride ions
near the RE surface provides constant potential over time. However, this statement is only
correct if the concentration of chloride reaches a steady state during the experiment. Thus,
the role of the PVB layer is to sustain a fixed concentration of chloride and create a bridge
between the electrolyte inside and outside the membrane.
The proposed RE was later successfully incorporated into a fully-fledged, drop-
casted commercial carbon fibre sweat sensor presenting a near-Nernstian response
(55.9 ± 0.8 mV/log [Na+ ]) in artificial sweat containing sodium levels between 10−3 M
and 10−1 M [49], leading the scientific community to further develop the proposed scheme
by taking advantage of DC simplicity or adapting the structure via screen printing. As a
result, when compared to a pseudo-reference electrode, the new RE scheme includes an

additional layer, a reservoir, responsible for controlling the RE chloride concentrations and,
as a result, ensuring a stable half-cell potential (Figure 3c).
3.1.2. Reference Electrodes: Ongoing Advances

Numerous proposals have adapted the RE structure in Figure 3c, introducing slight dif-
ferences to optimize its electrochemical performance [38–45]. A summary of the drop-casted
and screen-printed reference electrodes discussed is provided in Table 2 for a better compar-
ison amongst them. The table includes performance and fabrication differences highlighted
with their advantages and disadvantages and their suitability for certain applications.
Table 2. Summary of the REs proposed in the recent literature. Notation: AGTPB—silver
tetraphenylborate, TBATPB—tetrabutylammonium tetraphenylborate, PVA—poly(vinyl acetate),
PDMS—polydimethylsiloxane, SG—silicone gel, PVdF—poly(vinylidene difluoride), MM—methyl
methacrylate, BM—butyl methacrylate, PBA—poly(butyl acrylate), NaTFPB—sodium tetrakis [3,5-
bis(trifluoromethyl)phenyl] borate, TDMA—tridodecylmethylammoniumchloride.
Structure Method Advantages Disadvantages Ref.

Suitable structure for solid state and Time/cost differences between the
flexible REs. Remarkable lifetime and development of reservoir layers
Ag|AgTPB|PVC +
SP, DC negligible potential drift towards with hydrophilic or organic [38]
TBATPB
various electrolytes even under insoluble salts require
mechanical stress. further addressing.
PDMS junction suppresses electrolyte Electrolyte stability comes at the
Ag|AgCl|KCl +
SP leaking, conferring potential stability expense of long hydration times [39]
PVA|PDMS
over a month and ion insensitivity. (30 min).
Stable Nernstian response even after Crosslinked PVA increases
Ag|AgCl|KCl + PVA DC five months over a wide pH hydration time. There are no assays [40]
range (1–10). regarding sensitivity to ions.
Negligible potential drift under a wide PVB cocktail requires a mix of four
Ag|AgCl|KCl + PVB IJP range of pH and Cl− concentration solvents. A limited library of [41]
ranges. Three-month stability. interferents has been evaluated.
Paper-based SPRE as an alternative to Complicated structural assembly.
Ag|AgCl|SG + PVdF SP counterparts developed in Very limited lifetime and stability [42]
plastic substrates. during measurements.
Insensitive near-Nernstian
Requires extended conditioning
potentiometric response to a wide range
Ag|AgCl|PVC+ NaTFPB IJP, DC and may suffer from unwanted [50]
of pH (±0.02 mV) and
structural changes in the process.
Cl− concentrations.
Not enough experimental data have
Negligible potential drift (±4 mV)
been presented to define whether
Ag|AgCl|KC + MM:BM SP under mechanical stress suitable for [43]
the proposed electrode is a
in-vivo pH sensing.
pseudo-RE or an RE.
Negligible potential drift under light Considerable technical and cost
exposure and a wide range of pH. requirements given the
Ag|AgCl|KCl + PVA DC [44]
Successful integration into a characteristics of the
miniaturized chip wearable device. electrochemical device.
Polymeric and lipophilic salt reservoir Short lifetime and high potential
Ag|AgCl|PBA +
SP confers low interference (9 mV) against drift under the presence of [45]
NaTFPB:TDMA
a wide spectrum of anions. perchlorate ions.
As detailed in Table 2, the literature review indicates that the development of reference
electrodes has gradually increased in both structural and functional sophistication. Starting
with those electrodes that present a similar structure to the RE introduced in Figure 3c,
Moya et al. [41] adapted the drop-casted RE proposed by Andrade et al. [48,49] via inkjet
printing, obtaining a stable and reusable RE composed of Ag|AgCl|KCl + PVB. The
electrode was produced by consecutive printing of several layers: a silver ink, which
was further chlorinated with NaClO printing, and a reservoir/protection layer, obtained
by printing a Cl− saturated polyvinyl butyral membrane (Figure 4A). It is pertinent to
Chemosensors 2023, 11, x FOR PEER REVIEW 11 of 47

note that, compared to the simplicity of the PVB cocktail used in DC, the reported PVB
cocktail required a mix of four solvents (methanol, butanol, xylene, and diacetone alcohol)
to successfully control both the inkjet printing and the drying processes. It has often been
Alternatively, Manjakaal [43] developed an SPRE on a polyethylene terephthalate
found that functional electrodes produced by drop casting can later be inkjet printed;
substrate, the reservoir of which consisted of KCl + MM:BM. The SPRE was sequentially
however, the requirements of inks in terms of surface tension, viscosity, and drying require
prepared by alternating stages of printing and chlorination to obtain a reproducible layer
modifications to their formulation, such as mixes of solvents or the addition of additives [51].
of Ag|AgCl to which the reservoir layer was added later (Figure 4D). The reported results
Regarding its performance, the Ag|AgCl|KCl + PVB presented negligible potential drift
indicated that the produced RE exhibited negligible potential drift (±4 mV) under mechan-
under a wide range of pH (3–10) and Cl− conditions (KCl, NaCl and CaCl2 ) compared to a
ical stress (bending states between 3 mm and 7 mm). However, the lack of experimental
double junction reference electrode, in addition three-month stability. Additional sensitivity
data regarding potential stability outside of limited experimental conditions (NaCl,
essays focused on ions suspected to be responsible for potential changes in the outer pH
H3PO4) does not allow for dismissing the possibility that the RE is operating as a pseudo-
values could be considered a pending aspect.
RE.
Figure 4.4.(A)(A)
Figure Fabrication process
Fabrication of an IJPRE
process of an protected by a printable
IJPRE protected by a PVB-based
printable membrane
PVB-based[41]. mem-
(B) Wearable sensor platform based on a solid-state ISE gold nanodendrite
brane [41]. (B) Wearable sensor platform based on a solid-state ISE gold nanodendrite electrode electrode and a
Ag|AgCl|KCl + PVA membrane-coated RE [44]. (C) (a) Photograph of Ag/AgTPB/PVC-TBATPB
and a Ag|AgCl|KCl + PVA membrane-coated RE [44]. (C) (a) Photograph of Ag/AgTPB/PVC-
under no bending and 60 and 90° bending. ◦(b,c) Scanning electronic microscopy images of Ag/Ag-
TBATPB under no flexible
TPB/PVC-TBATPB bendingSCREand 60 and 90 bending.
developed (b,c)
by Gan [38]. Scanning
(D) Image ofelectronic microscopy
the as-obtained images
thick-film
of Ag/AgTPB/PVC-TBATPB
Ag|AgCl|KC + MM:BM SCREflexiblereported SCRE developed[43].
by Manjakaal by Gan [38]. (D)and
(E) Structure Image of the as-obtained
composition of the
thick-film Ag|AgCl|KC
SPRE proposed + MM:BM
by Dawkins SCRE
et al. [39]. reported
Inset: ionic by Manjakaal
exchange [43]. (E)within
pathways Structure
the and composition
electrode and
between
of the proposed
the SPRE electrode by
andDawkins
the analytes.
et al. (A)
[39].Reprinted with
Inset: ionic permission
exchange from [41].
pathways Copyright
within (2019),and
the electrode
Americanthe
between Chemical Society.
electrode (B)analytes.
and the Reprinted (A)with permission
Reprinted withfrom [44]. Copyright
permission (2017),
from [41]. American
Copyright (2019),
Chemical Society.
American Chemical (C)Society.
Reprinted(B) with permission
Reprinted from [38]. Copyright
with permission from [44].(2022), American
Copyright (2017),Chemical
American
Society. (D) Reprinted with permission from [39]. Copyright (2018), WILEY-VCH Verlag GmbH &
Chemical Society. (C) Reprinted with permission from [38]. Copyright (2022), American Chemical
Co. KGaA, Weinheim. (E) Reprinted from [39], Copyright (2021), with permission from Elsevier.
Society. (D) Reprinted with permission from [39]. Copyright (2018), WILEY-VCH Verlag GmbH &
Co. KGaA, Weinheim.
Targeting another(E)type
Reprinted from [39],
of substrate for Copyright
this sensing (2021), with permission
platform, Wang et al.from[44]Elsevier.
devel-
oped a wearable solid-state ISE and RE on a miniaturized chip using a gold nanodendrite
(AuND) array electrode as the solid contact and a poly(vinyl acetate)/inorganic salt
Introducing slight differences to the formulation of the reservoir layer,

Macedo et al. [40] and Bananezhad et al. [50] described the construction of a novel RE with
exceptional long-term stability. In the former case, an RE was constructed by electrochemi-
cally coating a silver wire with AgCl in a 1 M KCl solution and further drop casting the
KCl + PVA membrane. This initial construction scheme was found to undesirably leak
Cl− from the reservoir layer to the measurement solution, leading researchers to add a
stage to the preparation process, consisting of a crosslinking step using ethylene glycol
dimethacrylate (EGDMA). The obtained RE demonstrated a performance comparable to
that of a commercial reference electrode, including a stable Nernstian response even after
five months over a wide pH range (1–10), being a structural trade-off diminishing the
Cl− leak ratio by 30% against moderate to long hydration times due to the crosslinking
process. Furthermore, the RE performance was compared to a that of a commercial RE in
electrochemical set-ups to determine pH, showing differences of less than 2% in complex
samples, such as tap water, sea water, and coffee.
On the other hand, Bananezhad et al. [50] developed a flexible solid-state RE for which
inkjet printing was used to fabricate the conductive path and silver patterns that were
chemically transformed into the Ag|AgCl electrodes. The as-obtained electrode was further
drop-casted with a membrane containing PVC, KCl, and NaTFPB. This IJPRE exhibited a
near-Nernstian potentiometric response to a wide range of pH (3–10) and seven different
electrolytes (NaNO3 , KNO3 , NaCl, NaF, CaCl2 , KBr, and KCl) over a broad concentration
range (10−6 to 10−1 M). Despite the aforementioned figures of merit and the reported four-
week shelf life, the IJPRE presented resistive electrochemical behaviour, requiring up to 3 h
to reach a stable potential without preconditioning. As a counterpart to the requirement of
extended preconditioning in 1 M KCl, this process could cause unwanted changes in its
porous arrangement, affecting the ion exchange on the membrane-solution boundary.
Alternatively, Manjakaal [43] developed an SPRE on a polyethylene terephthalate
substrate, the reservoir of which consisted of KCl + MM:BM. The SPRE was sequentially
prepared by alternating stages of printing and chlorination to obtain a reproducible layer
of Ag|AgCl to which the reservoir layer was added later (Figure 4D). The reported results
indicated that the produced RE exhibited negligible potential drift (±4 mV) under mechan-
ical stress (bending states between 3 mm and 7 mm). However, the lack of experimental
data regarding potential stability outside of limited experimental conditions (NaCl, H3 PO4 )
does not allow for dismissing the possibility that the RE is operating as a pseudo-RE.
Targeting another type of substrate for this sensing platform, Wang et al. [44] devel-
oped a wearable solid-state ISE and RE on a miniaturized chip using a gold nanoden-
drite (AuND) array electrode as the solid contact and a poly(vinyl acetate)/inorganic salt
(PVA/KCl) membrane-coated RE. The Ag|AgCl|KCl + PVA RE was fabricated through the
sequential drop-casting of a Ag|AgCl commercial ink, followed by a KCl + PVA composite
(Figure 4B). Potentiometric results of the obtained RE indicated a lack of potential drift
under light exposure, a wide range of pH, and different ions (Cl− , NO3 − , SO4 2− ) over a
broad concentration range from 10−1 to 10−6 M. Additionally, on-body trials performed us-
ing a sweat headband indicated that the proposed sensor constitutes a reliable platform for
Na+ monitoring, requiring 20 min of pre-conditioning in 3 M KCl and standard calibration
in a concentration range of 10−4 –10−1 M to avoid initial potentially noisy signals.
While Komoda et al. [42] successfully fabricated a paper-based, planar-type RE using
an Ag|AgCl commercial ink alongside a silica gel-poly(vinylidene difluoride) ink, the latter
fulfilled the role of a hydrophilic liquid junction and electrolyte layer. The as-prepared
Ag|AgCl|SG + PVdF SPRE presented a pseudo-Nernstian response in NH4 Cl, KNO3 ,
NaHCO3 , and K2 CO3 solutions, reaching a stable potential within 5 min. Unfortunately,
due to either computer-aided design requirements or limited results regarding lifetime and
stability, there is a lack of recent proposals continuing this line of work.
After this first instance in the literature review, the abundance of reports of REs
based on successive Ag|AgCl and polymer/electrolyte composite layers may indicate
either a solved case or a lack of suitable experimental alternatives. However, the works of
Alva et al. [45], Gan et al. [38] and Dawkins et al. [39] indicate that there are other viable
strategies outside introducing slight changes to the polymeric composition of the reservoir
layer to enhance the analytical figures of merit of an RE. Alva et al. aimed to replace the
standard composition of the reservoir layer by screen-printing an ion-permeable membrane
consisting of a photocurable acrylic ink that contained immobilized lipophilic Na+ and
Cl− salts (NaTFPB and TDMA-Cl). By optimizing the ratio between the two salts (1:1), the
authors reported that the RE presented low and time-dependent potential drift towards
KCl, ranging from 10−6 M to 1 M, changing the potential drift value from 1 to 10 mV
after 7 h. This phenomenon was attributed to an uneven charge distribution inside the
reservoir layer, an unwanted by-product of using lipophilic salts. Despite the reported
issues, the SPRE exhibited remarkable insensitivity towards NO3 − , Br− , and SO4 2− within
a concentration range of 0.1 M to 10−8 M.
Gan et al. [38] focused their efforts on minimizing the risk of electrolyte leakage, re-
placing a hydrophilic electrolyte in the reservoir layer polymer matrix with a silver organic
insoluble salt (AgTPB). The SPRE consisted of an Ag|AgTPB substrate and a plasticized
PVC membrane containing TBATPB, another hydrophobic organic salt (~0.26 µM in water),
to further prevent leaking (Figure 4C). Functionally, the proposed electrode worked follow-
ing the basic principle of a standard Ag|AgCl-based SPRE, in which the fixed potential is
given as a product of an electron-coupled ion transfer reaction mechanism that occurs at the
Ag|AgTPB and AgTPB|PVC + TBATPB interfaces (AgTPB (s) + e−
Ag (s) + TPB− (PVC)).
The reported results indicate that the Ag|AgTPB SPRE potential stability was tested
against various electrolyte solutions (~4.8 mV), light (~0.5 mV), gas (N2 , O2 and CO2 ,
~5 mV), and redox interference (K3 [Fe(CN)6 ]/K4 [Fe(CN)6 , ~5 mV), showing negligible
potential drift as indicated respectively in parentheses. As for potential responses to
electrolytes (NaCl, LiCl, KCl, NH4 Cl and KNO3 ) within a concentration range from
10−5 to 10−1 M, the electrodes showed potential variations of 4.6 mV ± 0.8 mV and
10.6 mV ± 1.8 mV, respectively, with the significant drift increasing under high ionic
strength, adjudicated to the unstable phase boundary potential between PVC|TBATPB
and water interfaces due to differences in ion distribution in the two immiscible phases.
Furthermore, the SPRE, alongside a mini-potentiometer and a mobile battery, was tested as
a wearable sweat sensor, presenting a Nernstian response towards Cl− , allowing for high
accuracy Cl− determinations, with relative error of 7.0% compared to other standardized
methods, such as ion chromatography.
Finally, Dawkins et al. [39] prioritized solving Cl− leaking issues from the reservoir
spectrum, adding an additional layer to the traditional structure of SPREs. In this case,
SCREs were fabricated on PET substrates printing sequential layers containing an Ag|AgCl
commercial ink, an electrolyte composite containing KCl and PVA, and a PDMS membrane
(Figure 4E). The latter, due to balanced permeability and hydrophobicity, acts as a liquid
junction and dielectric protective coating able to increase the RE lifetime and stability
through electrolyte retention. The previous statement was supported via a thorough
report containing a structural characterization via SEM micrographs and EDS scans for the
analysis of the electroanalytical figures of merit of the SCRE.
Results concerning the potential stability and lifetime through OCP measurements
in 0.1 M K2 SO4 indicated low drift rates (0.25–0.05 mV/day) at up to three months being
observed only when an additional PDMS layer was present, while Ag|AgCl|KCl + PVA
SCRE failed to present a consistent Nernstian response owing to KCl particle loss and
compositional changes. Moreover, the disclosed SCRE insensitivity to various interferents,
such as halide/alkali ion species (up to 5 mV of potential drift), and a wide range of
pH conditions (+0.29 mV/pH), further highlights, compared to the recent literature, the
effectiveness of the PDMS layer. Withal et al., as well as Macedo et al. [40], proposed an
SPRE used in combination with a commercial pH probe to determine the pH of complex
samples, such as tap water, creek water, and coffee, showing good agreement with a
commercial pH meter. However, it required long hydration times that could reach up to
90 min depending on the sample characteristics, an expected disadvantage attributed to the
addition of a PDMS layer that should be weighed, depending on the sensing requirements,
against improvements in potential stability and lifetime.
3.1.3. Reference Electrodes: Lingering Challenges

As presented in this section, considerable work has been performed regarding the
development of reference electrodes. Regarding the aforementioned publications, many of
them came both functionally and structurally as close as needed to replace the conventional
glass silver/silver chloride gel RE. Moreover, a small number have managed to make the
leap from proof of concept to application in context.
However, there is still room for development in this field if we consider the current
state of the art in three interconnected requirements. The first one refers to the reduction in
costs linked to the manufacturing process, considering that works up to 2021 continued
to ground within their novel aspects the presentation of a low-cost experimental design.
According to recent literature [34], alternatives to the macroscale conventional RE should
be at least an order of magnitude cheaper, while presenting remarkable potential stability
and an extended lifetime. The latter constitute the two additional requirements and can be
further improved, reducing the overall costs in the process.
From the prior works, it is possible to state that, from the chloride concentration on
the reservoir layer to the composition and thickness of a polymeric layer to reduce ion
leakage, many structural elements have significant effects on the RE’s potential stability
and lifetime. Regarding potential stability, Sophocleous [34] presented extensive research
regarding the influence of the KCl to polymer weight ratios on the reservoir layer of REs,
identifying significant differences in both base potential at physiological pH and potential
drift towards chloride concentrations. Despite indicating that the optimization of said
layer is a crucial aspect, not all recent proposals that used diverse polymeric reservoirs
performed this needed characterization. The proposed layer optimization study is even
more relevant considering the recent alternatives to hydrophilic reservoirs by the addition
of insoluble organic salts. Furthermore, given the reported results after chlorination of
a silver layer [41], researchers may even evaluate intervening in previous stages of its
assembly, such as studying the possible effects over potential drift of replacing the use of
highly reproducible commercial Ag/AgCl inks with the controlled synthesis of composites
such as Ag@AgCl [52], reducing overall costs.
From a durability standpoint, while optimizing conditions for the reservoir layer can
also enhance the RE lifetime, the presence of an additional permeable and hydrophobic
epoxy-based junction layer on top of the RE was reported to dramatically increase its
lifetime [39]. Interestingly, this assembly proposal with an additional layer has been
previously described as a suitable alternative for RE development [34,35], but to date, a
majority of the reported reference electrodes have adhered to the structure proposed in
Figure 3b.
In closing, it is important to emphasize that the alternatives proposed for the future
development of RE could present a trade-off with other functional characteristics, such as
hydration times, which should be evaluated considering the requirements of the targeted
field of application. For instance, some applications resolved through disposable sensing
platforms may require an RE with high selectivity and short hydration times instead of
long-term stability. Therefore, considering the imbalance between proof of concept and
field-applied works, future RE structures should be designed from square one to meet the
requirements of a specific application, instead of prioritizing the development of an RE that
excels in all functional domains.
3.2. Ion-Selective Electrodes

Ionophore-based ion-selective electrodes (ISEs) are widely used for the determination
of ion activity in applications employed in fields such as environmental monitoring, life
science, process control, and, in particular, routine clinical analyses. These ISEs deliver
long-term stable potentials, and their electrode-to-electrode reproducibility is excellent, but
the miniaturization is not straightforward. Therefore, much effort has been devoted in past
years to the development of solid-contact ISEs.
3.2.1. ISEs Structural Overview

Solid-contact ISEs are comprised of an electric contact, an ion-to-electron transducer,
and a selective membrane that only allows the diffusion of the target ion to be sensed.
Regarding the most targeted ions in sweat, sodium and potassium, the component
of the membranes has the following components: a polymer, a plasticizer, a counterion
to the target ion, and an ionophore. The polymer provides a hydrophobic matrix with
a low glass transition temperature, forming a stable surface and providing mobility of
organic salts and sufficient electric conductivity of the membrane. The plasticizer decreases
the glass transition temperature, increasing therefore the ion mobility and acting as a
solvent for ionophores, preventing their phase separation. Then, the counterion provides
ionic sites for the target ion, and it is usually a hydrophobic molecule that remains in the
hydrophobic polymer matrix, preventing its release into the medium. Finally, the function
of the ionophore is to complex the target ion, increasing its solubility in the membrane [53].
For other analytes, e.g., hydrogen ions, there are electrodes in which the sensitive
component is the ion-to-electron transducer, such as in the case of polyaniline (PANI) [54],
or oxides [55].
3.2.2. ISEs: Ongoing Advances

In recent years, much research has been directed towards the development of novel
ion-to-electron transducers, aiming to increase the sensitivity, by either improving con-
ductivity or by increasing the surface area on which the selective membrane is applied.
Additionally, there has been much work in the development of conductive substrates to im-
prove integration into flexible or small devices, such as textiles, wearables, or microfluidic
devices. A collection of some of the latest reported ISEs is presented in Table 3.
Table 3. Summary of the ISEs proposed in the recent literature. Notation: LOD—limit
of detection, LR—linear range, PET—polyethylene terephthalate, PVC—polyvynilchloride,
KTPFB—potassium tetrakis(pentafluorophenyl)borate, DOS—dioctyl sebacate, KTClPB—
potassium tetrakis(4-chlorophenyl)borate, NiCAT—nickel triphenylene-fused metal catecholate,
NaTFPB—sodium tetrakis[3,5-bis(trifluoromethyl)phenyl] borate, NaBARF—sodium tetrakis[3,5-
bis(trifluoromethyl)phenyl]borate, NPOE—nitrophenyl octyl ether, PANI—polyaniline, PEDOT:PSS—
poly(3,4-ethylenedioxythiophene) polystyrene sulfonate.
Ion-To-Electron Ion-Sensitive
Analyte Substrate Figures of Merit Ref.
Transducer Membrane
pH: sensitivity = 54 mV/dec
Mix: β-cyclodextrin
Commercial conductive K+ : valynomicin, KTPFB, LOD = pH 10
K+ and rGO [56]
ink printed on PET PVC, DOS K+ : sensitivity = 56 mV/dec
(pH-sensitive)
LOD = 10−6.2
Stencil-patterned carbon K+ : sensitivity = 56.1 mV/dec
K+ : valynomicin,
K+ electrode on PET, carbon LOD = 10−5 M [57]
KTClPB, PVC, DOS
black-modified LR = 10−4 to 10−1 M
Na+ : sensitivity = 58.7 mV/dec
CNT fibres on an elastic NiCAT coated with Na+ : sodium ionophore
Na+ LOD = 10−6 M [58]
band Nafion X, NaTFPB, PVC, DOS
LR = 10−5 to 10−1 M
Nafion-covered Na+ : Na ionophore X,
Na+ Gold electrodes on PET Na+ : sensitivity = 65.1 mV/dec [59]
porous 3D graphene NaBARF, PVC, NPOE
Crown-ether-
Screen-printed Na+ and K+ : 42 mV/dec, but
Na+ and K+ functionalized graphene [60]
commercial electrode no selectivity
quantum dots
Table 3. Cont.
Ion-To-Electron Ion-Sensitive
Analyte Substrate Figures of Merit Ref.
Transducer Membrane
pH: 4-nonadecylpyridine,
H+ RGO dry-spun fibres Ferrocene pH: 55 mV/dec [61]
PVC
Screen-printed carbon
Cl− PANI, acrylic binder pH: sensitivity = 66 mV/dec [54]
electrodes
Na+ : Na ionophore X,
pH: PANI pH: sensitivity = 51.5 mV/dec
Laser-induced-graphene NaTFPB, PVC, DOS
H+ , Na+ and K+ Na+ and K+ : Na+ : sensitivity = 45.4 mV/dec [62]
on Kapton K+ : valynomicin,
PEDOT:PSS K+ : sensitivity = 43.3 mV/dec
KTClPB, PVC, DOS
Graphite-
Na+ Leather Na0.44 MnO2 Na+ : sensitivity = 58 mV/dec [63]
mix
Among the works presented in Table 3, there is a group of sensors, the electrodes of
which are manufactured applying layers on a substrate, such as PET [56,57,59], Kapton [62],
and even leather [63]. These substrates provide flexibility, although the integrity and
functionality of the sensors must be checked during or after the bending to assure flexibility.
In the other group of sensors, the electrodes are manufactured using conductive fibres as a
substrate, e.g., carbon nanotube fibre (CNTF) [58] or RGO fibre [61].
Even though there are various combinations of membranes, transducers, and con-
ductive electrodes, most of the sensitivities reported are around the Nernstian behaviour
(59 mV/dec).
The simplest electrode to measure potassium is the one presented by Henry et al. [57],
comprising a carbon electrode applied with a stencil on PET and then covered by differ-
ent cocktails of valynomicin, PVC, plasticizers (dioctyl sebacate (DOS), or nitrophenyl
octyl ether (NPOE)) and additives (potassium tetrakis(4-chlorophenyl)borate (KTClPB)
or sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB)) in THF. This work
compares the addition of carbon black prior to the sensitive membrane, showing that
this approach increases the surface area of the electrode and hence its sensitivity. For the
membrane cocktail with the highest sensitivity (56 mV/dec), the standard deviation for
five sensors is about 1.25%. This work also shows that the limit of detection (LOD) for
potassium is as low as 10−5 M with a linear range between 10−4 and 10−1.1 M, compatible
with normal values in human sweat.
Subsequently, Gao [59] presented a sensor for sodium in sweat. To enhance the
sensitivity, they synthesized 3D porous graphene by chemical vapour deposition (CVD).
This graphene has a gradient-like porosity that increased dramatically the electrochemically
active surface area. Then, this carbonous material was deposited on a gold electrode, and a
sodium selective ionophore solution was casted on top of that. Using this approach, they
claimed to achieve the highest sensitivity for sodium ions, 65.1 mV/dec.
Another carbon-based potassium sensor was created by Wu [64], without an ion-
electron transducer. In this work, the conductive electrode was of porous carbon resulting
from laser engraving of an adhesive layer of PI. On top of this layer, a selective membrane
of valynomicin, PVC, sodium tetraphenylborate (NaTPB), and DOS was casted. This work
is interesting because they evaluated the effects of different bending angles, up to 60◦ , and
tested the performance after 8000 bending cycles. The sensor experienced no significant
reduction in sensitivity (58.1 mV/dec before bending and 57.4 mV/dec afterwards). A
drawback of this sensor is that the calibration curve is displaced when the sensor is bent,
so the measurement must be performed at a constant angle to be reliable.
Another group of sensors involves the use of ion-electron transducers applied on
top of the conductive electrode. Sun et al. [56] worked on a flexible sensor in which a
β-cyclodextrin functionalized graphene (β-CD/RGO) was printed on a conductive layer
with a commercial ink on PET. The combination of β-CD with the graphene provides
many oxygen functional groups, and it possess good capacitance, giving this material
pH sensitivity ranging from 48 mV at a 0◦ bending angle to 51 mV/dec at a 90◦ bending

angle. This material also acts as ion-electron transducer, which was tested by applying
a potassium selective membrane, with a response of 55 mV/dec regardless of a bending
angle between 0◦ and 90◦ . These sensors were used to build a wearable device for on-body
sweat analysis. The linear response had no significant fluctuations before or after the test,
indicating that the on-body analysis data were reliable.
Conductive polymers were used as ion-electron transducers for several analytes.
Moreover, Lin et al. [62] proposed a platform using laser-induced graphene electrodes
covered with PEDOT:PSS and ion-selective membranes to measure Na+ and K+ and PANI
to measure pH. In this work, they built a multiplexed device for real time sweat analysis
with Bluetooth communication using an app in a smartphone. The sensitivities achieved
for pH, Na+ , and K+ were 51.5 mV/dec, 45.4 mV/dec, and 43.3 mV/dec, respectively. The
sensors showed no apparent change in the potential response upon bending, and they were
tested by a subject on a stationary bicycle for almost 2 h.
PANI was also used by Bilbao et al. [54] to measure the pH of synthetic sweat. This
work presents a scalable process to manufacture sensors via screen-printing and inkjet-
printing techniques, and the PANI is mixed with acrylic binders to increase the wear
resistance. While the authors did not perform on-body testing, the adhesion and friction
resistance were measured and compared to electrodeposited PANI, showing that the addi-
tion of binder increased the resistance without compromising high sensitivity (62 mV/dec)
and selectivity.
Khoshsafar [63] presented a sensor for sodium detection using a mix of graphite
and Na0.44 MnO2 . To achieve flexibility at low cost, this paste was applied on a leather
band. Na0.44 MnO2 was used because it has a crystalline structure that allows for Na+
accommodation and provides sensitivity. Graphite is a conductor, and PVDF is used as
a binder, with no effect on the potentiometric behaviour. This sensor has sensitivity of
58 mV/dec, and the authors tested it both off-body and on-body using a headband. This
band uses a hydrophobic coating to direct the collected sweat towards the sensing area.
A different approach for sensor manufacturing and integration consists of building
the sensor on a conductive fibre. In this regard, Omenetto et al. [61] used dry-spun fibres of
pure reduced graphene oxide (RGO) as a substrate. They reported the functionalization
of these fibres with ferrocene, which increased the ion-electron transduction, enhancing
the sensitivity. The fibres were functionalized with a membrane consisting of PVC and a
pH ionophore, 4-nonadecylpyridine, applied by spray coating. The sensitivity increased
from 48.3 mV/dec without ferrocene to 55.0 mV/dec when it was present. The variation in
sensitivity from sensor to sensor was low for both types of sensors, suggesting that fibre
fabrication and functionalization produced sensors with high reproducibility.
Finally, Wang et al. [58] presented another fibre-based sensor constructed of carbon
nanotube fibre (CNTF) and a metal-organic framework (MOF), as shown in Figure 5.
Vertically aligned nanowires of nickel triphenylene-fused metal catecholate (NiCAT) were
grown in situ through a one-step hydrothermal reaction. The nanowires were coated
with a thin layer of Nafion and then covered with an ion-selective membrane. In this
work, a sodium-selective membrane was used, providing sensitivity of 58.7 mV/dec. The
combination of Nafion and NiCAT nanowires yielded a well-defined capacitive behaviour
for efficient ion-electron transduction. Additionally, the Nafion prevented the accumulation
of water between the MOF and the ion-selective membrane.
3.2.3. Ion-Selective Electrodes: Lingering Challenges

According to the reviewed literature, large numbers of works have shown multiple
sensors with proven abilities to measure ions in sweat with on-body testing or at least
constructed of flexible substrates that ease integration into wearable devices.
A next step in research and development could be directed towards the life cycle of
these devices, aiming for long-term usability. Some aspects that could be worked on are
wear resistance and endurance studies when sensors are exposed for long periods of time to
sensitivity from sensor to sensor was low for both types of sensors, suggesting that fibre
fabrication and functionalization produced sensors with high reproducibility.
Finally, Wang et al. [58] presented another fibre-based sensor constructed of carbon
nanotube fibre (CNTF) and a metal-organic framework (MOF), as shown in Figure 5. Ver-
tically aligned nanowires of nickel triphenylene-fused metal catecholate (NiCAT) were
Chemosensors 2023, 11, 244 grown in situ through a one-step hydrothermal reaction. The nanowires were coated with 17 of 46
a thin layer of Nafion and then covered with an ion-selective membrane. In this work, a
sodium-selective membrane was used, providing sensitivity of 58.7 mV/dec. The combi-
nation of Nafion and NiCAT nanowires yielded a well-defined capacitive behaviour for
sweat or washing solutions. While these aspects are considered in the short-term on-body
efficient ion-electron transduction. Additionally, the Nafion prevented the accumulation
tests, it could be interesting to perform a more systematic analysis.
of water between the MOF and the ion-selective membrane.
Figure
Figure5.5.Schematic
Schematicillustrations
illustrationsof of
thethe
fabrication andand
fabrication structure of the
structure of NiCAT@CNTF-based
the NiCAT@CNTF-based ISE ISE and
and RE [58]. (A) One-step in-situ growth of NiCAT on CNTF in a liquid containing the organic
RE [58]. (A) One-step in-situ growth of NiCAT on CNTF in a liquid containing the organic linker of linker
of HHTP and Ni(II). (B) Schematic profile of NiCAT@CNTF-based ISE and RE in an electrochemical
HHTP and Ni(II). (B) Schematic profile of NiCAT@CNTF-based ISE and RE in an electrochemical cell.
cell. Reprinted from Ref. [58]. Copyright (2022), with permission from Elsevier.
Reprinted from Ref. [58]. Copyright (2022), with permission from Elsevier.
3.2.3. Ion-Selective Electrodes: Lingering Challenges
4. Sensing Metabolites in Sweat with Amperometric Enzyme Electrodes
According to the reviewed literature, large numbers of works have shown multiple
Amperometric
sensors enzymatic
with proven abilities biosensors
to measure are
ions in usually
sweat withemployed to detect
on-body testing or at small
least organic
molecules, such as metabolites, in biological fluids, such as sweat,
constructed of flexible substrates that ease integration into wearable devices. tears, exhaled breath,
and Asaliva, for diagnosis, treatment, control of medical conditions, and
next step in research and development could be directed towards the life cycle of fitness applica-
tions devices,
these [65]. A biosensor
aiming for consists
long-termofusability.
three main components:
Some aspects that acould
bioreceptor,
be workeda transducer,
on are and
wear resistance and endurance studies when sensors are exposed for long periods
a signal processor [66]. The bioreceptor is composed of a biological recognition element (anof time
to sweat oran
enzyme, washing solutions.
antibody, While
a protein these aspects
receptor, DNA,areor considered
whole cells)in the
thatshort-term
recognizes on-the target
body tests, it could be interesting to perform a more systematic analysis.
analyte. The transducer converts the recognition event into a measurable signal (electrical,
optical, thermal, electrochemical, or piezoelectric). Biosensors aiming for the determination
4. Sensing Metabolites in Sweat with Amperometric Enzyme Electrodes
of metabolite levels in sweat usually employ an enzyme that specifically recognises it
and catalyse a reaction, the rate of which can be effectively followed by amperometric
or voltammetric techniques. Enzyme electrodes have been used to detect glucose [67],
lactate [24,65,68,69], ethanol [29], and levodopa [70], among other substances. Table 4
summarizes the metabolites detected by amperometric sweat sensors, their physiological
concentrations, enzyme types, electrode characteristics, detection limits, sensitivity, linear
ranges, operation, storage stability, and related diseases.
Redox mediators are utilized to transfer electrons between the enzyme and the elec-
trode. Mediators can be immobilized onto the surface of the transducer, together with the
enzyme. Prussian blue is a widely used redox mediator in sweat sensors for the detection
of various analytes, including glucose [33], lactate [68], and ethanol [29]. However, direct
electron transfer is also possible between the active side of the enzyme and the transducer
without the need for a mediator [71]. Mediator-free sweat sensors have also been used
in sensors to determine analytes, such as glucose [72] and ascorbic acid [73]. Regarding
the construction of the electrodes, similar approaches to those used for potentiometric
measurements have been followed, with the extensive use of screen-printed electrodes
modified by drop casting or inkjet printing. Fully inkjet-printed biosensors are particularly
adequate for sweat biosensing since they can be printed on flexible substrate, although
the integration with the electronic processing circuit still presents some challenges (see
Section 6 on electronic instrumentation).
Table 4. Enzyme electrode biosensors for sweat analysis. References: ACA: anisotropic conductive adhesive; AOx: alcohol oxidase; HRP: horseradish peroxidase;
LOx: lactate oxidase; MWCNTs: carboxy-functionalized multiwalled carbon nanotubes; PB: Prussian blue; PDMS: polydimethylsiloxane; PP: polypropylene; PPD:
poly-m-phenylenediamine; SPCE: screen-printed carbon electrodes; SPPB: screen-printed Prussian blue conductive carbon; 3D-PMED: 3D paper-based microfluidic
electrochemical integrated device; NA: not available.
Operation
Relative Linear
Redox Electrode Detection Data Response Stability; Disease
Analyte Content in Enzyme Electrode Material Range; Sample Ref.
Mediator Substrate Limit Acquisition Time Storage Correlation
Sweat Sensitivity
Stability
CNT fibber electrode 0–200 µM; Sweat after
GOx Prussian Blue (CE, WE), Ag/AgCl Fabric 2.15 nA NA 10 min of Bluetooth 30 s NA [67]
10–200 mM
Glucose fibre (RE) µm−1 exercise Diabetes
[2]
Prussian
0–1.9 mM; Sweat from
Blue/graphite ink
Paper 35.7 forehead
GOx Prussian Blue (WE), SP graphite 5 mM Wires 60 s NA [33]
(3D-PMED) mAmM−1 during
ink (CE) Ag/AgCl
cm−2 cycling
(RE)
0–600 µM;
PPD/PtNP/Au/ACA
GOx Mediator-free ACA 15.1 0.9 µM Iontophoresis Bluetooth 60 s 10-h; NA [72]
(WE), Ag/AgCl (RE)
µA/mMcm−2
Carbon/GMgOC 0–50 mM Wires and
1,2- Artificial Cystic
LOx (WE), carbon (CE), PDMS (36.2 µA 0.3 mM sticker-based 2–6 min NA [69]
naphthoquinone sweat
Lactate 5–20 mM [2] and Ag/AgCl (RE) mM−1 cm−2 ) connector fibrosis, stress
Ag/AgCl (RE), ischemia,
Sweat from lactic acidosis
graphite paste (CE, 0–25 mM
LOx, forehead Intervals during
Os-complex WE), and WE PP (0.74 µA 0.04 mM Wires 60 s [24]
HRP during 6-h; NA
modified with mM−1 )
cycling
MWCNTs
1–222 µM
Sensitivity
(40.6 µA Sweat from
remained 88.3%
Ag/AgCl (RE), Flexible mM−1 cm−2 ) forehead
LOx Prussian Blue 0.8 µM Bluetooth 100 s after multiple [68]
SPCE (CE) substrate 0.222–25 mM while
use in 20 days;
(1.9 µA walking
NA
mM−1 cm−2 )
Table 4. Cont.
Operation
Relative Linear
Redox Electrode Detection Data Response Stability; Disease
Analyte Content in Enzyme Electrode Material Range; Sample Ref.
Mediator Substrate Limit Acquisition Time Storage Correlation
Sweat Sensitivity
Stability
SPPB conducting Alcoholism,
carbon, AOx, Sweat by hepatitis B,
0–40 × 10−3 At least 10
Ethanol 2–30 mM AOx Prussian Blue chitosan (WE), SPPB Tattoo NA 5-min Bluetooth 60 s diabetes, [29]
M; NA repetitions; NA
conducting carbon iontophoresis drunk
(CE), Ag/AgCl (RE) driving
Tumours,
cancer,
Ag/AgCl (RE), Sweat 2 h after
Ascorbic Ascorbate Tattoo 0−1000 µM; kidney
10–50 mM [2] Mediator-free SPCE (CE), and NA stimulation Wires 60 s ingesting [73]
acid oxidase polyurethane NA disease,
Rh-SPCE (WE) of forearm vitamin C; NA
thrombosis,
stones
Parkinson’s
Au nano dendrites
Sweat by disease
Dose Thionine on Au/Cr tyrosinase 1–1000 mM;
Levodopa Tyrosinase PET 1 µM 5-min Wires 200 s NA monitoring [70]
dependent acetate (WE), Ag/AgCl 1.7 nAmM−1
iontophoresis and
(RE), and Au (CE)
optimization
Several drawbacks, such as enzyme immobilization, high cost, low operation stabil-
ity due the changes in pH, temperature, and humidity dependence, are currently the main
issues
Chemosensors 2023,to be resolved, especially for the development of continuous monitoring sensors.20 of 46
11, 244
Hence, researchers are also currently working to develop high performance nonenzymatic
sensors [74].The current challenges presented by amperometric sweat sensors are related
Several drawbacks, such as enzyme immobilization, high cost, low operation stability
to the operational stability and long-term
due the changes stability of
in pH, temperature, functional
and materials, the
humidity dependence, are effect of pH
currently the main
variations in sweatissues on the sensing
to be device,
resolved, signal
especially fluctuation
for the developmentcaused by themonitoring
of continuous bending sensors.
of
wearable sensors, chemical interference, and the use of suitable materials in continuous
Hence, researchers are also currently working to develop high performance nonenzymatic
monitoring and sampling sensors [74].
[4]. The current challenges presented by amperometric sweat sensors are related
to the operational stability and long-term stability of functional materials, the effect of
pH variations in sweat on the sensing device, signal fluctuation caused by the bending of
4.1. Immobilization Strategies in the Development
wearable sensors, of Enzymatic
chemical interference, and theBiosensors
use of suitable materials in continuous
monitoring and sampling [4].
Enzymes that are immobilized onto solid substrates exhibit improved resistance to
harsh conditions, such as low or high
4.1. Immobilization pH,intemperature,
Strategies the Development and dryness,
of Enzymatic both during their
Biosensors
use in biosensors and during
Enzymesstorage,
that areand they alsoonto
immobilized have enhanced
solid substratesstability over time
exhibit improved [74]. to
resistance
Various immobilization
harsh strategies are usually
conditions, such as low oremployed on sweat sensors.
high pH, temperature, and dryness, both during their
Figure 6 summarizes the main
use in biosensors andstrategies used
during storage, andfor bioreceptor
they immobilization
also have enhanced stability over[51]:
time [74].
Various immobilization strategies are usually employed on
adsorption, covalent immobilization, and entrapment. In some cases, enzyme immobili- sweat sensors.
Figure 6 summarizes the main strategies used for bioreceptor immobilization [51]: ad-
zation protocols aresorption,
based covalent
on the immobilization,
combination and of several immobilization methods. Phy-
entrapment. In some cases, enzyme immobilization
sisorption can be attained by simple exposure of the electrode to a bioreceptor
protocols are based on the combination of several immobilization suspension;
methods. Physisorption
however, the interaction may bebyweak
can be attained simpleand unspecific,
exposure and usually
of the electrode other means
to a bioreceptor are pre-
suspension; however,
the interaction may be weak and unspecific, and usually other
ferred for the immobilization of molecular bioreceptors based on covalent bonds or highlymeans are preferred for the
specific and strong immobilization
interactions. of molecular bioreceptors based on covalent bonds or highly specific and
strong interactions.
Figure 6. Schematic
Figure 6. Schematic representation of representation
the most commonof the most commonused
methods methods used to immobilize
to immobilize bioreceptors in
bioreceptors
in the development ofthe development
sweat of sweat
biosensors. biosensors. Physisorption
Physisorption relies on intermolecular
relies on intermolecular interactions
interactions between between
the bioreceptor (red) and the electrode (black). Most common methods for covalent immobilization
the bioreceptor (red) and the electrode (black). Most common methods for covalent immobilization
rely on the formation of amide or imine bonds. Bioreceptors can be also entrapped by encapsulation
rely on the formation of amide or imine bonds. Bioreceptors can be also entrapped by encapsulation
within a hydrogel (light blue).
within a hydrogel (light blue).
Enzyme physisorption onto solid substrates represents the easiest method of physical
immobilization.
Enzyme physisorption ontoPhysical adsorption consists
solid substrates representsof thethe
simple deposition
easiest method of anofenzyme
physi-onto a
surface and its attachment through weak bonds. The adsorption mechanisms are based on
cal immobilization. Physical adsorption consists of the simple deposition of an enzyme
weak bonds, such as Van der Waal forces and electrostatic and/or hydrophobic interactions.
onto a surface and This
its attachment
technique may through weak
not involve any bonds. The adsorption
functionalization mechanisms
of the substrate are does
and generally
based on weak bonds, sucha as
not have Van dereffect
significant Waalon forces and activity.
the enzyme electrostatic and/or physisorption
Nevertheless, hydrophobicimmo-
interactions. This technique may
bilization may havenotpoor
involve any functionalization
operational and storage stabilityof the substrate
because and by
it can be affected
fluctuations in temperature, pH, and ionic strength, leading
generally does not have a significant effect on the enzyme activity. Nevertheless, phy- to the desorption of loosely
bound enzymes.
sisorption immobilization may have poor operational and storage stability because it can
Enzymes can also be electrostatically immobilized onto charged surfaces, taking ad-
be affected by fluctuations
vantage ofinthe
temperature, pH,charge
intrinsic surface and ionic
of thestrength,
material orleading
coatingtothethe desorp-
electrode with a
tion of loosely bound enzymes. with a charge opposite to that of the enzyme at a certain pH. Layer-by-layer
polyelectrolyte
immobilization
Enzymes can also based on electrostatic
be electrostatically immobilized adsorption is widely surfaces,
onto charged used in thetaking
development
ad- of
enzymatic biosensors. For example, wearable fabrics for real-time glucose monitoring were
vantage of the intrinsic surface charge of the material or coating the electrode with a poly-
developed using sensing fibres of CNT with Prussian blue as a redox mediator. Then, the
electrolyte with a charge opposite
CNT electrode wasto that with
coated of the enzyme
chitosan, andatglucose
a certain pH.(GOx)
oxidase Layer-by-layer
was immobilized
immobilization based on electrostatic
by electrostatic adsorption
interactions is al.
[67]. Cao et widely useda paper-based
developed in the development
microfluidic of
electro-
enzymatic biosensors. For example, wearable fabrics for real-time glucose monitoring
were developed using sensing fibres of CNT with Prussian blue as a redox mediator.
Then, the CNT electrode was coated with chitosan, and glucose oxidase (GOx) was im-
mobilized by electrostatic interactions [67]. Cao et al. developed a paper-based microflu-
idic electrochemical device for glucose detection, coating the carbon electrode with a
chemical device for glucose detection, coating the carbon electrode with a layer-by-layer of
Nafion/chitosan/GOx/chitosan/Nafion [33]. This repeatable number of layers provides
a strategy for the rational design of the properties of immobilized films; for instance, a
Nafion layer is critical to removing interferences present in sweat. Kim et al. [29] developed
a temporary tattoo for simultaneous monitoring of ethanol and glucose. In this work, they
used chitosan to immobilize alcohol oxidase (AOx) and glucose oxidase onto screen-printed
Prussian blue carbon electrodes.
Covalent coupling of enzymes to different substrates is a popular chemical immo-
bilization method. The enzymes are bound to the surface through functional groups,
which are not essential for their catalytic activity. Covalent immobilization is more reliable
and specific than adsorption and provides more stability to the recognition element. The
binding of the enzymes to the solid support is generally conducted by initial activation
of the surface using multifunctional reagents, such as carbodiimide [75] or crosslinking
with glutaraldehyde [76], followed by enzyme coupling to the activated substrate with the
removal of excess and unbound biomolecules.
In the development of sweat sensors, various authors have chosen covalent coupling
as an immobilization method. Shitanda et al. [69] developed a lactate sensing system
using a screen-printed sensor based on graft-polymerized MgO-templated carbon, onto
which lactate oxidase was covalently bound. Sempionato et al. [73] developed flexible
printable tattoo electrodes for monitoring changes in the vitamin C level in sweat, in which
the enzyme ascorbate oxidase was immobilized on the Rh-metalized carbon electrode
via glutaraldehyde crosslinking. Glutaraldehyde has also been used to immobilize ty-
rosinase, an enzyme that facilitates the electrochemical oxidation of levodopa excreted in
sweat [70]. Monitoring and optimization of levodopa dosage are critical in patients with
Parkinson’s disease.
Entrapment is a method of choice to immobilize enzymes in three-dimensional matri-
ces, such as electropolymerized films, amphiphilic networks composed of polydimethyl-
siloxane (PDMS), photopolymers, silica gel, polysaccharides, or carbon pastes [77]. In
these cases, enzymes, mediators, and additives can be simultaneously deposited on the
same sensing layer. There is no modification of the bioreceptor, so the activity of the
enzyme is preserved during the immobilization process. Biosensors based on physically
entrapped enzymes are often characterized by increased operational and storage stability.
However, limitations such as leaching of the bioreceptor and possible diffusion barriers
can restrict the performance of the systems. Zhang et al. [24] developed an enzymatic
biosensor for eyeglasses aimed at the determination of lactate in human sweat during
physical exercise. Lactate oxidase (LOx) was immobilized on the working electrode surface
by electropolymeric entrapment in a poly(phenylenediamine) (PPD) film.
Finally, it must be noted that the type of immobilization method affects the activity
and stability of enzymatic biosensors, a subject treated in the next section.
4.2. Stability of Enzymatic Biosensors

Enzymes have been widely used bioreceptor molecules in biosensor applications [78].
Enzymes are able to recognize a specific substrate (or a class of substrates) and, therefore,
are able to catalyse only a specific chemical reaction [79]. Electrochemical enzyme-based
sensors are advantageous in several ways because they include the high specificity of the
enzyme, as well as the sensitivity of electrochemical transducers. In these sensors the
enzyme is immobilized on the electrode surface. The enzyme should be immobilized in
the active form, and the activity must be preserved without reduction in the specificity for
the analyte. It is known that immobilized enzymes frequently exhibit more stability than
native enzymes [80]. However, the immobilization of an enzyme (or other protein) does
not guarantee the stabilization of the enzyme structure. Stability generally results from
the molecular rigidification introduced by the attachment of the protein molecule onto a
solid support, with the concomitant creation of a protected microenvironment [81]. Factors
such as accuracy of measurements, reproducibility, and operational lifetimes are drastically
influenced by enzyme stability, even more so in long-term application. Since the analytical
performance of a biosensor is strongly affected by the immobilization process, intensive
efforts have been undertaken to develop successful immobilization strategies to assure
greater sensitivity and stability of biosensors [77]. Enzymes are sensitive to changes in pH,
ionic strength, chaotropic agents, among other changes in the reaction medium, such as
the presence of polymers, polysaccharides, surfactants, etc. [82]. Moreover, changes in the
macroenvironment during any process could affect the enzyme stability involving cooling,
freezing, or desiccation. Despite this fact, enzyme stability is the main challenge when
achieving viable biosensors because it affects the shelf life and the operational stability
of the sensor, amongst other variables. The enzyme stability has to be maintained or
preferably increased to ensure the stability of the biosensor in terms of reusability and long
term storage [83]. To preserve the enzyme activity is essential to obtaining a high response
signal, principally in sensors for continuous monitoring.
Most of the biological responses converted by sensors require at least one to two years
of storage stability, known as shelf stability, whereas operational stability depends on the
type of biosensor. Operational stability varies from several minutes to several months [84].
Wearable sensors incorporate one or several inner or outer layers, in addition to the
reacting layer or membrane. These layers or membranes serve three important functions: a
protective barrier, a diffusional outer barrier for the substrate, and biocompatibility [84].
In past years, several authors have worked to improve the enzyme stabilization in
wearable sweat sensors. Motosuke et al. [69] developed a microfluidic lactate-sensing
system for the continuous monitoring of sweat lactate. The screen-printed sensor based on
graft-polymerized MgO-templated mesoporous carbon (GMgOC) had the enzyme lactate
oxidase and the redox mediator 1,2-naphthoquinone immobilized. The authors developed
carbon materials with controlled pore sizes (with a diameter of approx. 100 nm) using
MgO as a template. Then, the MgOC surface was modified with glycidyl methacrylate,
and the enzyme was covalently bound to the graft-polymerized MgO-templated carbon
via GMA polymers through epoxy groups that reacted with the amine groups of the
enzyme. The large surface area of mesoporous carbon and the covalent binding of the
enzyme translated into high response currents. To improve the stability of the sensor, they
added sucrose, catalase, and PEGDGE. Sugar addition enhanced the thermostability of the
enzyme. The sucrose stabilized the enzyme structure, thus extending the storage stability.
Catalase increased the operational stability of oxidase-based sensors by deactivating the
hydrogen peroxide produced by the oxidase when oxygen was used as the electron acceptor.
Catalase was applied on the electrode, either together with LOx or in a separate subsequent
step. Finally, the cross-linker PEGDGE enhanced the immobilization efficiency of LOx
and catalase.
Particularly for sweat lactate monitoring, there are other parameters that affect the
enzyme stability, such as the substrate concentration and sweat pH variations. The enzyme
lactate oxidase (LOx) immobilized onto electrodes exhibited stronger substrate inhibition,
resulting in the formation of large and inactive LOx aggregates. Cunha-Silva et al. [85]
observed these phenomena with LOx crosslinking on modified screen-printed electrodes.
In the lactate range from 4 to 50 mM, the biosensors showed a decrease in current signal
due to a substrate inhibition process. In addition, the authors found that the substrate
additions resulted in slightly acidic supporting electrolytes, generated by the dissocia-
tion of lactic acid in lactate and protons (pKa 3.86). The change in the pH affected the
crosslinked LOx activity, giving a place to lower rates of substrate catalysis. They solved
this problem using concentrations greater than 0.5 M of phosphate buffer with a pH of 7 as
a supporting electrolyte, which allowed for observing an enzymatic saturation extended
up to 50 mM. The phosphate buffer was dripped onto the electrode and dried under a
vacuum, demonstrating that such an inhibition mechanism was related to a pH variation,
rather than to a substrate inhibition. Special attention has been given to sweat pH because
lactate constitutes on average 0.28% of sweat and contributes to its acidity.
Wang et al. [68] proposed a wearable biosensor based on lactate sensing for skin
Bluetooth real-time monitoring of perspiration in vivo. They evaluated the effects of
pH variations of sweat on the sensing device. To solve this problem, they covered the
electrode surface with extensive and uniform Prussian blue and reduced the graphene
oxide membrane, which increased the sensor stability. They evaluated the long-term
stability of the biosensor by measuring the sensitivity every four days over 20 days and the
sensitivity remained 88.3% of the initial value after multiple uses in 20 days. Additionally,
the membrane was formed by urchin-like Au nanoparticles and lactate oxidase (LOx). The
urchin-like Au particles improved electron transport from the enzyme active centre to
the electrode.
To improve the sensor operational response time, especially for continuous monitoring
biosensors, Hojaiji et al. [72] presented a compact multicompartment wearable system to
study diurnal variations in sweat glucose levels in relation to daily food intake. The en-
hancement of this work was the use of the multicompartment, in which each compartment
could be activated to autonomously induce or modulate sweat secretion via iontophoretic
actuation and analyse sweat at set time points. This ability allowed for improvement of
the sensor operational response time. This system was developed following a hybrid-flex
design and a vertical integration scheme, integrating the required functional modules:
miniaturized iontophoresis interfaces, an adhesive thin film microfluidic-sensing module,
and control/readout electronics. Each compartment could be programmatically activated
at intermittent time points to obtain biomarker data.
Motosuke et al. [6] developed a microfluidic lactate sensing system for the continuous
monitoring of sweat lactate. The screen-printed sensor, based on graft-polymerized MgO-
templated mesoporous carbon (GMgOC), had the enzyme lactate oxidase and the redox
mediator 1,2-naphthoquinone immobilized. The large surface area of mesoporous carbon
and the easy covalent binding of the enzyme translated into high response currents.
To improve the stability of the sensor the authors added sucrose, catalase and PEGDGE.
The sucrose stabilized the enzyme structure, thus extending the storage stability; catalase
increased the operational stability of oxidase-based sensors by deactivating the hydrogen
peroxide produced by the oxidase when oxygen was used as the electron acceptor; and the
cross-linker PEGDGE enhanced the immobilization efficiency of LOx and catalase. They
also emphasized the use of PDMS as a substrate for the sensor because this material is soft
and comfortable to wear for extended periods of time (6 h) without causing skin irritation.
Furthermore, the PDMS prevented sensor chip bending when the sensing system was
attached to slightly curved body parts, such as the upper torso (front or back) or upper
limbs (arms or legs).
Qi et al. [4] developed smart glasses with an enzymatic biosensor for low-noise and
non-invasive determination of lactate in human sweat during physical exercise. A bi-
enzyme gel membrane was casted on a polypropylene (PP) substrate and was attached to
the arm of a pair of eyeglasses. Lactate was measured during cycling exercise for 30 min by
connecting the three-electrode biosensor with an electrochemical analyser.
4.3. Selectivity: Chemical Interferences

Sweat is a complex medium that consists of metabolites and electrolytes, and it usually
contains only traces of electroactive substances, such as ascorbic acid, acetaminophen, and
uric acid, which may disturb the electrochemical determination of analytes and lead to
inaccurate current readings [24,69]. Reports on sweat sensors have investigated the effects
of interfering electroactive substances only in the micromolar range. Thus, it is reasonable
to expect that negligible interference will distort the sensor signal during field testing on
healthy people. However, these electroactive substances can interfere with the sensor signal
when monitoring athletes during exercises or in unhealthy people.
Qi et al. [24] analysed common electroactive physiological interferents, such as ascorbic
acid, glucose, and uric acid in lactate sweat sensors. They found an insignificant current
interference, indicating excellent discriminatory selectivity of the biosensor.
Ye et al. [33] developed a wearable electrochemical sensor for real-time monitoring

of sweat glucose, using a three dimensional paper-based microfluidic electrochemical
integrated device. They analysed the presence of interferences, such as 0.1 mM NaCl,
2 mM uric acid, 2 mM ascorbic acid, and 5 mM lactate. The sensor did not present any
significant interference signals in the glucose signal, and they attributed this result to the
modification of the electrode surface. The working electrode was modified with Nafion;
and then a chitosan layer, the enzyme glucose oxidase, and the mediator Prussian blue
were immobilized on the modified electrode; and finally a layer of chitosan was casted.
They argued that the Nafion layer was critical to remove interferents present in sweat.
Molecules with a molecular weight higher than that of H2 O2 , such as ascorbic acid and uric
acid, could not penetrate the lattice and follow a redox reaction.
5. Sensing Biomolecules in Sweat with Affinity Electrochemical Biosensors

Immunosensors are biosensors that incorporate antibodies as biorecognition ele-
ments [66]. They have been used to detect the presence of intermediate-size organic
molecules and biomolecules, such as steroid hormones, drugs of abuse and their metabo-
lites [86], and macromolecules (proteins, interleukins, enzymes) [87]. The most usual elec-
trochemical transduction technique is either voltammetry or electrochemical impedance
spectroscopy. The main compounds present in sweat, which are being studied to be de-
tected with wearable immunosensors, are cortisol, cytokines, ethyl glucuronide, and drugs
of abuse [87]. Amongst these different analytes, a great amount of work has been espe-
cially performed to determine cortisol. Cortisol is a steroid hormone playing a key role
in homeostasis, and it has been related to stress-based diseases and disorders. On the
other hand, cytokines are a broad group of small proteins that play important roles in cell
signalling, modulating immune responses. Consequently, cytokine sweat sensors have
been developed for the diagnosis of diverse inflammatory processes. Finally, sweat im-
munosensors have been also developed for the detection of ethyl glucuronide (a metabolite
of ethanol) and drugs of abuse (such as methadone, methamphetamine, amphetamine, and
tetrahydrocannabinol).
Different approaches have been used in sweat analysis based on affinity biosensors,
most of them employing antibodies as biorecognition elements, although aptamers have
also been used. Figure 7 schematically shows two of the main strategies used for the
label-free detection of a given analyte acting as an antigen. One usual strategy is to detect
the formation of antibody-antigen complexes with the aid of a reversible redox probe
(Figure 7a). The electrode surface onto which antibodies are immobilized is accessible to
the redox probes, and they can conduct electron transfer reactions at the electrode. These
processes can be followed by diverse electrochemical techniques, such as chronoamper-
ometry, CV, DPV, and EIS. The formation of antigen-antibody complexes blocks the access
of the redox probes to the electrode surface, and consequently the current decreases in
amperometric or voltammetric signals (i.e., the electron transfer resistance increases).
Immunosensors often include the use of nanomaterials, which provide a larger elec-
troactive area, to improve sensitivity and the limits of detection. Santiago et al. [88]
developed an immunosensor based on graphene oxide, which was further modified by
covalent linking of protein A, which in turn was employed to immobilize cortisol anti-
bodies. K4 Fe(CN)6 was used as a redox probe, and the decrease in the anodic and ca-
thodic peak in a cyclic voltammogram was sensitive to cortisol in the 0.1–150 ng/mL range.
Laochai et al. [89] developed an immunosensor based on L-cys/AuNPs/MXene conductive
thread electrodes with covalently immobilized cortisol antibodies. An enhanced area was
achieved with MXene and AuNPs, and chronoamperometry was used to determine cortisol
levels, employing Fe(CN)6 3−/4− as a redox probe, obtaining a linear range of 5–180 ng/mL
and an LOD of 0.54 ng/mL. Madhu et al. [90] developed a cortisol biosensor based on
carbon yarns coated with zinc oxide nanorods, presenting high mechanical stability and
superwettability. After cortisol antibodies immobilization, cortisol levels were determined
using CV and DPV, again with Fe(CN)6 3−/4− as a redox probe, obtaining a linear range
R PEER REVIEW 26 of
from 1 fg/mL to 1 µg/mL and an LOD of 0.45 fg/mL by CV and 0.098 fg/mL by DPV.
Additionally, Madhu et al. [91] employed a similar approach with TiO2 nanostructures
combined with carbon yarns, obtaining a linear range from 10 fg/mL to 1 µg/mL.
Figure 7. Schematic representation of two label-free immunosensors designs employed in sweat

Figure 7. Schematic biosensors.
representation of two label-free immunosensors designs employed in sw
(a) The formation of antibody-antigen (in yellow and blue, respectively) complexes
biosensors. (a) The formation of antibody-antigen
can be detected indirectly since they block (in
theyellow
electrode and blue,
surface to therespectively)
passage of a redoxcomplexes
probe c
be detected indirectly(red)
since they block
and diminish current the electrode
signals surface
due to electron transfer to the passage
reactions, ofcan
a process that a redox probe (r
be followed
by voltammetric or impedimetric techniques (e.g., SWV, as shown in the figure). (b) Field-effect
and diminish current signals due to electron transfer reactions, a process that can be followed
transistors allow for direct detection of the formation of antibody-antigen complexes (for instance, by
voltammetric or impedimetric
measuring the techniques (e.g.,
drain current under SWV, conditions).
appropriate as shown in the figure). (b) Field-effect tr
sistors allow for direct detection of the formation of antibody-antigen complexes (for instance,
Microfluidic channels have been included in several sweat sensors to improve sample
measuring the drain current
collection. under appropriate
Naik et al. [92] presented conditions).
a microfluidic platform for cortisol detection and contin-
uous glucose monitoring. Adhesive microchannels and an inkjet-printed graphene sensing
electrode, with the possibility of incorporating electrowetting valves, were integrated with
Immunosensors often include the use of nanomaterials, which provide a larger el
a synthetic skin to produce a “smart bandage”. Au nanoparticles were deposited onto
troactive area, to improve
the surfacesensitivity and the
of graphene electrodes and limits
cortisolof detection.
antibodies Santiago
were covalently et al. [88] dev
immobilized.
The cortisol levels were determined by chronoamperometry for a Fe(CN)6 3−/4− redox
oped an immunosensor based on graphene oxide, which was further modified by covale
probe, and an LOD of 10 pM was achieved for cortisol in sweat. Nah et al. [93] developed
linking of protein aA, which
wearable in turn that
immunosensor wasincluded
employed to immobilize
a microfluidic cortisol
channel and chamber. Ti3 C2antibodi
Tx
MXene-loaded laser-burned graphene flakes were transferred onto a PDMS substrate, and
K4Fe(CN)6 was used as a redox probe, and the decrease in the anodic and cathodic pe
cortisol antibodies were covalently immobilized. Cortisol concentrations in artificial sweat
in a cyclic voltammogram was sensitive
were determined to cortisol
by EIS, obtaining in ofthe
a linear range 0.1–150
0.01–100 nM andng/mL
an LODrange.
of 88 pM.Laocha
Cheng et al. [94] developed an immunosensor
al. [89] developed an immunosensor based on L-cys/AuNPs/MXene conductive based on flexible screen-printed elec- thre
trodes, onto which AuNPs were electrochemically deposited, and cortisol antibodies were
electrodes with covalently immobilized
covalently immobilized cortisollinker.
via a HOOC-PEG-SH antibodies.
Cortisol levelsAn
were enhanced
detected by DPV, area w
employing Fe(CN) 3−/4− as a redox probe, and an LOD of 7.47 nM was obtained. However,
achieved with MXene and AuNPs, 6 and chronoamperometry was used to determine c
the emphasis of the work was on the construction of a complete wearable cortisol sensor,
tisol levels, employing Fe(CN)
including
3−/4− as a redox probe, obtaining a linear range of 5–1
aspects 6not usually covered in publications on electrochemical sweat sensors,
ng/mL and an LOD of 0.54 ng/mL. Madhu et al. [90] developed a cortisol biosensor bas
on carbon yarns coated with zinc oxide nanorods, presenting high mechanical stabil
and superwettability. After cortisol antibodies immobilization, cortisol levels were det
Cheng et al. [94] developed an immunosensor based on flexible screen-printed el
trodes, onto which AuNPs were electrochemically deposited, and cortisol antibodies w
covalently immobilized via a HOOC-PEG-SH linker. Cortisol levels were detected
DPV, employing Fe(CN)63−/4− as a redox probe, and an LOD of 7.47 nM was obtained. Ho
ever, the emphasis of the work was on the construction of a complete wearable corti
sensor, including aspects not usually covered in publications on electrochemical sw
sensors, such as the need for a portable, miniaturized, low consuming electronic inst
such as the need for a portable, miniaturized, low consuming electronic instrumentation
mentation
capablecapable of processing
of processing the transduced
the transduced signal and communicating
signal and communicating the results
the results to the user
the user (Figure
(Figure 8). 8).
Figure 8. The
Figure integrated
8. The integratedwireless, battery-free,
wireless, battery-free, andand flexible
flexible detection
detection system
system with with NFC-enab
NFC-enabled
smartphone. (a) An
smartphone. image
(a) An imageofofin
in situ detection
situ detection of sweat
of sweat cortisol;
cortisol; (b) the(b) the flexible
flexible sensing
sensing system for system
electrochemical
electrochemical detection of
detection ofcortisol;
cortisol;(c) block diagram
(c) block of the wireless,
diagram of thebattery-free,
wireless, and flexible patch.
battery-free, and flexi
Reprinted from Ref. [94]. Copyright (2017), with permission from Elsevier.
patch. Reprinted from Ref. [94]. Copyright (2017), with permission from Elsevier.
Cortisol aptamers have also been used as biorecognition elements. Ganguly et al. [95]
Cortisol
developedaptamers have also
an impedimetric been used
aptasensor for theas biorecognition
determination elements. Ganguly
of sub-microlitre passively et al. [
developed an impedimetric
eluted sweat. Aptamers wereaptasensor for the determination
bound to a ZnO-modified electrode, andofcortisol
sub-microlitre
levels were passiv
determined by EIS. A dynamic range of 1 − 256 ng/mL was obtained.
eluted sweat. Aptamers were bound to a ZnO-modified electrode, and cortisol levels w
Another strategy is based on the use of field-effect transistors (FETs). This approach has
determined by EIS. A dynamic range of 1−256 ng/mL was obtained.
additional advantages compared to those systems employing an auxiliary redox probe, such
Another strategy
as direct detection of is
thebased
formationon the use of field-effect
of antigen-antibody transistors
complexes (FETs).
(Figure 7b). This
The use of approa
has additional advantages compared to those systems employing an auxiliary
transistors is especially attractive in biosensors because of the possibility of miniaturization red
probe,
andsuch as direct
achieving detectionand
high sensitivity of extremely
the formation
low LODsof when
antigen-antibody complexes
used in combination with (Figu
nanomaterials. Demuru et al. [96] developed an immunosensor based on a cortisol
7b). The use of transistors is especially attractive in biosensors because of the possibil antibody
modified organic transistor, obtaining sensitivity of 50 µA/dec (Figure 9).
of miniaturization and achieving high sensitivity and extremely low LODs when used
combination with nanomaterials. Demuru et al. [96] developed an immunosensor bas
on a cortisol antibody modified organic transistor, obtaining sensitivity of 50 μA/dec (F
ure 9).
OR PEER REVIEW 28 of 47
Figure 9. Organic transistor

Figure structure
9. Organic and sticker
transistor application.
structure and sticker(a) Schematic
application. (a) of the biosensing
Schematic prin-
of the biosensing
principle involved and structure of the OECT devices, including antibody-coated
ciple involved and structure of the OECT devices, including antibody-coated gate electrodes and gate electrodes and
the organic PEDOT:PSS channel for current amplification; the latter shows three OECT devices with
the organic PEDOT:PSS channel for current amplification; the latter shows three OECT devices with
different PEDOT:PSS channels of an increased width per length ratio. (b) 3D-image with two OECTs
different PEDOT:PSS channels of an increased width per length ratio. (b) 3D-image with two OECTs
integrated into a microfluidic system for direct sweat collection and zoom of the gate electrode with
integrated into a microfluidic system
the anticortisol for direct
antibody sweat collection
immobilized. andofzoom
(c) Application of sensing
a cortisol the gate electrode
sticker with
on the human
the anticortisol antibody
skinimmobilized. (c) during
for sweat collection Application of a cortisol
sports activities; sensing
the microfluidic partsticker
with theon the human
multiple inlets is
skin for sweat collection during
placed sports
in contact withactivities; the microfluidic
the skin. Reprinted with permission part
Ref.with the multiple
[96]. Copyright (2022), inlets
Americanis
Chemical Society.
placed in contact with the skin. Reprinted with permission Ref. [96]. Copyright (2022), American
Chemical Society. In the next section, we address issues such as electronic signal processing, wireless
communication, and power supply in connection to electrochemical sweat sensors.
In the next section, we address issues such as electronic signal processing, wireless
6. Electronic Instrumentation
communication, and power Oncesupply in connection
the sample to electrochemical
extraction, collection, sweat sensors.
transport, and detection stages have been
established, signal processing and transmission need to be performed. For portable, non-
invasive, rapid, and real-time monitoring applications in the field, the use of heavy, bulky,
6. Electronic Instrumentation
and non-portable laboratory equipment for signal processing is not suitable. For this reason,
Once the samplethere
extraction, collection,
is great interest transport,
in researching and detection
and developing thin and stages have been
light embedded es-
wearable
tablished, signal processing and
sensors with transmission
advanced electronicneed to be that
technologies performed.
incorporateFor portable,circuitry,
miniaturized non-
wireless transmission systems, and more efficient power modules [7,19,97–101].
invasive, rapid, and real-time monitoring applications in the field, the use of heavy, bulky,
and non-portable laboratory equipment
6.1. Integrated Electronics for signal processing is not suitable. For this rea-
son, there is great interest in researching
Electronic and processes
instrumentation developing thinsignals
the electric and light embedded
produced wear-
after the transduc-
tion of chemical or physical signals (physiological, biochemical, environmental, etc.). These
able sensors with advanced electronic technologies that incorporate miniaturized cir-
electrical signals are conditioned by going through different amplifiers and filters to reduce
cuitry, wireless transmission systems,
spurious signals andThis
and noise. more efficient
process powerformodules
is responsible [7,97–101].
signal processing and data log-
ging management using microcontrollers (µC), memory systems, and analogue-to-digital
6.1. Integrated and digital-to-analogue converters (ADC/DAC) that work together to adapt and control
Electronics
Electronic instrumentation processes the electric signals produced after the transduc-
tion of chemical or physical signals (physiological, biochemical, environmental, etc.).
These electrical signals are conditioned by going through different amplifiers and filters
Chemosensors 2023, 11, x FOR PEER REVIEW 29 of 47

the flow of signals. In addition, they must handle the communication with different devices
and the data transmission for subsequent analysis and visualization [102,103]. The main
components of a wearable sensor and their interaction can be represented schematically
detailed introduction,
in a block diagram, ashow theyinwork
shown and10.
Figure theColburn
most important characteristics
et al. [104] explained, of
inthese com-
a detailed
ponents to correctly understand the electrical signals resulting from experimental
introduction, how they work and the most important characteristics of these components meas-
urements.
to correctly understand the electrical signals resulting from experimental measurements.
Figure 10. Fundamental blocks of a wearable electrochemical sweat sensor.

Figure 10. Fundamental blocks of a wearable electrochemical sweat sensor.
To characterize (bio)sensors, typically using amperometric and voltammetric tech-
niques, the use of a potentiostat is required, which can be described as an instrument that
To characterize (bio)sensors, typically using amperometric and voltammetric tech-
permits control of the working electrode potential with respect to a reference electrode, by
niques, the use of a potentiostat is required, which can be described as an instrument that
applying a potential difference between the working electrode and an auxiliary electrode.
permits control of the working electrode potential with respect to a reference electrode,
Additionally, the current flowing through the working electrode must be measured.
by applying a potential difference between the working electrode and an auxiliary elec-
Focusing on such scenarios that require this type of portable device, a wide variety of
trode. Additionally, the current flowing through the working electrode must be meas-
potentiostats have been developed, prioritizing in each case the most relevant characteris-
ured.
tics, depending on the application or the use for which they were created. Some of them
Focusing on such scenarios that require this type of portable device, a wide variety
stand out for their measurement ranges and others for their precision, level of integration,
of potentiostats have been developed, prioritizing in each case the most relevant charac-
portability, ease of use, configuration possibilities, accessibility, cost, and so on.
teristics,
Theredepending on the
are portable applicationonorthe
potentiostats themarket
use forcapable
which they were created.
of conducting mostSome of
electro-
them stand out for their measurement ranges and others for their precision,
chemical techniques [105–107]. However, this type of equipment is not accessible for the level of inte-
gration,
widespread portability, ease of use,
use in research configuration
or education possibilities,
settings accessibility,
(>US $1000). For these cost, andcheaper
cases, so on.
There are portable potentiostats on the market capable of conducting
devices are required, which have limitations (resolution, ranges, etc.) but allow for the most electro-
chemical
most usedtechniques [105–107].
electrochemical However, this
characterizations type
to be of equipment
performed. Thereisisnot
even accessible
a trend infor the
open-
widespread use in research or education settings (>US $1000). For these
source portable devices of this type, which makes the knowledge generated available to thecases, cheaper
devices are required,
entire community. Thewhich have limitations
main characteristics (resolution,
of some of them ranges,
are shownetc.)inbut allow
Table for the
5 [108–112].
mostThe usedfirst
electrochemical
potentiostats characterizations
developed were to be performed.
based on analogue There is even
circuits witha different
trend in
open-source portable devices
discrete components, which of
werethisconfigured
type, whichasmakes
simpletheblocks
knowledge generated
that interact with availa-
each
ble to the entire community. The main characteristics of some of them
other without any type of programming complexity, requiring additional hardware to are shown in Table
5obtain,
[108–112].
send, and display the signals. Later, potentiostats that based their processing logic
on microcontrollers began to be developed, also incorporating amplifiers and ADC and
DAC converters to digitize and condition the signals to subsequently interact with digital
communication protocols. The whole system was mounted on a printed circuit board
(PCB) or another kind of substrate, thus forming a final device capable of performing
electrochemical measurements [108,111–113].
Table 5. Characteristics of open-source potentiostats.
Name Power Communication User Interface Range Resolution Ref.

Wireless with
Lithium polymer 67 µV (40 µV noise);
protocol Smart
UWED rechargeable ±1.5 V, ±180 µA 6.4 nA (30 nA noise [109]
“Bluetooth Low phone/tablet
battery (LiPo) + nonlinearity)
Energy” (BLE)
Wired connection PC— ±1.5 V; lowest limit of
DStat USB via USB/serial multiplatform detection 600 fA 46 µV; ~pA [111]
port software (7 ranges)
2990 mV a 990 mV
Wired connection (~±1 V);
PC and three-line
CheapStat USB via USB/serial ±100 nA and ±10 µA ~mV; ~nA [112]
display + joystick
port (2 ranges);
1 to 1000 Hz
(DAC: 20 bits/ADC:
22 bits) 15.3 µV;
USB-controlled Wired connection PC— ±8 V, ±20 mA,
12 nA, 120 pA and
potentio- USB via USB/Serial multiplatform ±200 µA and ±2 µA; [110]
1.2 pA and noise:
stat/galvanostat port software sample rate: 90 ms
88 nA,
1.1 nA and 9.9 pA
Wired connection PC—
External 15 volt
MYSTAT via USB/serial multiplatform ±12 V; ±200 mA - [108]
DC
port software
Jenkins et al. proposed a topology in which an AD5933 was used to measure the
impedance of the electrochemical cell, in addition to working as a signal generator, while
some authors [29,114–116], looking for a higher level of integration, decided to use an
analogue front end (AFE) integrated on a chip as a potentiostat, e.g., LMP91000/LMP91002
from Texas Instruments [117]. These devices allowed for working in a current range
between ±5 µA and ±750 µA, operating in a voltage range of 2.7 V to 5.25 V, and being
able to apply a bias programming voltage in the range of 0 to 24% of its reference voltage
(VREF) or VDD, in steps of 2%. Therefore, considering a VREF of 3.3 V (a very common
supply voltage value), the voltage sweep would be ±792 mV with steps of 66 mV. If
higher resolution is required, an external DAC with a higher bit count must be added.
Physically, it comes in a 4 × 4 mm WSON package with 14 connection pins, pad dimensions
of 250 × 600 µm, and a 500-µm pitch. Finally, with a higher level of integration and
design specificity, there are potentiostats based on application specific integrated circuits
(ASICs) [118,119], such as ADuCM355 from Analog Devices [120], in which the AFE and
all of the digital processing blocks were integrated into a single chip. This design has
the advantage of reducing the noise produced by the electrical connections between the
components and a size reduction of the entire system in general, presenting a package
of 6 × 5 mm, a72-lead LGA, and 250-µm and 500-µm pitch. This model also allows for
achieving an ultra-low consumption configuration, while maintaining high processing
performance. It can be noted that this kind of ASICs can achieve measurement currents
from ±0.00005 µA to ±3000 µA and apply voltages of ±2.2 V with a 12-bit resolution
(<1 mV).
In cases in which EIS is the transduction technique, as a complement of the microcon-
troller, a high-precision impedance converter system is used, such as the AD5933 (Analog
Devices) [121], which generates the voltage sweep signals (27-bit DDS: Direct Digital Syn-
thesis) with frequencies from 1 kHz to 100 kHz. On the other hand, if it is required to work
with lower frequencies, it is necessary to add an external clock [122,123].
In wearable-type applications, the electronics should go along with the sensing system,
generating a comfortable user experience with good mechanical performance, light weight,
easiness to adapt to the body, and an extended shelf life, using suitable materials to ensure
biocompatibility. These types of devices are those that Ling et al. called “next-generation
wearables” or “wearables 2.0” [99].
Before achieving the characteristics proposed by Ling, we can mention the works of
Gao et al. and Vinoth et al. as part of the state of the art of multi-parameter wearable
devices [7,124]. To attain this multiplicity of information without exceeding the num-
ber of components and physical volume, Vinoth et al. benefitted from the use of a µC
(CC2640, from Texas Instruments), which integrates a signal processing module and a BLE
(Bluetooth Low Energy) communication module. It also uses a strategy combining DAC
and Op-amp configured in a way that allows for amperometric measurements (e.g., for
the amperometric determination of glucose and lactate concentrations), together with the
differential amplifiers used as micro-voltmeters to measure pH signals and Na+ and K+
sensors. Gao used a similar strategy, adding a resistor that also measures body temperature
by voltage difference.
6.2. Power Supply

It is critical to have proper power control in portable devices since they must operate
continuously without being connected to a power supply and, in many cases, for long
periods of time. As we increase the number of functionalities, the detection of multiple pa-
rameters at the same time, a complex data analysis, sampling rates, different communication
protocols, and data transmission, power management becomes a critical issue [125,126].
Krorakai et al. [127] used an alternative approach to create a near-field potentiostat
in a credit card-like design (5.5 cm × 8.6 cm). Based on a SiC4341 NFC chip (Silicon
Craft Technology PLC) and using a connector for the electrodes and an NFC antenna for
connection with smart devices (phones, tablets, etc.), this device is an example of a passive
device that operates without batteries that remarkably minimizes and optimizes intercon-
nections. In this case, the passive device is powered from the wireless NFC connection
from the smart device, and it is operated remotely from a user-friendly Android mobile
application. The application allows for establishing the conditions for the electrochemical
characterizations to be conducted in the device within a limited range of current (±20 µA)
and voltage (±0.8 V) generated from the DAC, which has a full-scale voltage value of
1.28 V with steps of 5 mV. A similar approach was used by Bandodkar et al., although it
attained a lower level of integration. In a 32-mm diameter area and with an approximate
weight of 1 g, they developed a skin-interfaced microfluidic/electronic system with a
microcontroller (F430FRL152H from Texas Instruments) and other discrete components,
which were powered through a coil fabricated in a flexible printed circuit board (FPCB) by
NFC technology [128].
With respect to the power source, rigid lithium-ion batteries (Li-ion) are most com-
monly used in portable devices, with a proper performance and shelf life and fast charge
and discharge cycles. On the other hand, these kinds of batteries are not as adaptable
to flexible wearables devices, which require quite demanding physical and mechanical
operating conditions, such as bending, twisting, folding, and stretching. It must be taken
into account that, normally by reducing the physical size of the device, the necessary
storage energy is also reduced; therefore, it is possible to use other alternatives to batteries.
A typical example for disposable, non-rechargeable use is the CR2032 lithium-ion coin-cell
battery (commonly used in watches and portable devices), which has a charge of 225 mAh
and an operating voltage of 3 V, and it is widely used due to its small size (approx. 20 mm
in diameter, 3.1 mm in thickness and a weight of 2.9 g) and low cost.
The rapid development and evolution of wearable sensor technology, which has spread
to many applications in recent years, have required a huge effort in terms of research and
development of rechargeable and flexible batteries, seeking to improve power density,
charge cycling, and energy efficiency, as well as achieving a greater degree of flexibility
to obtain a more adaptable and comfortable device. New materials (flexible, stretchable,
more efficient, and environmentally friendly) and processing techniques were tested and
implemented [129,130]. On the other hand, flexible substrates, such as elastomers, textiles,
and paper, have been studied, as well as their application in new energy accumulators
(batteries, supercapacitors, etc.) and energy harvesting (solar cells, biofuel cells, movement
systems, etc.) [126,131–134]. The review of Kong et al. [135] summarizes the most recent
advances in terms of flexible batteries, giving a complete characterization regarding their
mechanical properties and how they affect electrochemical performance, describing at a
constructive level some of the most significant achievements in their critical components
(electrolytes, current collectors, etc.). It also presents some cases of functional prototypes,
highlighting design considerations to achieve their implementation at the industrial level.
Gai et al. [131] and Song et al. [132] proposed an energy harvesting system that used
the energy coming from the motion of the human body during exercise by means of so-
called nanogenerators, offering powers of a some milliwatts, quite enough to process
and wirelessly transmit the sensing data continuously or increase the charge of an energy
accumulator (supercapacitors or batteries). Gai et al. reported that power generation
systems can obtain about 60 V and 15 mA from movements at frequencies as low as 5 Hz.
It is important to emphasize that, in all these cases, a critical condition is that the
energy produced by the generator must be higher than the consumption of the entire
system. For this purpose, it is very important to select ultra-low consumption components
and optimize both the design of the circuits and the criteria used in signal sampling and
communication methods.
As we mentioned before, a substantial drawback in wearable applications is that,
by reducing the size of the devices, they are limited in energy harvesting, have short
lifetimes, and variations in energy efficiency. Something similar happens with biofuel
cells. Supercapacitors are becoming an increasingly strong alternative for portable energy
storage since they have the ability to store more energy per unit volume than batteries and
have high charge/discharge cycles (>100,000), low discharge rates, low electrical resistance,
higher performance, and good resistance to mechanical stress [133]. Studies in chemistry,
physics, and materials science have allowed for progress with greater possibilities of
fabricating robust supercapacitors with better and more appropriate materials (electrolytes,
electrodes, binders, and additives), as well as packages and electrical characteristics (charge
time, lifetime, leakage current, degradation, etc.). Some nanomaterials based on carbon
or graphene, due to their greater surface area, good electrical conductivity, and chemical
inertness, are of great interest for this type of application. Other complementary proposals
that have been tested for self-powered sensors and actuators are some portable devices
based on origami techniques [136]. Nevertheless, one of the great challenges that remain in
this type of technology is the reduction in size and biocompatibility.
6.3. Wireless Communication

Another function that the electronic instrumentation of the device must provide is com-
munication with the user to clearly and precisely transmit the information obtained from the
sensors. In the case of the first portable devices, this transmission was usually accomplished
through a wired connection using USB ports or analogue audio interface [110,111,137].
With the advance of technology and the growth of smartphones and internet of things (IoT)
applications, wireless connectivity began to be used instead, as wired connections made
the usability of portable and wearable devices more complicated [109]. For this type of
communication, a large number of wireless protocols that respond to current IoT demands
are available: Bluetooth Low Energy (BLE) [138–140], ZigBee [141], radio frequency (RFID)
and near field communication (NFC) [35,142–144], Wi-Fi [145,146], and narrowband inter-
net of things (NBIoT) [147], of which BLE and NFC are perhaps the most widely used in
wearables. However, these protocols could have limitations to being used in more complex
applications that require higher information density and energy consumption. We can see
in Figure 11 a comparison graph showing the relationship between speed and the range of
data transmission for different communication protocols.
most widely used in wearables. However, these protocols could have limitations t
used in more complex applications that require higher information density and
consumption. We can see in Figure 11 a comparison graph showing the relations
tween speed and the range of data transmission for different communication prot
Figure 11. Data rate vs. range of data transmission for different communication protocols.
Figure 11. Data rate vs. range of data transmission for different communication protocols.
In the case of NFC communication, for example, it works at a frequency of 13.56 MHz
with a data rate of 106 kbps to 424 kbps. Regarding the transmission distance, the devices
In the case ofshould
NFCbecommunication,
quite close to communicate forwith
example,
each otheritsince
works at a frequency
the wireless range is 20 cmof 13.5
with a data rate ofor 106
less. This
kbps technology
to 424can work Regarding
kbps. in two different themodes: passive mode, indistance,
transmission which the the d
reading device initiates the communication, and the other NFC device (tag) uses the energy
should be quite close to communicate
of the electromagnetic with each
field to energize other
itself and thensince
exchange theinformation;
wirelessand range
the is 2
active mode, in which both devices have their own power
less. This technology can work in two different modes: passive mode, in which the r sources for data communication.
The advantage in the case of active mode is that, in addition, the distances between
device initiates the
the communication,
reader and the tag can be and theThis
longer. other
kindNFC device
of wireless (tag) uses
communication theinenergy
is used
electromagnetic devices
field with simpler electronics that only need to transfer a little information to other NFC
to energize itself and then exchange information; and the
compatible devices (readers, smartphones, etc.) and do not require extra power. Among the
mode, in which both devices
most popular uses have their own
of this technology power
are smart sources
labels, for devices,
access control data communicatio
contactless
payment systems, ID documents, and smart signage. However, due to its simplicity, it
advantage in the iscase of active mode is that, in addition, the distances between the
also widely used in medical applications for health monitoring, to record and monitor
and the tag can be longer.
different This kind
vital parameters of wireless
of patients autonomously. communication
In this case, the proximityis used
requiredin bydevic
NFC technology makes it possible to guarantee the correct reading of data, thus reducing
simpler electronics that only need to transfer a little information to other NFC com
the chances of error or information theft. Sweat sensors are also an example of devices that
devices (readers,have smartphones,
incorporated NFCetc.) and taking
technology, do not require
advantage of the extra power.
stable operation andAmong
small th
size of devices with low weight and packaging options that allow for good mechanical
popular uses of this technology
compatibility [148,149]. are smart labels, access control devices, contactle
ment systems, ID documents, and smart
In terms of improving signage.
the efficiency However,
of this type of communication,due different
to its simplicity,
strategies it
can be used to optimize its operation from the electronic control unit, such as reducing
widely used in medical
sampling timesapplications for health
based on the application monitoring,
requirements or turning offtocommunication
record andwhile monitor
ent vital parameters
the deviceof patients
is inactive orautonomously.
even combining different In communication
this case, the proximity
technologies (RFID, requ
ZigBee, Wi-Fi, Bluetooth, etc.), taking advantage of each of them [126,150,151].
NFC technology makes it possible
Regarding Bluetooth, itto
is aguarantee the correct
short-range, high-data reading
rate, low-power of data,
wireless communi- thus re
the chances of error
cation or information
technology commonly foundtheft. Sweat
in most sensors
portable are asalso
devices, such an example
smartphones, smart- of d
watches, and computers. It works in the ultra-high frequency band of 2.4 to 2.485 GHz and
that have incorporated NFC technology, taking advantage of the stable operati
small size of devices with low weight and packaging options that allow for good m
ical compatibility [148,149].
(RFID, ZigBee, Wi-Fi, Bluetooth, etc.), taking advantage of each of them [126,150,151].
Regarding Bluetooth, it is a short-range, high-data rate, low-power wireless commu-
nication technology commonly found in most portable devices, such as smartphones,
smartwatches, and computers. It works in the ultra-high frequency band of 2.4 to 2.485
Chemosensors 2023, 11, 244 GHz and supports a maximum data rate of 2 Mbps [139]. In particular, the Bluetooth 33 of 46Low
Energy (BLE) protocol was designed specifically for short communications between de-
vices for IoT applications and devices with very limited power consumption [7,130]. BLE
supports aless
consumes maximum
than 15data
mA,rate
hasofa2data
Mbps [139].
rate fromIn 125
particular,
kbps tothe Bluetooth
1 Mbps (BLELow
4.0)Energy
[152], and
(BLE) protocol was designed specifically for short communications between
supports a maximum distance of 100 m from the device to the receiving station. Typicaldevices for
IoT applications and devices with very limited power consumption [7,130]. BLE consumes
applications of this technology are personal healthcare, fitness training, entertainment,
less than 15 mA, has a data rate from 125 kbps to 1 Mbps (BLE 4.0) [152], and supports a
etc.
maximum
On thedistance of 100 the
other hand, m from the devicewith
technologies to the receiving
the station. consumption
lowest energy Typical applications
are NFC,
of this technology are personal healthcare, fitness training, entertainment, etc.
Bluetooth, ZigBee, and Lora, and despite having a low data transmission rate, they are
On the other hand, the technologies with the lowest energy consumption are NFC,
generally suitable for the instrumentation of this type of sensor. Finally, we can therefore
Bluetooth, ZigBee, and Lora, and despite having a low data transmission rate, they are
say that the choice of the most convenient communication protocol for each application
generally suitable for the instrumentation of this type of sensor. Finally, we can therefore
will depend
say that on technological
the choice availability,communication
of the most convenient costs, frequency bands,
protocol fordata
eachrates, coverage,
application
and
will energy
depend consumption [99].
on technological availability, costs, frequency bands, data rates, coverage, and
energy consumption [99].
7. Machine Learning Signal Processing of Electrochemical Sweat Sensors
7. Machine
Signal Learning
processing Signal Processing of tool
is a fundamental Electrochemical
for enhancing Sweat
theSensors
performance of electro-
Signal processing is a fundamental tool for enhancing the performance
chemical sweat sensors. It consists of various steps that involve noise filtering, of electrochem-
identifica-
ical sweat sensors. It consists of various steps that involve noise filtering,
tion and characterization of relevant signal events, and interpretation of these signals identification and(Fig-
ure 12). This tool, along with other strategies (i.e., the use of nanomaterials), allows12).
characterization of relevant signal events, and interpretation of these signals (Figure for the
This tool, along with other strategies (i.e., the use of nanomaterials), allows for the enhance-
enhancement of figures of merit of the sensors, such as LOD, sensitivity, and selectivity.
ment of figures of merit of the sensors, such as LOD, sensitivity, and selectivity. However,
However, signal processing plays a key role for establishing correct relationships between
signal processing plays a key role for establishing correct relationships between a primary
a primary signal (current, potential) and analyte concentrations, a health condition, or
signal (current, potential) and analyte concentrations, a health condition, or even prediction
even prediction
of a possible of event.
future a possible
This future
goal canevent. This goal
be achieved can
using be achieved
traditional using traditional
algorithms (filters
algorithms (filters for noise reduction or deconvolution, calibration etc.),
for noise reduction or deconvolution, calibration etc.), machine learning (ML) algorithms, machine learning
(ML) algorithms, of
or a combination orboth.
a combination of both.
The difference betweenThe traditional
difference and
between traditionalis and
ML algorithms that ML
algorithms
the former is based on known parameters and models and gives the same output for a the
is that the former is based on known parameters and models and gives
same
givenoutput
datasetfor a given
from dataset
the first from thethe
run, whereas first run,iswhereas
latter based onthe latter is parameters
unknown based on unknown
that
parameters
are acquiredthat
and are acquired
refined throughanda refined
trainingthrough
process. a training process.
12. Four steps

Figure 12.
Figure steps for
forsignal
signalprocessing:
processing:(1)(1)noise filtering;
noise (2) (2)
filtering; signal identification;
signal (3) signal
identification; (3) signal
characterization; (4)
characterization; (4)signal
signalinterpretation.
interpretation.Adapted
Adapted with permission
with permission from Ref.Ref.
from [153], Copyright
[153], 2019.2019.
Copyright
ML algorithms
ML algorithms are
arelimited
limitedbybycomputational
computational capabilities andand
capabilities data availability
data for for
availability
training, but they are able to adapt to a particular sensing system and predict relationships
training, but they are able to adapt to a particular sensing system and predict relationships
not previously defined by a given model. Since the internet of things and cloud computing
not previously defined by a given model. Since the internet of things and cloud computing
are currently solving the limitations of ML, these algorithms are gaining ground in many
are currently solving the limitations of ML, these algorithms are gaining ground in many
sensing applications and demonstrating their advantages.
sensing applications and demonstrating their advantages.
ML algorithms can be classified as supervised and unsupervised learning. Supervised
learning relies on training data already labelled, and a predictive model is given. On the
other hand, unsupervised learning algorithms can be trained using unlabelled data and
no training model. Regarding models for supervised learning, they can be classification
or regression models. The former is more suitable for dividing data into classes (i.e., low,
On the other hand, unsupervised learning algorithms can be trained using unlabell
and no training model. Regarding models for supervised learning, they can be cla
tion or regression models. The former is more suitable for dividing data into class
low,2023,
Chemosensors normal,
11, 244 or high sweat glucose levels), whereas the latter is designed 34 offor
46 tren
casting (i.e., prediction of abnormal glucose levels in the future).
Regarding chemical sensors for health applications, supervised learning algo
normal, or high sweat glucose levels), whereas the latter is designed for trend forecasting
are by far the most (i.e.,used because
prediction they
of abnormal canlevels
glucose operate with a relatively small training d
in the future).
Regarding chemical sensors for health applications, supervised learning algorithms
the training data are can be easily labelled using state-of-the art clinical signs and sym
by far the most used because they can operate with a relatively small training dataset,
and the computational the trainingcost
datais
canrelatively low.
be easily labelled Astate-of-the
using model classification
art clinical signs andfor supervised
symptoms,
and the computational cost is relatively low. A model classification for supervised learning
ing is shown in Figureis shown 13.in Figure 13.
Figure 13. Classification of Classification

Figure 13. ML algorithms. Ensemble
of ML algorithms. methods
Ensemble methodscombine two
combine two or more
or more models
models.
Examples of ensemble methods are random forest and gradient boosting.
ples of ensemble methods are random forest and gradient boosting.
Electrochemical sweat sensors possess many advantages over other alternatives, such
as ease of miniaturization and integration. These features allow for their implemen-
Electrochemical sweat sensors possess many advantages over other altern
such as ease of miniaturization and integration. These features allow for their imp
tation in wearable sensor technologies at a massive scale and continuous moni
tation in wearable sensor technologies at a massive scale and continuous monitoring.

Therefore, these sensors are able to collect a huge amount of data that can be processed
by ML algorithms.
On the other hand, many sweat electrochemical sensors and biosensors are intended
for continuous analyte monitoring due to their ability for miniaturization and integration.
Electrochemical sweat sensors for continuous monitoring (ESSCMs) are able to provide
a large amount of data automatically without the patient’s intervention and can detect
and predict health issues anytime. However, ESSCMs suffer from several limitations that
condition their accuracy during their lifetimes, such as the dynamic correlation of analyte
concentrations in sweat compared to other fluids that constitute a clinical gold standard,
such as blood; changes in their figures of merit due to physical or chemical interferences or
electrode degradation over time; and patients’ personal characteristics or health condition.
From the analytical point of view, the aforementioned limitations can be circumvented
by periodic external calibration. However, external calibration is a severe restriction for the
application of ESSCMs because it causes discomfort to the patient and cuts down on the
main advantage that ESSCMs have in terms of user experience.
The case of continuous glucose monitoring is very relevant because of the high in-
cidence of diabetes among the global population (9.3%) [154] and the huge market for
portable devices for glucose testing. For instance, the FreeStyle Libre® Flash Glucose
Monitoring System was developed by Abbot for the continuous monitoring of glucose
in interstitial fluid. This sensor did not show significant subject-dependent variations in
sensitivity and mean absolute relative difference (MARD). The overall MARD compared to
blood glucose reference was 11.4%. The correlation with blood glucose had a slope of 1.02,
an intercept of −6.4 mg/dL, and a correlation coefficient of 0.95 within a range of 23 to
498 mg/dL. This system has a factory calibration and allows algorithmic correction for the
measured analyte data during its 14 day use period [155]. Moreover, this sensor is able to
predict short-term trends in glucose levels, which is very important for patients to minimize
the occurrence of hyperglycaemia and hypoglycaemia. Although this sensor monitors
glucose levels in interstitial fluid, it is relevant in that it performs continuous monitoring
and has been developed on a commercial scale. For the case of this sensor, long-term
predictions were made employing machine learning algorithms, which are very relevant
for the prediction of nocturnal hypoglycaemia in diabetic patients [156]. The FreeStyle
Libre® sensor data were combined with data from a wearable physical activity tracker.
The main problems regarding FreeStyle Libre® and similar sensors are that they are
still invasive, and their application to preventive medicine is still limited. Regarding the
former, ESSCMs can be used as a non-invasive alternative for continuous monitoring of
glucose, as well as many other analytes. Regarding the latter, ML algorithms are very useful
tools for establishing trends in a patient’s condition and prescribing preventive treatments.
A major issue with sweat sensors is that there are many interferences that affect the
measurement of analytes, such as movement, temperature, and humidity. Regarding
accuracy, a multisensor approach has been proposed by many research groups [157]. The
problem with using various sensors is they provide increased noise levels. For this reason,
there are many noise-cancellation algorithms, such as Kalman filter, Fourier analysis, or
moving average filter.
Clark error grid analysis has been extensively used as a gold standard for clinical
validation of glucose meters. According to this analysis, the accuracy of glucose meters
should be within 20% of the true value. Accuracy of this order of magnitude is expected for
other analytes, including those present in much lower concentrations compared to glucose,
such as cytokines [158].
Application of ML algorithms to electrochemical sensors as solutions for enhancing
their selectivity and LOD, calibration, continuous monitoring, establishing trends, and
predictions of many clinically relevant analytes from measurements of body fluids have
been reported in recent literature. Some of these recent works are summarized in Table 6.
Table 6. Recent applications of ML for the optimization of monitoring relevant analytes from body
fluids using electrochemical sensors. EIS: electrochemical impedance spectroscopy.
Calibration/Prediction
Type of Sensor Monitored Parameters Monitored Fluids Reference
Strategy
Chloride, skin External calibration, principal
Potentiometric temperature, core Sweat component analysis, [159]
temperature, heart rate random forest
Principal component analysis,
synthetic minority
Ethanol, methanethiol, oversampling technique,
Chemoresistive (MOS
ammonia, Sweat (gas phase) support vector machine, [160–162]
type gas sensor)
trimethylamine decision tree, K-nearest
neighbours, naïve
Bayes classifier
Glucose, skin
Linear regression, ensemble
EIS temperature, relative Sweat [163]
regression, decision tree
humidity
Cortisol, skin
External calibration,
EIS temperature, relative Synthetic sweat [164]
K-nearest neighbours
humidity
EIS, Amperometric, Glucose, pH, relative External calibration,
Sweat [165]
Chemoresistive humidity K-nearest neighbours
External calibration, algorithm
Amperometric Creatinine, heart rate Sweat [166]
not specified
Linear regression, K-nearest
neighbours, decision tree,
Potentiometric pH Wound random forest, gradient [167]
boosting, artificial
neural network
Linear regression, support
vector regression, Bayesian
Amperometric Tyrosine, uric acid Sweat, saliva regression, K-nearest [168]
random forest
Linear regression, K-nearest
Amperometric Creatinine Tears random forest, gradient [169]
boosting, artificial
neural network
Support vector regression,
Potentiometric Na+ , K+ , Li+ , Pb2+ Emulated dataset [170]
artificial neural network
For instance, Choi et al. proposed a multisensor approach combined with machine
learning for the prediction of dehydration during exercise. They used a potentiometric chlo-
ride sweat sensor combined with a heart rate sensor and a skin temperature sensor [159].
Sabilla et al. used a set of commercial MOS-type gas sensors to build an electronic
nose for sweat analysis [160–162]. The gas phase of sweat from the armpit was sampled
using a vacuum pump searching for volatile metabolites produced by bacteria present in
the axilla region (ethanol, methanethiol, ammonia, trimethylamine). It should be noted that,
in this case, the target analytes were not analysed individually, and the sensors had many
cross-sensitivities. The aim of this work was to train this electronic nose for classification of
human gender using ML algorithms. The best accuracy (96.15%) was obtained using the
naïve Bayes classifier.
The use of ML algorithms allows for the monitoring of analytes in sweat by correlation
with other electrochemical measurements than selective potentiometric or amperometric
response. For instance, Sankhala et al. developed a sweat glucose sensing platform based
on electrochemical impedance spectroscopy combined with skin temperature and relative
humidity [163]. In this case, the decision tree algorithm provided better predictions of
glucose levels.
Another example of the application of ML to electrochemical impedance spectroscopy
was described for the monitoring of cortisol levels in sweat [164]. In this case, a label-
free immunosensor containing gold nanoparticles with the immobilized cortisol antibody.
Zmod was used as the only input signal, and the K-nearest neighbours algorithm was
used. The sensor was calibrated and then trained and tested using synthetic sweat samples,
showing accuracy for distinguishing low, medium, and high cortisol concentrations within
the range 8–140 mg/mL.
Sensors based on the same material can be used for simultaneous detection of different
parameters. For example, a flexible wearable sensor platform based on ruthenium sulphide
nanoparticles as a sensing element was developed for the monitoring of the pH, glucose
levels, and relative humidity of skin [165]. The sensor was calibrated externally and was
able to quantify glucose within the range of 10 Nm–1 mM and measure the pH of sweat
between 4 and 8.5. The K-nearest neighbours algorithm was used for data processing and
provided simultaneous monitoring of these three parameters.
ML algorithms can be used for the prediction of stress based on sweat monitoring. For
instance, Kalasin et al. [166] developed a wearable electrochemical sweat sensor for contin-
uous monitoring of creatinine. This sensor was based on poly(3,4-ethylenedioxythiophene)
polystyrene sulfonate (PEDOT:PSS) and cuprous oxide nanoparticles and was able to quan-
tify creatinine in sweat within the range 0.4–960 µM. Creatinine concentration was used in
combination with heart rate for the prediction of heat stress.
In another ML-assisted approach for the detection of creatinine, a cotton fibre electrode
system was fabricated and integrated to eyeglasses [169]. The working electrode consisted
of a cotton fibre with carbon black, cuprous oxide nanoparticles, a copper-based metal-
organic framework and graphene oxide. The use of ML in this case was trained to learn
the relation between creatinine concentrations in serum and in tears and gain the ability to
discriminate between low, normal and high concentrations of creatinine in patients using a
non-invasive method and a wearable device.
In another work from Kalasin et al. [167], a wearable pH sensor was used for wound
monitoring. The potentiometric working electrode was based on polyaniline and cuprous
oxide nanoparticles. The measured electrode potential was processed using ML and
deep learning algorithms to predict wound healing stages (inflammatory, proliferative,
remodelling). As expected, the deep learning algorithm performed better than tested ML
algorithms. Interestingly, this sensor also included flexible electronics and a 13.56 = MHz
antenna for energy harvesting and sensor readout.
One common problem regarding electrochemical detection of multiple analytes and
complex matrices is the superposition of signals and mutual interference. ML algorithms
can be used for signal discrimination. For instance, a flexible electrode made from laser-
induced graphene and molybdenum sulphide was fabricated and used for the simultaneous
detection of tyrosine and uric acid in sweat and saliva [168]. In this case, various ML
algorithms were trained using data from various electrochemical techniques, such as
cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, and large
amplitude AC voltammetry. In this case, the application of ML algorithms was useful not
only for the discrimination of both analytes but also for lowering the LOD to 100 nM and
10 nM for tyrosine and uric acid, respectively.
Regarding potentiometric simultaneous detection of ions in sweat, an emulator for
providing data for training a ML algorithm was developed by Hanitra et al. [170]. This em-
ulator was developed using, for the generation of simulated data, ion selective electrodes
for Na+ , K+ , Li+ , and Pb2+ at different mixed concentrations considering ion activities,
selectivity coefficients, and offset parameters extracted from real ISE calibration curves.
This emulator was able to provide 10 datasets of 68 points for training ML algorithms of dif-
ferent complexity. In this work, a multivariate vector regression model performed equally
accurately and much faster compared to a more computationally complex neural network.
ML algorithms are useful tools for enhancing the performance of electrochemical sweat
sensors, especially ESSCMs. Their main application in sweat sensing is the classification of
health status and prediction of abnormal analyte concentrations in a possible future event,
rather than contributing to extremely accurate concentration measurement. Supervised
learning algorithms are most used for applications for sweat electrochemical sensors. These
algorithms are used in combination with other strategies, such as use of nanomaterials,
multisensory approaches, and non-ML signal processing, to enhance their sensor accuracy,
LOD, and ability for correct classification and prediction. Training data and computational
availability are still a limitation regarding electrochemical sweat sensors at a research
scale, but these limitations can be overcome once the sensors are used at a larger scale in
combination with the internet of things and cloud computing.
8. Regulatory Aspects
According to the United States Food and Drug Administration (FDA) “General Well-
ness: Policy for Low-Risk Devices” guide, wearables should be considered medical devices
if they are intended to diagnose or treat a health condition or disease. Wearable medical
devices (WMDs), as any other medical devices, are regulated by health authorities over the
entire life cycle to ensure their safety, efficacy, and constant quality level.
ISO 14971 (“Medical Devices: Application of risk management to medical devices”) is
an FDA-recognized standard that provides medical device manufacturers with a framework
to systematically manage the risks to people, property, and the environment associated with
the use of medical devices. Risk management impacts the design and testing phases of the
manufacturing process. Application of ISO 14971 helps to develop devices free of structural
defects that could mean a risk to the patient or could compromise the correct technical
functioning. Ravizza et al. [171] recognized as a main risk for WMDs software malfunc-
tion, hacking of data, loss or alteration of data, electrical hazards, and electromagnetic
compatibility (IEC 60601), biocompatibility (ISO 10993), and physic and chemical stability.
Additionally, the FDA classifies WMDs according to their risk as low-risk Class I,
moderated-risk Class II and high-risk Class III. Most WMDs can be considered Class I or
Class II and require general or special controls, respectively. However, some products (e.g.,
wearable automated external defibrillators) are considered high-risk Class III WMDs and
require premarket approval.
Before the manufacturing of medical devices, it is imperative to evaluate which ma-
terials will be used and their interactions with the human body. ISO 10993: “Biological
evaluation of medical devices” intends to protect humans from potential biological risks
from the use of medical devices. For a complete biological safety evaluation, it classi-
fies medical devices according to the nature and duration of their contact with human
tissues. According to ISO 10993, WMDs can be categorized as intact skin surface de-
vices, which are indispensable for evaluating cytotoxicity, sensitization, and irritation or
intracutaneous reactivity.
Polymers used in medical devices must be biocompatible; i.e., they must be able to be
in contact with living systems without producing an adverse effect [38]. A list of the most
used biocompatible polymers in flexible electronics is presented in Appendix A. [39,40].
Several authors have proposed the use of polydimethylsiloxane, a soft and non-irritating
material often employed in microfluidic systems [69,172]. PDMS is soft and comfortable to
wear for extended periods of time (at least 6 h) without causing skin irritation. Furthermore,
PDMS prevents sensor chip bending when the sensing system is attached to slightly curved
body parts, such as the upper torso (front or back) or upper limbs (arms or legs) [69].
Another commonly used substrate is fabric. Fibre sensors can be woven into a fabric,
and compared with the traditional thin-film sensor, fabric sensors can bear various com-
plexes and even support severe deformations, such as twisting, while providing an effective
and comfortable interface with human skin (i.e., the fabric is breathable and comfortable),
and the sensor could be used for the long term without discomfort to users [67].
9. Concluding Remarks
The development of reliable electrochemical sweat sensors requires sensitive, highly
selective detection of targets, the electrochemical transduction of which must be integrated
into associated electronic circuits for signal processing and external communication. Many
efforts have been directed in the biosensors community to attain lower LODs and higher
sensitivity, along with an extended lifetime under working conditions. Many challenges
must still be solved, especially regarding collecting representative sweat samples and
improving signal processing in wearables, considering the signal drift due to sensor aging,
interference, and noise introduced by the user. The processing of signals by machine
learning could offer a new approach to improve the reliability of sweat sensors.
Author Contributions: Writing—original draft preparation, all authors; writing—review and edit-
ing, all authors; supervision, G.Y. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Instituto Nacional de Tecnología Industrial (INTI),
Argentina.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Polyethylene, cellulose, silk, shellac, polylactic acid, polycaprolactone, polyethylene
glycol, albumen, polyglycerol-co-sebacate, polylactic-co-glycolic acid, polypropylene, pol-
ytetrafluoroethylene, polymethylmethacrylate, polyethylene-co-vinyl acetate, polydime-
thylsiloxane, low MW polydimethylsiloxane, polyether urethanes, polyethylene tereph-
thalate, polysulphone, polyethylene oxide, polyethylene oxide co propylene oxide, poly-
vinyl alcohol, polyaniline, polypirrole, poly(3,4 ethylenedioxithyophene), melanin, PDPP-
DP, polyvinylidene fluoride.
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chemosensors

1 Functional Nanomaterials Group, INTI-Micro and Nanotechnologies,

Chemosensors 2023, 11, 244. https://doi.org/10.3390/chemosensors11040244 https://www.mdpi.com/journal/chemosensors

of electrochemical determination of the concentrations of small organic molecules (e.g.,

Especially attractive is the possibility of minimally invasive analysis provided by

1.2. Key Health-Related Examples of Sweat Biochemical Analysis

obtained is a interferent-free biofluid, and the normal evaporative process is maintained

Figure 1. Schematic diagram of an iontophoretic-sensing tattoo device, containing the iontophoretic

Second, surface wettability can be modified to collect, accumulate, and d

al. [31] developed a band aid that combines superhydrophobic and s

Figure 2. Schematic diagram of the three-dimensional

3.1.1. Reference Electrodes: Structural Overview

concentration of chloride ions (M). Under this thermodynamic correlation, a conventional

3.1.1. Reference Electrodes: Structural Overview

result, when compared to a pseudo-reference electrode, the new RE scheme includes an

3.1.2. Reference Electrodes: Ongoing Advances

Structure Method Advantages Disadvantages Ref.

Chemosensors 2023, 11, x FOR PEER REVIEW 11 of 47

Introducing slight differences to the formulation of the reservoir layer,

3.1.3. Reference Electrodes: Lingering Challenges

3.2. Ion-Selective Electrodes

3.2.1. ISEs Structural Overview

3.2.2. ISEs: Ongoing Advances

pH sensitivity ranging from 48 mV at a 0◦ bending angle to 51 mV/dec at a 90◦ bending

3.2.3. Ion-Selective Electrodes: Lingering Challenges

4.2. Stability of Enzymatic Biosensors

4.3. Selectivity: Chemical Interferences

Ye et al. [33] developed a wearable electrochemical sensor for real-time monitoring

5. Sensing Biomolecules in Sweat with Affinity Electrochemical Biosensors

Figure 7. Schematic representation of two label-free immunosensors designs employed in sweat

Figure 9. Organic transistor

Chemosensors 2023, 11, x FOR PEER REVIEW 29 of 47

Figure 10. Fundamental blocks of a wearable electrochemical sweat sensor.

Table 5. Characteristics of open-source potentiostats.

Name Power Communication User Interface Range Resolution Ref.

6.2. Power Supply

6.3. Wireless Communication

12. Four steps

Figure 13. Classification of Classification

tation in wearable sensor technologies at a massive scale and continuous monitoring.

120. Analog Devices. ADuCM355. Available online: https://www.analog.com/en/products/ADUCM355.html#product-overview

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