Wearable electrochemical sensors for forensic and clinical applications

Trends in Analytical Chemistry 119 (2019) 115622

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Wearable electrochemical sensors for forensic and clinical applications Patrick Cesar Ferreira a, 1, Vanessa Neiva Ataíde b, 1, Cyro Lucas Silva Chagas b, 1, ~o b, 1, Lúcio Angnes b, 1, Wendell Karlos Tomazelli Coltro c, 1, Thiago Regis Longo Cesar Paixa a, *, 1 William Reis de Araujo a b c

~o Paulo, Brazil Instituto de Química, Departamento de Química Analítica, Universidade Estadual de Campinas, 13083-970, Campinas, Sa ~o Paulo, 05508-000, Sa ~o Paulo, Brazil Instituto de Química, Departamento de Química Fundamental, Universidade de Sa s, Campus Samambaia, 74690-900, Goia ^nia, Goia s, Brazil Instituto de Química, Universidade Federal de Goia

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 9 August 2019

Electrochemical sensors are powerful analytical tools that, in the last few years, have attracted tremendous attention in coupling with wearable devices due to their incomparable properties, such as instrumental simplicity, low cost, flexibility, and miniaturization. These outstanding characteristics fit with the desired features for continuous on-body analyses. Wearable electrochemical sensors enable obtaining insights into individuals' health status through the noninvasive monitoring of clinically relevant biomarkers in different biofluids (saliva, tears, sweat, and interstitial fluids) without complex manipulation, sampling, and treatment steps. The electrochemical system can be fabricated in different substrates and transferred to the human body or coupled to common utensils to monitor (bio)chemical species or potentially hazardous compounds surrounding the users without disturbing their usual activities. In this work, we critically review the recent advances, the main technological and chemical challenges identified in wearable electrochemical sensors for forensic and clinical applications, highlighting the remarkable trends, needs, and challenges for future studies. © 2019 Elsevier B.V. All rights reserved.

Keywords: Wearable devices Electrochemical sensors Forensic chemistry Clinical analysis Portable chemical sensors

1. Introduction Personal clinical monitoring out of medical facilities enables a fast disease diagnostics and, consequently, the suitable treatment. From the point of view of the health industry, these devices could decrease the healthcare cost, and for the scientific community, we are pushing the limit to perform technological advances to increase life expectancy. The glucometer is a success case in homecare monitoring sensing which highlights these two points reported previously. However, since from the first report envisioned advantages for the real-time measurements of Parkinson's disease in 1990 [1], technological limitations were some of the obstacles to the development of this on-site and real-time measurement device. Some of the limitations are how to transmit the extracted information and how to miniaturize or power these devices.

* Corresponding author. E-mail address: [email protected] (W. Reis de Araujo). 1 All authors contributed equally to this work. https://doi.org/10.1016/j.trac.2019.115622 0165-9936/© 2019 Elsevier B.V. All rights reserved.

In this scenario, wearable chemical sensors (WCS) have attained the market, predominantly by the fitness monitoring gadgets, but also by commercialization of devices dedicated to health and clinical monitoring, showing a large growth in the last 5 years (see Fig. 1). According to Grand View Research Inc. [2], the wearable sensor market will reach USD 2.86 billion in 2025, an increase of 38.8% during the forecast period, with big companies like AppleⓇ and GoogleⓇ looking very carefully at these new devices. By definition, wearable sensors are devices that can be worn by someone to track their health and fitness using chemical or physical techniques to extract the desired information without any external pretreatment of the samples, i.e., all the necessary sample preparation needs to be performed by the device platform. Electrochemical techniques have been widely used to develop wearable sensors aiming at the real-time monitoring of relevant biomarkers directly on the human body without human decision, color changes, and due to the potentialities like portability, miniaturization, and low limits of detection compared with color detectors. Hence, the use of electrochemical detection in wearable technologies is a hot topic in Analytical Chemistry and other diverse

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Fig. 1. Progress of the citations and articles published per year by the scientific community using the keywords “wearable chemical sensor” (WCS, black bars), “wearable electrochem* sensor” (WES, red bars), and “wearable health* chemical sensor” (WHCS, blue bars) using the Web of Science® search engine.

scientific fields due to the significant contribution to healthcare management, portable medical tools mainly to assist people with disabilities or the elderly and provide a reduction in the analysis steps, helping analytical chemists. Notwithstanding, some challenges for the application of wearable sensors have to be addressed for continuous growth of this area, especially when nonenzymatic approaches are used because strategies to increase selectivity and sensitivity have to be proposed for real applications, as well as, inherently reversibility and stability of the sensor to circumvent fouling and nonspecific binding for complex matrix samples. In addition, the amount of sample and sampling strategies are problems that need to be circumvented too, as well as mechanical material degradation or failure due to stress. In recent years, wearable devices have been used in forensic sciences to understand absorption/excretion pathways, toxicology, drug abuse monitoring, as well as detection of dangerous compounds surrounding the user. Therefore, we believe that it is timely to perform a critical and concise review of these relevant topics. The purpose of this review is to present and discuss a critical view of the recent advances (<5 years) and the state of the art in wearable electrochemical sensor (WES) technology pointing out the main applications focused on clinical and forensic analyses trying to show approaches to circumvent problems enabling real-time measurements directly on the individual's body. 1.1. Historical remarks At the beginning of the development to implement these onbody sensors, many of the efforts were made in the use of physical sensors to extract the desired information. According to an excellent review published by Heikenfeld and coauthors [3], the development of wearable sensors started with the Apollo Space Program trying to monitor humans' exposure to physical extremes using wearable sensors capable of extracting electrocardiogram (EKG) information, breathing, and body temperature monitoring, which resulted in the wireless EKG in 1977 [3]. A pioneer sensors proposed by Asada and coauthors in 2001 [4] which was a ring sensor to monitor beat-to-beat pulsation transmitting the optical extracted information via radio frequency. The information was extracted optically using a light emitting diode (LED) and a photodiode detector at the ring sensor and this approach opened a

new possibility that allowed smartwatches to measure heart rate. Other approaches monitoring different body parts appear in the literature like an in-ear optical sensor [5] to measure cardiovascular functions. These studies opened a new field to develop devices for fitness, wellness, and activity monitoring purposes using physical parameters like respiration rates [6], blood pressure [7,8], body temperature [9,10], oxygen [11], and motion sensors [12e15], in which microelectronics help to develop accelerometers, gyroscopes, magnetometers, and others for these purposes and smartphones technologies. Radio frequencies [4,16] emerged as the first technology used to transmit the information by someone or somewhere, but new technologies were embarked on the wearable devices to transmit the desired information through Bluetooth [17], wireless [18], and ZigBee connections [19]. With the advent of smartphones, this every day and worldwide available item enables the implementation of multiple detectors and has become a valuable and costeffective analytical tool for continuous monitoring and quick decision-making [20]. This point needs to be highlighted because wearable devices are used to track elderly people remotely with or without any diseases or chronic conditions to put them in a safe condition based on this real-time monitoring. For example, vital signs monitoring and motion, and signal transmission of the data collected by wearable devices can help to manage treatment protocols and follow up. Notwithstanding, chemical sensors appear to attract less attention than physical detectors embedded in the wearable devices as highlighted by Wang and coauthors in a review about wearable electrochemical sensors in 2012 [21]. Since then, large advances have been made using different chemical detectors in wearable technologies like optical and colorimetric (bio)sensors measurements [22] for healthcare [23,24] and electrochemical (bio)sensors for the detection of illicit drugs, explosives, nerve agents, and pathogens [25e27] and sweat monitoring for exercise and clinical purposes [22,28,29], or using hybrid detection, color detection, and electrochemistry [30] for clinical evaluation tools. 2. Fabrication methods and main strategies This section describes the main approaches for the WES fabrication, as well as the trends in sensing platforms and materials/ modifications used to enhance the performance for on-body analyses. There are numerous substrate types for fabrication of WES, such as gloves [31], temporary tattoos [32], mouthguard [33,34], contact lenses [35], wristband [36], smart bandage [37], and textiles [38]. The materials used and fabrication approaches for these devices depend on the desired application [39]. Hence, we discuss some of the most common approaches to building up the sensing device, based on the unique and clever ideas of the authors. Fabrication methods reported in the literature include templatebased methods, such as screen printing [26,40], roll-to-roll (R2R) gravure printing [41], stamp transfer electrodes (STEs) [42]; nontemplate based methods, such as inkjet printing [43], 3D printing [44,45], and lithographic methods, including soft-lithography [46], photolithography [47e49], and electron-beam evaporation [50]. 2.1. Screen printing The screen-printing method has been widely used in the development of wearable devices due to its unique characteristics for fabrication, like large-scale manufacture in different geometries, low cost, robustness, and suitable electrochemical performance [21]. However, some important factors must be taken into account when the devices will be used for electrochemical measurements because its performance could be compromised, for instance, the

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properties of the substrate, the composition of the ink, and the manufacturing conditions [39] are some of these parameters. Windmiller and coauthors [40] demonstrated the first example of a temporary electrochemical tattoo sensor for clinical and forensic detection, Fig. 2A. Temporary tattoo transfer paper was used as a

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substrate. In the first step, Ag/AgCl conductive ink was used to make the electrical contacts and the reference electrode. In the next step, the working and counter electrodes were screen printed using a carbon ink, and in the last step, an insulator ink was printed on that surface to delimit the electroactive area. After printing each layer,

Fig. 2. A) Picture of screen printed tattoo-based electrodes, (B) screen printed glove-based electrochemical sensor. Image (A) was adapted with permission from Ref. [31] and (B) was adapted with permission from Ref. [40]. (C) Printing of the three-electrode arrangement on PET (flexible substrate) using R2R gravure printing (a). Three layers of inks are deposited: silver, carbon, and insulation; Image of the R2R system for each printer unit (b), Fabrication steps of the device and subsequent functionalization of its surface (c). The working electrode can be functionalized depending on the desired application; the device can be used as a “smart” wristband for continuous and real-time monitoring of target molecules present in sweat (d). These sensors can be integrated with printed circuit boards that provide processing and transmission of information collected in situ. Reproduced with permission [41]. (D) Schematic representation showing the fabrication steps involved of stamp transfer electrodes (STEs): (a) Ag/AgCl conductive layer, (b) carbon layer, (c) insulation layer, and (dee) application of STEs on different substrates. Adapted with permission [42].

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the substrate was cured in a temperature-controlled oven at 60
C for 40 min, followed by cutting into single-use tattoos. The printed electrodes' tensile strength was improved by the addition of carbon fibers to the carbon ink. This work proposed a fabrication method of WES compatible with the characteristics of human skin, allowing the monitoring of chemical constituents that can provide important information about a person's health. Another approach using a screen-printing technique to produce WES was proposed by Barfidokht and coauthors [31]. They described a wearable glove-based sensor (“Lab-on-a-Glove” sensors) for electrochemical detection of the synthetic opioid fentanyl, Fig. 2B. The finger worked as a sample collector and the index finger, containing the electrodes, as the sensing device. The working and counter electrodes were screen printed in carbon ink, while the reference electrode with a Ag/AgCl-based ink, on the index finger. A circular carbon pad on the thumb was printed through the same procedure. 2.2. Roll-to-roll gravure printing Another printing method that has been widely employed in the fabrication of flexible electrodes is R2R technology [51]. Bariya and coauthors [41] successfully demonstrated the manufacturing of a noninvasive biosensor using the R2R gravure printing technique aimed at its application for health and fitness monitoring. Because the carbon films printed by R2R engraving are not as thick as those produced by screen printing, the electrical conductivity of the surface is compromised, therefore, it was necessary to print a silver ink layer prior to the carbon layer. The three-electrode setup was printed on poly(ethylene terephthalate) (PET) substrate. The working and counter electrodes were made of carbon ink and the reference electrode of silver ink, a layer of insulating ink was also used to separate the detection zone from the electrical contacts (Fig. 2C). The total printing time of the device, taking into account the printing of the three layers (silver, carbon, and insulation inks) and curing stage, was about 30 min, making possible the fabrication of 150,000 electrodes in a roll of 150 m PET web. The authors reported that the use of the R2R gravure printing is a technique that allows the production of devices on an industrial scale, with a large area, and in a fast way. 2.3. Stamp transfer An alternative fabrication method over printing techniques was proposed by Windmiller and coauthors [42] and consists in the production of electrodes using stamp transfer. This method allows the fabrication of electrochemical sensors in nonplanar substrates (e.g., skin). As proof-of-concept, STEs were used in the detection of physiologically important compounds, such as dopamine and ascorbic acid, and also for compounds of forensic interest, as for example copper (residue of firearm discharge), and safety matters, such as explosives (TNT). In addition, the device was applied directly on the skin surface and levels of uric acid were monitored. An elastomeric stamp was patterned with the electrode layout and in the first step was applied in a stamping pad with Ag/AgCl-based ink and then pressed onto the desired substrate, creating a conductive layer and the reference electrode. In the second step, a stamp with another layout was used to fabricate the working and counter electrodes with diluted carbon ink. In the last step, an insulation paint was used to delimit the detection zone (Fig. 2D). The electrical contact between the STEs and the electrochemical analyzer was implemented through lead wires connected with adhesive tape. The carbon ink used in STEs applied to the skin was not diluted to maintain a suitable viscosity for epidermal transfer. In this case, the electrical contact was made with a conductive elastic strap.

2.4. Inkjet printing Inkjet and 3D printing methods are classified as non-template methods, owing to the ink being distributed freely on the substrate surface. These printing methods rely on ink dispensing systems, such as pneumatic, piezoelectric, aerosol, electrohydrodynamic, and thermal [52]. Inkjet printing has several advantages, including an accurate deposition of micro- and nanomaterials in functional arrangements, affordable, easy-to-change digital patterns, compared with screen-printing technologies, consuming small amounts of materials [53]. Qin and coauthors demonstrated the development of a pH sensor on flexible and rigid platforms employing the inkjet printing technique [43]. The fabrication process of the electrochemical monitoring system is represented in Fig. 3A. A Pd organoamine complex was used as a precursor ink of the Pd metallic layer. Hence, this complex was solubilized with toluene (Pd/PhMe) and the bifunctional Pd/PdO metallic electrodes were fabricated by the oxidation of the printed Pd film. The PdO surface acts as the pHdependent layer and the surface of the Pd (the conductive layer underneath) as paths that allow electron conductivity. The oxidation process of Pd was carried out by thermolysis and annealing at 200
C for 48 h. The reference electrode was printed with silver nanoparticle ink. A commercial SU-8 ink was used to fabricate an interface between the working and reference electrodes. Pd/PdO printed sensing electrodes demonstrated sensitivity, rapid response, stability, and accuracy in measuring water samples, showing potential in environmental applications and in human health conditions. 2.5. 3D printing 3D printing is a good alternative in the fabrication of WES among other printing techniques, allowing printing of highly complex parts in different substrates. In addition, the manufacturing process is simple, cost effective, and it may be explored for large-scale production depending on printing technology [54]. For instance, Nesaei and coauthors [44] demonstrated two-step 3D printing of Prussian blue ink and the enzyme glucose oxidase for electrochemical glucose biosensing. The technique used was Direct-Ink Writing (DIW), which consisted of dispensing inks out of the nozzle in a range of mm to mm, depositing the inks with great spatial control and resolution. The DIW platform features a three-axis positioning system, enabling the substrate to be positioned with sub-micron accuracy within the work area. The inks were inserted into the stainless-steel nozzle, which was attached to the pump outlet, using a needle and dispensed by a positive pressure pump. The working electrode was fabricated in two stages; in the first one, the electrodes were printed using carbon modified with Prussian blue ink onto the tattoo paper surface, followed by drying in a vacuum oven at 100
C for about 10 min. In the second step, the enzyme-containing ink was printed on the carbon electrodes (Fig. 3B). The electrochemical cell was assembled using a Ag/ AgCl reference electrode and a platinum counter electrode. An important point to highlight in the work described is the need to integrate the printing of the three electrodes to enable their application as a wearable electrochemical sensor. In addition, as pointed out by the authors, integration of the proposed biosensor with other electronic components is required, aiming to obtain signal processing and wireless communication. 2.6. Lithographic methods The lithographic methods, including photolithography, e-beam, and ion-beam lithography, produce high performance and reproducible wearable electrochemical devices. However, the high cost of this technique over the printing methods previously mentioned

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Fig. 3. A) Schematic representation of fabrication steps of Pd/PdO printed sensing electrodes. (a1) Cleaning of the surface of the glass or polyimide (PI); (a2) Drying the surface of the PI using air plasma; (b) Inkjet printing of the Pd ink; (c) Oxidation of the Pd film employing thermolysis and annealing; (d) Inkjet printing of SU-8 ink; (e) Inkjet printing of the reference electrode with silver nanoparticles; (f) Chlorination of the Ag surface using NaOCl ink; (g) Pipette printing of PVC/KCl/AgCl ink to form a solid electrolyte on the reference electrode, electric contacts painted with silver paste. Reproduced with permission from Ref. [43]. (B) Schematic representation of the fabrication processes of the 3D-printing glucose biosensor: (a) Direct-Ink-Write (DIW) system; (b) DIW of carbon modified with Prussian blue ink onto the surface of the tattoo paper; (c) DIW containing enzyme ink printed on the carbon electrodes; (def) summary of the device's production stages. Reproduced with permission from Ref. [44]. (C) Schematic illustration fabrication steps of nanopillar electrode (NPE) (a), Scheme showing the configuration of three electrodes and photograph of NPE (b), Images of the NPE bending and twisting (c), USB connection (d), and Scanning Electron Microscopy (SEM) images of NPE (e). Reproduced with permission [46].

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represents a noticeable disadvantage, limiting its use to a few research groups. Clean-room facilities, expensive chemicals, and laborious processes drive up fabrication costs [52]. Park and coauthors [46] reported the development of flexible electrochemical sensors based on nanopillar array electrodes for analysis of pathogens in food. The nanopillar array was used as a matrix for the sensor fabrication because it offers high surface area, increased mass transfer, easy control of surface functionalization, among others. The flexible nanopillar electrode (NPE) was produced using polyurethane as a substrate. In the first stage, Si wafers were used as a mold and the nanopillars were patterned by softlithography. Then, the polymer was detached from the mold. In the second step, titanium and gold layers were deposited on the polymeric substrate using electron-beam evaporation (Fig. 3C). The gold layers patterned on the NPE were used as working and counter electrodes, while a silver paste was printed on the nanopillar, creating a reference electrode. The device demonstrates the potentiality of being applied as a wearable sensor, although this feature was not explored in the paper. Table 1 summarizes some advantages and disadvantages of the main fabrication methods aforementioned. 3. Clinical applications The impressive advances in the development of wearable sensors is largely associated with people's longing to monitor vital functions and perform clinical diagnoses in real time without sophisticated instrumentation requirements. Considering the advances reported in the last five years, a noticeable increase in the number of publications on wearable chemical sensors for health applications (WHCS) has been observed based on the data extracted from the ISI Web of Knowledge database (Fig. 1). Most of the published studies have demonstrated in vitro tests only. For this review, the discussion has focused on the studies containing bodyworn electrochemical sensors. In addition, the analytical techniques adopted as well as the target analytes will be discussed considering the ease-of-use, biocompatibility, application relevance, and analytical validation. The WES dedicated to clinical diagnosis are mostly based on potentiometric, amperometric, and voltammetric measurements. Considering the reports found in the literature, the wearable devices with potentiometric detection are extensively applied for the analysis of alkali and alkaline earth metal ions, such as Naþ, Kþ, and Ca2þ, as well as detection of NH4 þ, Cl, and pH measurement [48,49,55e63]. Another important point to note is the fact that the main target biological fluid has been the sweat, except for the

studies published by Mostafalu and coworkers, who successfully measured the pH in mice interstitial fluids [64] and by Lee and coworkers, who monitored Naþ concentration levels in saliva [65]. The determination of Naþ and Cl ions, mainly in sweat, is intended to diagnose and monitor the evolution of cystic fibrosis, a disease of genetic origin that causes a thickening of mucus secreted by the body and may cause diabetes due to changes occurring in the pancreas. In addition, individuals affected by cystic fibrosis have a life expectancy reduced to 15 and 25 years old for children and adult patients, respectively. The devices for sweat analysis are based on three basic types: sweatbands [48,49,55,66e68], wristbands [28,41], and epidermal patches [50,58,60,62,69e73]. However, studies using carbon fiber [74], textile [75], and even the use of eyeglasses [63] have already been reported in the literature. Although many devices have been tested in vivo, only a few studies have performed accuracy tests, which serves to tell us how reliable the proposed device is, when compared with reference methodologies [41,47e49,62,68,71,75]. More details and discussion about wearable potentiometric sensors can be found in recent review articles published by Crespo and coauthors [76,77]. While wearable potentiometric sensors have focused on the analysis of small ions, the amperometric devices have applications dedicated to clinically relevant organic molecules, such as glucose [29,35,46,48,57e59,61,63,73,78e85], lactate [30,49,63,85,86], and uric acid [34,37]. According to the data from the World Health Organization (https://www.who.int/diabetes/en/), the current estimate is approximately 422 million adults living with diabetes and approximately 1.6 million deaths per year caused by the disease [87]. Considering these alarming data, the major published studies using wearable sensors have successfully explored glucose as a target analyte, either with a single test or with real-time monitoring in several biological fluids such as sweat, interstitial fluid, blood, saliva, and tears. One of the main advantages of wearable devices is that the collection of biological fluids occurs usually in a noninvasive or minimally invasive way. However, there are still some obstacles to be overcome because the commercially available glucometers are quite attractive for monitoring blood glucose levels, despite the need for a drop of blood. Recently, Bae and coworkers reported a study using a WES to monitor glucose in sweat made with nanoporous gold electrodes in a poly(dimethylsiloxane) (PDMS) substrate [81]. Fig. 4A shows the wearable device attached to the volunteer skin, the current response for glucose in sweat before and after a meal, and the assembly steps of the wearable sensor. The proposed sensor presented ease-of-use features, revealed good skin adaptation and satisfactory accuracy when compared with commercial sweat and blood glucose tests, thus

Table 1 Advantages and disadvantages of the main WES fabrication methods.

Template-based methods

Fabrication methods

Advantages

Disadvantages

Screen-printing

Low-cost; large scale manufacture; robustness; design flexibility; good reproducibility; wide choice of materials Mass production; can cover a large area

Incompatibility with nonplanar and oversized substrates

Roll-to-roll gravure printing Stamp transfer Non-template methods

Inkjet printing

3D printing

Lithographic methods

Soft-lithography Photolithography Electron-beam evaporation

Allow the use of nonplanar substrates; simple; robust Accurate deposition of micro and nanomaterials; affordable; easy to change digital patterns and consumes a small amount of materials Allow print highly complex components using different materials; simple manufacturing process; cost-effective and large-scale production Reproducibility; micro/nanoprocessing of flexible thin-film materials

Register errors (machine and cross directions); surface topography Fabrication efficiency and devices performance consistency are not well ensured Ink formulation (suitable tension surface and viscosity needs optimization) Nozzle clogging; resolution limited materials for flexible device printing Clean-room facilities; expensive chemicals and laborious fabrication processes

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Fig. 4. A) Wearable device placed on the volunteer's skin and the respective monitoring of the glucose level in the sweat before and after a meal (a). Schematic representation of the sensor with the detail of its parts demonstrated through micrographs (b). Reproduced with permission [81]. B) Photo of the device with the flexible circuit board (a). Representation of the arrangement of electrodes for the detection of glucose, Naþ, Kþ, and Hþ (b). Representation of all the layers that make up the electrochemical sensor and the flexible circuit board as well as the model of power and data acquisition via wireless by a smartphone (c). Reproduced with permission [57]. C) Representation of the multiplexed analysis with the wearable sensor of chloride, pH, glucose, and lactate present in sweat. (a) Photo showing the volunteer using the device during physical exercise. (b) Graph showing the influence of the reading distance of the NFC antenna. Colorimetric analysis of chloride (purple color) and pH (yellow/green color) present in the sweat collected during the physical exercise of the volunteer. (c) # 1 and (f) # 2. Real-time response graphs for glucose and lactate detection in the sweat of volunteers (d and e) # 1 and (g and h) # 2. Reproduced with permission [30]. D) Detection of metals present in sweat through a wearable electrochemical device. (a) Photo of the sensor fixed on the individual's arm. (b and c) Detection of zinc and copper in sweat. (d) Comparison between the levels of zinc and copper obtained with the wearable sensor versus ICP method. Reproduced with permission [88].

demonstrating it to be a promising and powerful device compared with commercially available tests. The combination of electrochemical techniques for the simultaneous determination of various analytes in wearable devices is quite common. Potentiometric techniques combined with amperometry are the most commonly used together, generally in the determination and monitoring of inorganic cations and anions as well as organic molecules. The possibility of performing multiple analyses simultaneously in a single device has received growing attention from the scientific community, providing to the patient conditions to obtain helpful clinical information through noninvasive or minimally invasive procedures. Fig. 4B displays a flexible

wearable device reported by Xu and coworkers composed of an arrangement of electrodes printed in PDMS [57]. The developed device has two different detection regions, one for glucose amperometric measurement and the other for potentiometric readout of Naþ, Kþ, and Hþ cations. In addition, the electrochemical sensor has a flexible electronic circuit with wireless and batteryfree communication, requiring only a smartphone to feed and collect data from the wearable device without the use of an external power source. This approach is very promising being possible also to conduct a multiplexed analysis. Pulsed electrochemical techniques are also often explored in wearable devices, such as square-wave stripping voltammetry

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[32,88] and differential pulse voltammetry [89]. These techniques enable the analysis of heavy metals [32,88], cortisol [90], and other organic molecules [89]. The monitoring of metals present in the body is of great clinical interest, such as zinc and copper, which play fundamental roles in strengthening the immune system and it is responsible for the energy production and functions of various enzymes in the body, respectively. In this regard, Gao and coworkers developed a wearable electrochemical sensor for the simultaneous analysis of Zn2þ and Cu2þ in sweat (Fig. 4D) [88]. The same device was also tested for Cd2þ, Pb2þ, and Hg2þ in sweat and urine samples, demonstrating the sensor's ability to determine possible contamination by metals. In a previous study, Kim and coworkers demonstrated the detection of Zn2þ in the sweat of three volunteers using a tattoo-based sensor during physical exercise [32]. In order to compare the analytical performance of the WES, some analytical parameters such as linear concentration range, sensitivity, repeatability, selectivity and accuracy were summarized in Table 2. The linear concentration range was the basic parameter to verify the feasibility of the wearable devices for clinical applications. All the studies presented calibration curves comprising the clinical ranges of interest for each analyte, with concentration intervals very similar to each other. However, the sensitivity values achieved by the wearable sensors have showed significant differences between them. The sensor proposed by Wang and coworkers [59] provided good sensitivity for Ca2þ (52.3 mV dec1) when compared to the report from Nyein and coworkers (32.7 mV dec1) [48]. On the other hand, the sensor described by Wang's group exhibited sensitivity values slightly lower for Naþ, Kþ and glucose

in comparison with other sensors described in Table 2. In addition, this sensor presented a narrower working pH range (4e7). However, among the summarized studies, this sensor was the most comprehensive considering the number of analytes simultaneously analyzed (Naþ, Kþ, Ca2þ, pH and glucose), thus demonstrating its potential for multiplexed assays. From Table 2, it is possible to highlight the expressive use of wearable potentiometric ion sensors to supply physiological information in sweat samples during certain human activities. The simplicity, portability and usual robustness (avoiding the requirement of constant recalibration) of potentiometric devices associated with wearer platforms (sweatbands, epidermal patches, textiles, etc) provide a simple way to continuous monitoring of clinical biomarkers in sweat. It is important to note that the analytes assessed by potentiometric sensors usually present high concentration in the biologic fluids (~mmol L1 range) and when is necessary detect very low quantity, the use of amperometry or voltammetric techniques associated with preconcentration steps are almost mandatory (Table 2). The sensors proposed by Javey's group [48,49,88] and by Parlak and coworkers [90] successfully described analytical reliability of on-body tests by comparative standard methods, which presented excellent results in terms of repeatability, selectivity and accuracy. On the other hand, most of the WES cited in Table 2 performed partial analytical reliability experiments only, highlighting mostly selectivity analysis. In summary, there is still a certain difficulty to correlate the real-time results (dynamic events) obtained with the wearable sensors to other robust and well established analytical techniques. The integration of these devices with the human body

Table 2 Comparison of the analytical performance of some WES for clinical applications. Analyte 2þ

Technique

Sample

Ca pH Glucose Lactate Naþ Kþ Naþ Cl Glucose Naþ Kþ Glucose Lactate Naþ Kþ pH Glucose Naþ Kþ Naþ Kþ Ca2þ pH Glucose Kþ Lactate pH Glucose Naþ Uric Acid Uric Acid Zn

Potentiometry Potentiometry Amperometry Amperometry Potentiometry Potentiometry Potentiometry Potentiometry Amperometry Potentiometry Potentiometry Amperometry Amperometry Potentiometry Potentiometry Potentiometry Amperometry Potentiometry Potentiometry Potentiometry Potentiometry Potentiometry Potentiometry Amperometry Potentiometry Amperometry Potentiometry Amperometry Potentiometry Amperometry Amperometry Square-wave stripping voltammetry

Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Sweat Interstitial fluid Interstitial fluid Saliva Saliva Wound fluid Sweat

Zn Cu Caffeine Cortisol

Square-wave stripping voltammetry Square-wave stripping voltammetry Differential pulse voltammetry Amperometry

Sweat Sweat Sweat Sweat

Linear Range 0.125e2 mM 3e8 0e200 mM 0e30 mM 10e160 mM 1e32 mM 10e160 mM 10e160 mM 0e100 mM 10e160 mM 1e16 mM 10e200 mM 5e25 mM 10e160 mM 1e32 mM 3e8 100e500 mM 0.1e200 mM 0.1e100 mM 10e160 mM 2e32 mM 0.5e2.53 mM 4e7 0e200 mM 0e100 mM 0e14 mM 3e8 0e20 mM 0.0001e1 M 0e1 mM 100e800 mM 0.1e2.0 mg mL1 0e300 mg L1 0e300 mg L1 10e40 mM 0.01e10.0 mM

Sensitivity

Repeatability

Selectivity

Accuracy

Ref.

32.7 mV dec e 2.35 nA mM1 220 nA mM1 64.2 mV dec1 61.3 mV dec1 63.2 mV dec1 55.1 mV dec1 2.1 nA mM1 0.031 nF mM1 0.056 nF mM1 0.5 mA mM1 0.0075 mA mM1 60.1 mV dec1 64.5 mV dec1 60.0 mV dec1 0.714 nA mM1 46 mV dec1 60 mV dec1 45.8 mV dec1 35.9 mV dec1 52.3 mV dec1 e 2.15 nA mM1 58.0 mV dec1 e 59.63 mV pH1 e 188 mV dec1 2.32 mA mM1 2.4 nA mM1 23.8 mA mL mg1

Yes Yes Yes Yes Yes Yes Yes Yes e e e e e Yes Yes Yes Yes Yes Yes e e e e e Yes e e e Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes e e e e e e e Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes e e Yes Yes Yes e

Yes Yes Yes Yes Yes Yes e e e e e e e e e e e e e Yes Yes Yes Yes Yes e e e e e e e e

[48]

10.4 nA L mg1 4.1 nA L mg1 110 nA mM1 2.68 mA dec1

Yes Yes e Yes

Yes Yes Yes Yes

Yes Yes e Yes

1

[49]

[55]

[56]

[57]

[58] [59]

[63] [64] [65] [34] [37] [32] [88] [89] [90]

P.C. Ferreira et al. / Trends in Analytical Chemistry 119 (2019) 115622

and verification of the precision of the proposed methods is undoubtedly one of the main challenges of the WES. Besides electrochemical devices, wearable sensors with colorimetric detection have gained considerable visibility in recent years. These colorimetric sensors offer several advantages such as ease of data acquisition without the need for sophisticated equipment, no requirement for using power supply, and the possibility to perform analyses of a wide variety of compounds of clinical interest [23,24,91,92]. Fig. 4C shows a wearable device integrated with dual electrochemical and colorimetric detection for lactate, glucose, pH, and chloride in sweat, developed by Bandodkar and coworkers [30]. In addition, the developed device demonstrated the ability to monitor the rate of sweat loss volumetrically requiring only a smartphone to collect and analyze the data recorded with the wearable sensor. The sensor was tested on two volunteers and the on-body results revealed an excellent correlation with those obtained by commercial tests. 4. Forensic applications Considering that the main focus of the development of wearable sensors since its inception has been for applications in health and well-being of individuals, there is still a large field of research to demonstrate the potential of such sensors in various other research and industrial fields, such as environmental, food, and forensic analyses [93]. In the forensic scenario, there is limited development of wearable smart devices addressed to this type of application. A search in the literature (Web of Science®) shows that in the last 5 years, less than 20 articles have been associated with keywords like “wearable” and “forens*,” and only one of these results when searched by “wearable electrochem*” and “forens*”. The low number of results can be associated with the choice of authors' keywords and the use of more specific terms because the main results were obtained with “wearable security,” for example. However, in all cases, few research studies have been published until now regarding wearable devices applied to forensic chemistry. These few results may be related to the high control for obtaining analytical standards associated with legal aspects in operating these kinds of studies on the human body and even in a very specific public, such as users of illicit drugs, for example. In the field of forensic chemistry, we mainly referred to the detection of drugs, explosives, and other toxic/dangerous compounds for safety issues, different types of adulterations, analyses of physicochemical and biological evidence at a crime scene, etc. [94,95]. In this review, the recent development of portable electrochemical sensors that work/measure directly on the body or in utensils commonly used near the human body to detect forensically relevant species are divided and critically discussed into two main subtopics: 4.1. Detection of licit and illicit drugs The detection of licit and illicit drugs in different biofluids is usually performed by several analytical techniques, mainly by gasand liquid chromatography combined with mass spectrometry [96]. The analysis by conventional methods requires the collection, storage, and delivery of biofluids to a specialized laboratory, which increases the possibility of sample contamination during the collection and preparation steps, risk of evaporation, and the possibility of degradation of biocompounds and/or unstable metabolites (short-life products), thus not providing a measure in real time, losing information of dynamic events. From the analytical point of view, the application of wearable sensors that allow rapid, real-time in situ analyses could be of great interest for forensic applications. On the other hand, as mentioned

9

earlier, the development of wearable (bio)sensors for the detection of drugs of abuse, for example, is still a relatively unexplored field of activity. This probably can be attributed to the difficulty of conducting studies in humans because of ethical and security issues, as it would be necessary to subject individuals to ingestion of harmful and illegal species to perform in vivo studies/optimizations of the protocols. Another challenging point to be highlighted is the typically low concentration of the drug and metabolites in samples of high complexity like saliva, sweat, and interstitial fluids that are the most common biofluids assessed by wearable sensors (minimally invasive methods). One possibility for future studies to avoid human ingestion of drugs for pharmacokinetic studies and development of analytical methods is the use of biomimetic systems such as organ-on-a-chip technology [97]. In view of these drawbacks, in vivo studies using wearable chemical sensors have so far been limited to analyses of licit drugs. Regarding the use of WES, we can highlight some work such as that of Tai and collaborators [89], who developed a wearable platform equipped with an electrochemical differential pulse voltammetry sensing module for caffeine detection in sweat. The authors fabricated flexible electrodes by the R2R printing process. The carbon working electrode was modified with carbon nanotubes and Nafion mixture through a drop-casting method aiming at antifouling protection and improves the sensitivity. Initially, they evaluated sweat extraction by an iontophoresis protocol (1 mA for 5 min using pilocarpine hydrogel as an inductor) and collected these samples for ex vivo quantification and verified a good correlation between ingested caffeine levels and those analyzed in collected sweat. Following the study, to demonstrate the sensor's ability to capture physiological trends of caffeine in human subjects, ergometer-based cycling experiments were conducted. To enable wearability, the R2R printed electrodes were connected with flexible printed circuit boards that were worn on a subject's wrist and sealed with a PDMS band, Fig. 5A. The main results demonstrated that when an individual drinks a single-shot espresso coffee (~75 mg of caffeine) and starts physical exercise 30 min after ingestion, the caffeine concentration increases, its peak value is 13 mmol L1 around 60 min after caffeine intake and decreases over time, Fig. 5A(c). The time corresponding to the maximum concentration falls within the expected range of 30e120 min. Concentration values were determined by sweat collection at defined interval times and analyzed ex situ and the authors concluded that the data from on-body experiment follows a similar pattern to the ex situ data. The weak point of this work was the lack of validation of the method or the performance of comparative analyses to guarantee the accuracy of the results. Although caffeine is not a compound of great forensic interest, we consider that this work opens the door to other studies for demonstrating as a proof-of-concept the possibility of detecting and monitoring drug intake and metabolism directly in sweat in a noninvasive and real-time way. The observed caffeine levels and metabolic trends are consistent with the physiological data reported in the literature. Thus, we can envision the development of new wearable methods for the detection of abuse of several other illicit drugs of forensic relevance, avoiding the need for sample collection and minimizing the possibility of sample adulteration. Regarding the detection of licit drugs, an important example of an application involving WES is the detection of alcohol. Alcohol consumption is common and widespread throughout society. However, its use has promoted a wide range of adverse effects on personal health and economic factors. Thus, the monitoring of alcohol abuse and effective real-time detection represents substantial concerns for personal and automobile safety as well as forensic applications [98]. Accordingly, there are tremendous needs

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P.C. Ferreira et al. / Trends in Analytical Chemistry 119 (2019) 115622

Fig. 5. A) In situ monitoring of sweat caffeine levels using a wearable electrochemical device during a cycling exercise (a) and the zoom-in image of a sensing platform packaged in a PDMS wristband (b). Caffeine monitoring through exercise-induced sweat and some differential pulse voltammetry (DPV) signals for the exercise trial and a summary plot of the caffeine levels over time (c). The exercise begins at 30 min after caffeine intake. Reproduced with permission of [89]. B) Schematic representation of a hollow microneedle (a), press fitting of Pt and Ag wires to the aperture of the hollow microneedle (b) and Pt and Ag wires integrated microneedle array (c). An image showing the microneedle array mounted on the fingertip (d). Optical micrograph of the microneedle array integrated with Pt and Ag wires (e). Schematics demonstrating the construction of an alcohol biosensor with its multilayer reagent coating along with the biocatalytic reaction of the immobilized alcohol oxidase (AOx) (f). Reproduced with permission of [99]. C) Schematic of the measuring procedure for suspicious powder samples at a wearable fingertip device. The fingertip exhibiting the surface of screen-printed electrode onto a flexible nitrile finger cot (bottom left inset), as well as a conductive gel immobilized upon the thumb (bottom right inset) (a), swiping method of sampling to collect the target powder directly onto the electrode (b), completion of the electrochemical cell by joining the index finger with electrodes to the thumb coated with the conductive gel electrolyte (c). Reproduced with permission of [101]. D) Schematic diagram of a wireless operation of the iontophoretic-sensing tattoo device for transdermal alcohol sensing. Reproduced with permission of [100]. E) Image of the integrated potentiometric electronic wireless biosensor system, placed on the mannequin, shows the wirelessly transmitted signals; a nebulizer was used to generate the fluorinecontaining organophosphates nerve-agent simulant diisopropyl fluorophosphate vapors throughout the vapor study. Reproduced with permission of [104]. F) Ring-based sensing platform with the screen-printed sensing electrodes and embedded electronic board for explosives/chemical vapors detection. Reproduced with permission of [25].

for an accurate easy-to-use alcohol measuring device for use by law enforcement personnel, the service/hospitality industry, or individual drinkers to provide a convenient means to monitor alcohol consumption [100]. Currently, the most consistent measure of alcohol intoxication is a direct evaluation of blood alcohol concentration or by the use of breathalyzers to estimate the level

indirectly in the blood through measurement of breath alcohol concentration. However, these portable methods suffer from low accuracy and selectivity due to significant interferences from humidity, temperature, physiological variation, contamination from biocompounds present in the mouth and environment, as well as by system calibration [98].

P.C. Ferreira et al. / Trends in Analytical Chemistry 119 (2019) 115622

The development of WES for alcohol monitoring occurs mainly with the use of the enzymatic system (alcohol oxidase, AOx) to increase the selectivity of the methods. An interesting sensor for minimally invasive detection of alcohol consumption has been reported by Mohan and collaborators [99] that described an attractive skin-worn microneedle sensing device for electrochemical monitoring of subcutaneous alcohol level. The device consists of an assembly of microneedle structures integrated with Pt and Ag wires, Fig. 5B. The microneedle aperture was modified by electropolymerized o-phenylenediamine onto the Pt wire transducer, followed by the immobilization of AOx and layers of chitosan and Nafion membrane. The resulting microneedle-based biosensor displays an interference-free ethanol detection in artificial interstitial fluid considering the presence of major interfering species commonly found in biofluids, a wide linear response range (0e80 mmol L1) with appreciable sensitivity (0.0452 nA/mmol L1) and adequate stability. However, this sensor was only tested in vitro using artificial interstitial fluid assays and the skin penetration ability and biosensor performance for subcutaneous alcohol monitoring were evaluated by ex vivo mice skin model analysis. Thus, the aspects that hinder the practical use and commercial implementation of this sensor are the lack of information about robustness and accuracy through analyses of real complex samples, as well as the integration with signal processing and wireless communication on the microneedle platform for true on-body analysis anywhere. An interesting wearable tattoo-based alcohol biosensing system for noninvasive monitoring in induced sweat was reported by Kim and coauthors [100]. The skin-worn alcohol platform was integrated with an iontophoretic-biosensing temporary tattoo system and flexible wireless electronics, Fig. 5D, and when applied for onbody analyses showed clear differences in the current response before and after alcohol consumption, indicating enhanced ethanol levels. The wearable device consisted of a screen-printed threeelectrode arrangement for the detection system and two additional electrodes for the process of sweat induction by iontophoresis. The working electrode was modified with Prussian blue as redox mediator and coated with a mixture of AOx and chitosan layer. The authors point out that to achieve the fully integrated system, with simultaneous iontophoresis, enzymatic reaction, and amperometric detection, the position of the iontophoresis electrode and its distance from sensing electrodes were crucial factors due to the small volume of electrical-induced sweat. On-body experiments demonstrated a good correlation between the blood alcohol content by breathalyzer and the current response of the proposed alcohol tattoo sensor after serial consumption of alcoholic beverages. A positive aspect to highlight was the use of external equipment (breathalyzer) for the analytical calibration/correlation and the authors emphasize the importance of performing a personalized/individualized calibration of the device because there is great variability in the skin permeability and sweat composition among individuals. Wang's group recently published a comprehensive review paper about recent advances in wearable electrochemical alcohol sensors, where more information on this subject can be found [98]. Wearable devices may aim for portable analyses with biases other than the detection of (bio)compounds excreted by the body. For the forensic scenario, a clever approach was proposed by Jong and collaborators [101] who reported a wearable fingertip sensor for on-the-spot identification of cocaine and its cutting agents in street samples. The electrochemical device consisted of screenprinted electrodes directly on a nitrile glove (index finger) and the use of ionic gel as electrolyte immobilized on the thumb, that when united formed the electrochemical cell for carrying out the voltammetric measurements, Fig. 5C. This approach is quite

11

interesting because it allows rapid on-site screening of suspicious drug samples and contaminated surfaces by simply swiping the electrode system over the suspicious samples, joining both fingers and starting the square-wave voltammetric measurement of microparticles without the need of sampling, preparation, treatment, or additional utensils. Regarding this approach, the authors did not describe the useful life (repeatability study) of the electrochemical system nor the working concentration range. Taking into account that the electro-oxidation of organic compounds such as the evaluated drugs can lead to the passivation of the bare carbon electrode surface, making it difficult to perform reliable successive analyses, probably this wearable sensor is a single use. In addition, future studies need to couple an electronic system of acquisition, treatment, and transmission of portable data. Recently, Barfidokht and coauthors [31] proposed a similar wearable glove-based sensor for rapid on-site detection of fentanyl, a potent synthetic opioid. The glove-based sensor consisted of flexible screen-printed carbon electrodes modified with a mixture of multi-walled carbon nanotubes and a room temperature ionic liquid, 4-(3-butyl-1-imidazolium)-1-butanesulfonate. The use of nanomaterial-modified electrode is a common strategy to enhance the electroanalytical performance [102]. The sensor shows direct oxidation of fentanyl in both liquid and powder forms with a detection limit of 10 mmol L1 using square-wave voltammetry. The Lab-on-a-Glove chemical sensors, combined with a portable electrochemical analyzer, Fig. 5C, provided wireless transmission of the measured data to a smartphone or tablet for further analysis. 4.2. Security applications: wearable detection of toxic and hazardous species Wearable devices that are capable of detecting chemical threats surroundings the users represent a powerful tool to safety and protection issues. These portable sensors with wearable features can be used mainly by at-risk individuals (military, people in war zone or hazard places, etc) to alert of an imminent risk, helping in a fast decision-making process. In this subsection, we present WES accessories that represent a technology in increasing development. Glove-based sensors are a promising technology for wearable monitoring of environmental surroundings because the “swipe, scan, sense, and alert” strategy brings chemical analytics directly to the user's fingertips and opens new possibilities for detecting/ screening target analytes in emergency situations or resourcelimited settings. Mishra and coworkers [103] developed a flexible glove-based electrochemical organophosphorus hydrolase-based biosensor with highly stretchable printed electrode system as a wearable screening tool for nerve agents' detection (defense and food security applications). The authors verified that mechanical stress (bending and stretching) of the printed electrodes does not significantly compromise the performance (electrical resistance) of the glove-based biosensor. The biosensor was fabricated using organophosphorus hydrolase and Nafion polymeric membrane and applied to indirect detection of methyl parathion and methyl paraoxon by the p-nitrophenol product of the biocatalytic hydrolysis reaction at 0.85 V. Some positive aspects of the proposed method to highlight are the mechanical robustness, the integration of stretchable printable enzyme-based biosensing system and active surface for swipe sampling of organophosphate nerve agent/pesticides on suspicious surfaces and agricultural products on different fingers, as well as the compact electronic interface for electrochemical detection and real-time wireless data transmission to a smartphone device. In this study, some deficiencies can be verified, such as the lack of quantitative assessment of the chemical threats level on contaminated surfaces (checked only one concentration,

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P.C. Ferreira et al. / Trends in Analytical Chemistry 119 (2019) 115622

200 mmol L1), absence of analysis in real samples and lack of selectivity study. Even using an enzymatic system the potential necessary for detection is relatively high and other concomitants in real samples could provide false positive and false-negative results. A fashionable method was reported by Sempionatto and coauthors [25] that described a wireless wearable ring-based multiplexed chemical sensor platform for rapid electrochemical monitoring of explosive and nerve-agent threats in vapor and liquid phases, Fig. 5F. The ring-based sensor system consisted of two main parts: a set of interchangeable screen-printed sensing electrodes and a miniaturized electronic interface, based on a battery-powered stamp-size potentiostat, for signal processing and wireless transmission of data. The attractive features of the wearable system were demonstrated for fast voltammetric and amperometric monitoring of nitroaromatic and peroxide explosives, respectively, along with amperometric biosensing of organophosphate nerve agent, methyl paraoxon. The concentration range of the chemical threats was in ppm and mmol L1 range for explosives and methyl paraoxon, respectively. For practical applications, these values need to be decreased considerably and some efforts to enhance the sensitivity need to be addressed. An important aspect to highlight is the toxicity of the nerve agents so, in this study, the experiments were realized using a mannequin arm with the ring sensor on its finger placed inside a doublelayered glovebox in the fume hood with continuous air flow. It is also worth noting that the ability of the miniaturized wearable sensor ring platform simultaneously to detect multiple chemical threats in both liquid and vapor phases and alert the wearer of such hazards offers considerable promise for achieving the demands of diverse defense and security settings. Wang's group recently demonstrated another smart wearable potentiometric tattoo biosensor for real-time on-body monitoring of G-type nerve agents simulant [104]. The skin-worn flexible organophosphate hydrolase-based biosensor was applied for detection of fluorine-containing organophosphate nerve-agent simulant diisopropyl fluorophosphate (DFP, a model analyte) in the mmol L1 range for both liquid and vapor phases. The epidermal potentiometric biosensor was evaluated on a mannequin arm due to safety aspects and the response mechanism relies on the pH-sensitive polyaniline (PANI) coating on a flexible printed transducer to monitor the proton release during the enzymatic hydrolysis of DFP. The sensing electrodes were screen printed on a temporary tattoo paper and the surface modified with electropolymerized PANI and enzymes. Positive aspects to highlight are the good resilience, stability, and reproducibility features of the device, as well as the integrated conformal electronic interface for wireless data transmission, Fig. 5E.

In the same scenario of chemical threats monitoring, Colozza and coauthors [105] fabricated a wearable origami-like paperbased electrochemical biosensor for sulfur mustard detection directly in the aerosol phase, to mimic chemical attack, by spraying these volatile agents into the air. The electrodes were screen printed onto a filter paper support and carbon black/Prussian blue nanocomposite was used as a bulk-modifier of the conductive graphite ink, constituting the working electrode. The authors explored the porosity of paper to preload all the needed reagents and choline oxidase enzyme into the cellulose network. Mustard agent detection was carried out by monitoring its inhibitory enzymatic effects, through the amperometric measurement of the enzymatic by-product hydrogen peroxide at very low potential (0.0 V), due to the electrocatalytic properties of Prussian blue for the reduction of hydrogen peroxide. The proposed method was a proof-of-applicability because no study of the integration of the device with the body seems to have been performed and the detection limit was 1 mmol L1 and 0.019 g min/m3 for liquid and aerosol phases, respectively. The sensitivity of the proposed method needs to be improved for practical wearable alarm systems aimed at use in high-risk war zones, allowing the immediate evacuation of affected areas. In order to summarize the analytical performance of the WES applied to forensic scenario, the Table 3 display some recent and important studies describing the platform and technique explored by the authors, as well as some analytical parameters and sample used. 5. Challenges and remarkable considerations The development of chemical sensors that allows the on-body operation and enhances the wearability requires several intrinsic characteristics, the main ones to be highlight are: (i) Use of stable, nontoxic, and biocompatible products; (ii) Non-invasive or minimally invasive procedures using flexible and lightweight device components avoiding disturbing the daily activities; (iii) The transducer should present excellent robustness to withstand small physicochemical changes like the sample composition, pH, temperature and mechanical stresses; (iv) Imperative need of coupling power-battery supply and electronic system for acquisition, treatment and wireless transmission of portable data; (v) The sensor should provide reliable, accurate and rapid analysis with easily interpretable results for decision making, among others.

Table 3 Comparison of the analytical performance of some WES for forensic applications. Analyte Ethanol Ethanol Cocaine and some cutting agents Fentanyl Methyl paraoxon and methyl parathion DNT

Sample

Platform

Artificial interstitial fluid Induced sweat Street cocaine

Microneedles biosensor Tattoo-based biosensor Glove-based sensor

Residues on contaminated surfaces Residues on contaminated surfaces Vapor and liquid phases

Glove-based sensor Glove-based biosensor Ring-based sensor

H2O2 Methyl paraoxon Diisopropyl fluorophosphate Sulfur mustard

Liquid and vapor phases Liquid and aerosol phases

Tattoo-based biosensor Origami paper-based biosensor

Technique Amperometry Amperometry Square-wave Voltammetry Square-wave Voltammetry Square-wave Voltammetry Square-wave Voltammetry Amperometry Square-wave Voltammetry Potentiometry Amperometry

Linear Range

Sensitivity

Ref. 1

0e80 mM 0e36 mM e

0.0452 nA mM 0.362 mAmM1 e

[99] [100] [101]

10e100 mM

e

[31]

e

e

[103]

4.55 mA ppm

10e100 ppm (Liquid-hase) 2e10 mM (Liquid-phase) 0.25e1.25 mM (Liquid-phase)

1.8 mA mM e

10e120 mM (liquid phase) 1e6 mM (Liquid phase)

e e

1

[25]

1

[104] [105]

P.C. Ferreira et al. / Trends in Analytical Chemistry 119 (2019) 115622

From the analytical point of view, the development and application of wearable chemical sensors represent an unprecedented tool for continuous and real-time monitoring of relevant target compounds directly on an individual's body. However, some challenges are associated with the real implementation of these devices, since several analytical steps must be taken into account, such as how to carry out the analysis directly on-body without complex sample preparation, analysis of representative sampling volume, presence of concomitants/interfering species in the biofluids, and need to verify the analytical reliability of the results [106]. An important aspect to consider is the quantification approaches that many times are necessary. It is important to have in mind that the electrochemical methods are not absolute, and the results need to be compared with a calibration curve or performed by the standard addition method. However, the latter is not viable for in vivo analysis because it would imply contaminating the individual/sample. Therefore, the usual strategy relies on sample collection and conducting comparative ex situ analyses to correlate the results obtained by the on-body sensor and that of the collected sample. This correlation in some cases can be performed considering a population mean, but ideally it should be customized/ personalized to take into account the differences between individuals (metabolic, excretion, sample production, etc.). Other critical issues to be considered are the robustness and stability of the electrochemical sensors due to biofouling/passivation of the electrode surface and the electrical risk during operation or when using the iontophoresis protocol to extract the interstitial fluid, for example. The use of biological recognition agents and (bio)chemical modification of the electrode surface to improve the selectivity and sensitivity are almost mandatory to allow direct on-body sensing, as well as the coupling of additional strategies and devices to collect, preconcentrate, and incubate the biofluids during on-body measurements. 6. Conclusions and future studies The WES development has grown and gained prominence in the last 10 years, in the sensing mobile devices scenario for continuous monitoring of parameters related to health, well-being, and user safety. Wireless health monitoring could revolutionize healthcare especially in places distant from large urban settings, centers lacking laboratory infrastructure or with high clinical demand. The ability to develop miniaturized, flexible, sensitive, selective, and robust devices for analysis directly on the human body is of the utmost importance for various scientific and industrial fields, and electrochemical techniques have proven to be a valuable tool due to the presence of these outstanding features. The electrochemical devices have been fabricated using several simple, low-cost, and scalable methods in different substrates that allow wearability and adequate analytical performance. It should be noted that most wearable chemical sensors still focus on clinical/health applications and with this review we would like to envision other approaches such as forensic applications that are still underexplored fields for wearable chemical sensor technologies, and until now there is any notification about commercial WES for this field. These sensors present great potentiality for better understanding pharmacokinetic phenomena, drug toxicology, and bioavailability, and development of new methods for applications to the health and safety of individuals, as well as tools for law enforcement, for example. In general, the literature presents a series of “proof-of-concept,” demonstrating only the potential of the proposed study, and some critical and fundamental steps for the advancement of these technologies from the point of view of application and commercialization,

13

such as the demonstration of applicability in real samples and in feasible scenarios to verify the robustness, selectivity, and stability of the sensor during its operation, and comparison with standard methods to ensure the reliability of on-body results, were mostly absent. Another important point is the common lack of the integration of flexible electronics for acquisition, processing, and wireless transmission of results to a portable device or a data center. We believe that WES for clinical and forensic applications will experiment an extraordinary evolution. The gaps even now present in the area are common at the beginning of a new theme. It is necessary to remember that this field is still in its infancy, but current advances correspond to not much more than 10 years ago. In the next decade, the growing of this field will be expressive and many of the lacks pointed here will be solved due to greater financial investments, nanoscience and nanotechnology advances, and cooperation of researchers from several areas of knowledge for the development of increasingly functional sensors that provide a set of information (sensor/detector arrays) to the detriment of a single analytical parameter. Clinical field will probably still be the flagship of this area, once the development of new and robust sensors and their association in arrays will provide a general clinical picture of the individual's health, for example. New applications envisioned/demanded by our scientific community, integrating platforms and reducing costs will promote a great advance and dissemination/commercialization of wearable technology providing increased quality of life for their users. Besides the forensic area will benefit from these sensors, once the advances in this field will provide more precise and sensitive sensors, with great simplicity of use and fast decision-making. Acknowledgments We would like to acknowledge to the Brazilian agencies CAPES -Forenses), FAPESP (Grant Numbers: 2016/ (Edital 25/2014 Pro 16477-9, 2017/10522-5, 2018/14462-0, and 2018/08782-1), and CNPq (Grant numbers 306504-2011-1, 438828/2018-6, 426496/ 2018-3 and 308140/2016-8) for supporting the research. References [1] J. Ghika, A.W. Wiegner, J.J. Fang, L. Davies, R.R. Young, J.H. Growdon, Portable system for quantifying motor abnormalities in Parkinson's disease, IEEE Trans. Biomed. Eng. 40 (1993) 276e283. https://doi.org/10.1109/10.216411. [2] Wearable Sensors Market Worth $2.86 Billion By 2025 j CAGR: 38.8%, (n.d.). https://www.grandviewresearch.com/press-release/wearable-sensorsmarket. (Accessed 17 May 2019). [3] J. Heikenfeld, A. Jajack, J. Rogers, P. Gutruf, L. Tian, T. Pan, R. Li, M. Khine, J. Kim, J. Wang, J. Kim, Wearable sensors: modalities, challenges, and prospects, Lab Chip 18 (2018) 217e248. https://doi.org/10.1039/C7LC00914C. [4] Sokwoo Rhee, Boo-Ho Yang, H.H. Asada, Artifact-resistant power-efficient design of finger-ring plethysmographic sensors, IEEE Trans. Biomed. Eng. 48 (2001) 795e805. https://doi.org/10.1109/10.930904. [5] L. Wang, B.P. Lo, G.-Z. Yang, Multichannel reflective PPG earpiece sensor with passive motion cancellation, IEEE Trans. Biomed. Circuits Syst. 1 (2007) 235e241. https://doi.org/10.1109/TBCAS.2007.910900. [6] C.R. Merritt, H.T. Nagle, E. Grant, Textile-based capacitive sensors for respiration monitoring, IEEE Sens. J. 9 (2009) 71e78. https://doi.org/10.1109/ JSEN.2008.2010356. [7] J. Espina, T. Falck, J. Muehlsteff, X. Aubert, Wireless body sensor network for continuous cuff-less blood pressure monitoring, in: 2006 3rd IEEE/EMBS Int. Summer Sch. Med. Devices Biosens, IEEE, 2006, pp. 11e15. https://doi.org/ 10.1109/ISSMDBS.2006.360085. [8] Y. Zhang, C.C.Y. Poon, C. Chan, M.W.W. Tsang, K. Wu, A Health-Shirt using e-Textile Materials for the Continuous and Cuffless Monitoring of Arterial Blood Pressure, in: 2006 3rd IEEE/EMBS Int. Summer Sch. Med. Devices Biosens, IEEE, 2006, pp. 86e89. https://doi.org/10.1109/ISSMDBS.2006. 360104. [9] S. Jung, T. Ji, V.K. Varadan, Point-of-care temperature and respiration monitoring sensors for smart fabric applications, Smart Mater. Struct. 15 (2006) 1872e1876. https://doi.org/10.1088/0964-1726/15/6/042. [10] W.S. Lee, S. Jeon, S.J. Oh, Wearable sensors based on colloidal nanocrystals, Nano Converg 6 (2019) 10. https://doi.org/10.1186/s40580-019-0180-7.

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