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Wearable potentiometric tattoo biosensor for on-body detection of G-type nerve agents simulants
Sensors & Actuators: B. Chemical 273 (2018) 966–972
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Wearable potentiometric tattoo biosensor for on-body detection of G-type nerve agents simulants
T
Rupesh K. Mishra1, Abbas Barfidokht1, Aleksandar Karajic1, Juliane R. Sempionatto1, ⁎ Joshua Wang, Joseph Wang Department of NanoEngineering, University of California San Diego, La Jolla, CA 92093, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Wearable devices Biosensor Nerve agents Security Flexible sensors Polyaniline Chemical warfare threats
A new wearable potentiometric tattoo biosensor for real-time on-body monitoring of G-type nerve agents simulant is described. The skin-worn flexible biosensor responds rapidly and selectively to the fluorine-containing organophosphates (OP) nerve agent simulant diisopropyl fluorophosphate (DFP, a model OP analyte) in both liquid and vapor phases. The epidermal potentiometric OP biosensor relies on the pH-sensitive polyaniline (PANi) coating on a flexible printed transducer for monitoring the proton release during the enzymatic hydrolysis of DFP by enzyme organophosphate hydrolase (OPH). The sensing electrodes are screen printed on a temporary tattoo paper and are interfaced to a conformal electronic interface that provides wireless data transmission. The skin-worn OP potentiometric sensor can withstand severe mechanical strains without compromising its analytical performance. The biosensor displays a wide dynamic range, fast response and high selectivity towards DFP (including efficient discrimination against organophosphate pesticides), and good reproducibility. The attractive performance of the new wearable biosensor indicates considerable promise for onbody threat detection towards rapid warning regarding potential exposure to G-series nerve agents.
1. Introduction The growing concerns regarding chemical warfare threats have led to urgent demands for rapid and sensitive on-site detection of such agents. Among the major chemical threats, organophosphates (OP) nerve agents, particularly the G-series agents sarin and soman, are extremely toxic and fast-acting warfare agents owing to their irreversible inhibition of acetylcholinesterase (AChE) [1–3]. These concerns have led to the development of analytical methods for detecting chemical warfare agents (CWA), including powerful chromatographic, mass spectrometry and capillary electrophoretic methods [4]. However, these techniques are limited to bulky and costly instrumentation. Hence, developing reliable field-deployable sensing platforms for detecting OP neurotoxins is of tremendous importance towards protecting soldiers and civilians [1]. Such warning against CWA threats could greatly benefit from recent advances in wearable electrochemical sensors [5]. Over the past decade we have witnessed a dramatic growth in wearable sensors and biosensors that provide real-time (bio)chemical information [6,7]. While the majority of these new wearable chemical sensors have been focused on healthcare and fitness monitoring, recent
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Corresponding author. E-mail address: [email protected] (J. Wang). Authors with equal contribution.
https://doi.org/10.1016/j.snb.2018.07.001 Received 6 May 2018; Received in revised form 26 June 2018; Accepted 1 July 2018
Available online 02 July 2018 0925-4005/ © 2018 Elsevier B.V. All rights reserved.
efforts have led to on-body sensors for security applications [8]. These sensors can be integrated into diverse wearable platforms such as, rings [9], gloves [10], patch [11,12], balloons [13] or microneedles [14]. In addition to these wearable threat-detection biosensors, other miniaturized devices have been recently developed for detecting OP nerve agents, including wireless hazard badges [15], chemiresistor devices [16], 3D printed stainless steel sensors [17], fluorescent molecular rotors [18], silicon field effect transistor [19], and paper-based devices [20]. The majority of these recently developed wearable sensors for security applications have focused on liquid-phase measurements of explosives and OP nerve-agent/pesticides. Still, a major challenge is the detection of odorless and colorless vapor of OP agents, considering their particularly low vapor pressure [4]. A recent study demonstrated a wearable OPH-based sensor for detection of OP threats in the vaporphase through voltammetric monitoring of the nitro-phenol product [12]. It is also possible to use potentiometric sensors for detecting OP agents [21,22]. In principle, the OPH enzyme is able to cleave PeO, PeF, PeS, and PeCN bonds, resulting in hydrolyzed products able to change the local pH [21,23]. This mechanism has been exploited for developing classical potentiometric biosensors for detecting of OP
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container was sealed by a double-layer protected glove box. A circular hole was drilled in the wall of the polymeric container to place the Mesh Nebulizer (mini Air 360+ A, LFS, Shenzhen, China) that is used to generate the DFP vapors. The electronic board was connected to the sensors and wrapped in parafilm to avoid any contamination from DFP vapor. The recorded data were automatically transmitted to laptop via Bluetooth. Different DFP concentrations (1–120 mM) were prepared by diluting 0.1 M DFP aqueous stock solution to the final concentrations. Both liquid and vapor phase experiments were conducted separately under the fume hood inside the glove box. Safety note: Diisopropyl fluorophosphate (DFP) is an extremely toxic organophosphate pesticide [26]. Poisoning effects may be delayed for up to 12 h; extreme caution is advised. Experiments were thus performed in a sealed air bag-based glove box (4 gal-capacity), containing an airtight (1gal-capacity) polymer box inside. This rigid box had a circular hole to insert the inlet of the Nebulizer. The complete set up was placed inside the fume hood with continuous air flow. The bagbased glove box was double layered protected with an additional bagbased glove box to provide further protection from the OP simulant vapors nebulized inside the fume hood. Proper attention should be taken to the toxicity of the acrylamide component [27]. Although the tattoo sensor is not facing the skin, possible toxicity of the PVA-acrylamide hydrogel membrane must be considered carefully before onbody testing. It should be noted that the risk of using acrylamide is considerably decreased when it is polymerized to form the PVA gel. Some hydrogel membranes containing acrylamide, e.g., Geliperm®, are commercially used as wound dressings [28].
compounds liberating protons via detection of the pH change. They have relied on conventional pH electrodes, pH-sensitive field effect transistors (pH-FETs), or pH-sensitive fluorescent dyes [22,24,25], but not in connection to wearable sensing devices. Herein, we demonstrate the first example of a wearable potentiometric OP tattoo sensor for selective real-time screening of a model Gtype nerve agent simulant. The attractive performance of the new skinworn potentiometric sensor was demonstrated for liquid- and vaporphase detection of diisopropyl fluorophosphate (DFP), a fluorine-containing organophosphate with structural similarity to the chemical warfare agent’s Sarin and Soman. DFP has thus been widely used as a common simulant for G-series nerve agents [1]. On-body detection of chemical agents, such as DFP, is essential for providing rapid warning regarding personal exposure to G-series nerve agents towards timely countermeasures. The present biosensor relies on the enzymatic hydrolysis of DFP by the OPH enzyme that results in proton release [25]. The resulting pH change offers direct detection of DFP in the liquid and gas phases by the skin-worn potentiometric pH-sensing transducer. Such solid-state pH transducer relies on the attractive pH-sensitive conductivity of poly(aniline) (PANi). The epidermal sensor offers a selective response to G-series nerve agents even in the presence of common OP pesticide residues. Such epidermal OPH–pH biosensor has been printed onto a temporary tattoo paper and integrated to a miniaturized conformal wireless electronic interface (Fig. 1). The transducer surface was covered with a PVA-acrylamide hydrogel which ensures surface distribution of the target DFP vapors. The DFP interaction with the immobilized OPH results in a rapid pH-induced potentiometric response which is wirelessly transmitted to a mobile device. This wearable platform offers considerable promise for on-body sensing of chemical threats towards personal exposure warning in field settings, and for alerting farmers about exposure to pesticides.
3. Results and discussion 3.1. Design of the potentiometric tattoo sensor
2. Experimental
The development of the new epidermal OP tattoo biosensor builds on our early introduction of temporary transfer tattoo-based printed electrochemical sensors [29]. Fig. 1A shows the concept of the potentiometric epidermal tattoo sensor for monitoring the fluorine-containing nerve agent simulant, DFP, using a PANi-coated working electrode. The sensor integrates commercially-available temporary tattoo paper with inexpensive screen-printing technique. The new potentiometric tattoo sensor was designed in a “skull face’’ layout with one printed carbon ‘eye’, serving as a working electrode, and the second, Ag/AgCl ‘eye’ (Fig. 1A, a), as a reference electrode. The two-upper printed ‘bones’ were employed as connectors for attaching the sensor to
The reagents, sensor preparation and the wireless transceiver are described in the supplementary Information section. 2.1. Nerve agent detection in liquid and vapor phase Diisopropyl fluorophosphate is acutely potent neurotoxin. Hence, to safeguard, practical scenarios were mimicked in the lab fume hood by using double layered glove box. The tattoo sensors were transferred onto a mannequin arm and placed inside a polymeric container. The
Fig. 1. Tattoo biosensor for detecting nerve agents; (A) Illustration of the sensor embedded with electronic board showing the reagents layers on the working carbon electrode. Schematics of the potentiometric tattoo sensor working mechanism showing DFP hydrolysis in the vapor phase using OPH based electrode upon spraying of OP simulant (DFP) is also shown. During this step, protons are released and protonate the PANi layer. The data was transmitted wirelessly. The Ag/AgCl reference electrode (left side of the sensor) was modified with mixture of the polymeric PVB membrane containing NaCl. (B) 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 DFP vapors throughout the vapor study. (C) Sensor printing on the tattoo paper. (D) Sensor transfer to the skin through removal of the protective layer (bottom image). 967
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shown (g). A visual inspection of the epidermal sensor, after repetitive deformations exemplifies its ability to endure numerous extreme strains while remaining attached to the forearm reflecting the good skin contact. No observable structural damage to the tattoo electrodes has been observed after such repeated stress cycles. The influence of such strains upon the electrochemical behavior was examined by cyclic voltammetry of a ferricyanide marker. Fig. 2h displays the electrochemical behavior of the tattoo sensor after 60 strains (20 of each stress shown in d–f). No substantial changes are seen in the CV before (black curve) and after (red curve) the mechanical distortion, indicating that the sensor can maintain its performance in the face of complex mechanical deformations. While no DFP measurements were performed on the human skin, the mechanical strain experiments were demonstrated on human skin.
the miniaturized potentiometric PCB transducer (Fig. 1B). Fig. 1C illustrates the steps of the printing process on the tattoo paper by using Ag/AgCl printing first and subsequently, carbon ink for completing the “skull design”. The printed tattoo sensor preparation was followed by electropolymerization of PANi on the working electrode and by drop casting both Nafion/OPH enzyme layer and PVA-based hydrogel (Fig. 1A). The pH-sensing PANi-layer is suited for developing biocompatible epidermal tattoo-based potentiometric sensors [30]. The solid-state reference electrode was fabricated by drop casting the reference cocktail (mixture of 78.1 mg PVB and 50 mg NaCl in 1 mL of methanol) and polyurethane (PU; protective membrane) on the printed Ag/AgCl surface, respectively. Fig. 1A shows the mechanism involved in the potentiometric DFP detection by the epidermal tattoo sensor. Once the DFP vapor reaches the outer hydrogel layer of the biosensor, it absorbs and diffuses towards the Nafion/OPH layer where the enzymatic hydrolysis of DFP takes place. The enzymatically catalyzed hydrolysis of the fluorine-containing nerve agent results in the generation of protons, originating from the dissociation of generated HF and eP−OH group [23]. The released protons then diffuse towards pH sensing PANi layer and protonate its active sites [31]. Once the PANi protonation reaches the equilibrium state, a difference between the potential of the working and reference electrode is recorded. Since the concentration of released protons is proportional to the amount of the hydrolyzed chemical-threat substrate, the difference of potential signal offers rapid and effective quantification of DFP. The bio-catalytic recognition of the nerve agent target thus generates a potentiometric signal which is transmitted wirelessly to a mobile device, (Fig. 1B). Such monitoring of the OPH-catalyzed hydrolysis product of DFP offers the advantages of simple and fast measurements of OP nerve agents [32]. The placement of the tattoo biosensor on the skin and removal of protective layer are illustrated in Fig. 1D (upper) while the complete epidermal tattoo on the human subject is depicted in Fig. 1D (bottom).
3.3. Potentiometric bio-sensing of DFP in liquid phase The stoichiometric production of hydrogen ions, resulting from enzymatic hydrolysis of DFP by OPH enzyme, offers an opportunity to detect the corresponding threat substrate by the change in pH. Before testing the modified tattoo biosensor (OPH/PANi) for nerve agent simulant, the response of the new potentiometric epidermal tattoo sensor was evaluated employing standard McIlvaine’s buffer solution. The main advantage of polyaniline is its conductivity and ability to sense the changes in the pH based on the transition between emeraldine salt (ES) and emeraldine base (EB) [31]. Fig. 3A, i illustrates cyclic voltammograms recorded during the electropolymerization of PANi film. During the first cycle of electropolymerization, two peaks that correspond to the initial oxidation of aniline on a screen-printed carbon are observed at +0.7 and +0.9 V vs. Ag/AgCl (1 M NaCl) (not shown). Subsequently, peaks at +0.24, +0.52 and +0.77 V emerges during the consecutive scans showing the reversible performance of deposited PANi layer as it has been previously reported for PANi electropolymerization in acidic media [33]. The initial performance of the potentiometric tattoo sensor was studied by recording the Open Circuit Voltage (OCV) in McIlvaine’s buffer solutions with different pH levels between 8.2 to 2.2 using 1.0 pH value intervals. Aliquot of 100 μL of the buffer test solution was dispensed on the sensor’s surface and then OCV was recorded until a stable response was obtained, following by repeating the process with the next test solution (with distilled water rinsing between each test). Fig. 3A, ii displays the response of McIlvaine’s buffer obtained using PANi-modified tattoo sensor during these tests, along with the corresponding calibration plot (OCV vs pH; inset). This figure illustrates that the PANi-modified pH sensitive tattoo sensor
3.2. Mechanical resiliency study The new flexible threat-sensing system withstands mechanical deformations anticipated from the wearer’s movement. The impact of mechanical stress was investigated for assessing the resiliency of the wearable sensor against mechanical strains. As illustrated in Fig. 2(a–h), firstly, the tattoo was transferred to the human subject (a), subsequently, the protective layer was removed (b), then stress fatigue tests were performed by applying continuous 20 stretching (with alternate relaxing and stretching) (c), 20 bending (d), 20 twisting (e), indentation (f); the tattoo sensor after all mechanical strains is also
Fig. 2. Resiliency of the skin-worn OP tattoo sensor to mechanical strains. Images obtained after different repeated stress cycles. Transfer of the tattoo sensor onto the skin includes: (a) removal of the protective paper cover, (b) adhesion onto the skin. The strain test consists of 20 repeated (c) stretching, (d) bending (e) twisting cycles, and (f) indentation events of the skin (g) Image of tattoo sensor after 60 consecutive strain tests. (h) Cyclic voltammograms for 1 mM ferricyanide (in 0.1 M KCl) before (black plot) and after (red plot) applying the 60 strains of.d–f. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 968
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Fig. 3. Potentiometric pH sensitive biosensor for DFP detection in liquid phase using OPH Tattoo modified with PANi layer. (Ai) Electrodeposition of PANi in 1 M HCl solution containing 0.1 M aniline, by cycling the potential from −0.2 V to 1.0 V at a scan rate of 100 mVs−1. (ii) Real time calibration curve for pH sensor obtained in McIlvaine’s buffer at different pH values by using PANi modified tattoo sensor; the inset illustrates the linear response of the calibration curve. (Bi) Real time potentiometric response of DFP (10–120 mM) in the presence (black line, a) and absence (red line, b) of OPH on the sensor; inset shows the linear trend obtained from DFP response in the presence and absence of OPH on the surface of the transducer. (Bii) Detection of DFP (sarin simulant) in lake and (Biii) pond water samples; samples were spiked with different DFP concentrations (10, 20, 40, 80, 120 mM). (Ci) Carry-over evaluation of the OP biosensor from 5 mM to 100 mM DFP concentrations, and (Cii) for High and Low (40 mM and 80 mM) DFP concentrations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the immobilized OPH in the biosensor operation. Sensor stability is a major requirement for a successful wearable sensor. The storage stability of the OP tattoo sensors was evaluated by assessing the potentiometric behavior of a set of freshly prepared tattoo sensors over different time periods. Fig. S2 illustrates the stability performance of the sensor for up to 5 days without losing significant activity. The good stability signifies the judicious and systematic preparation of the sensors. To assess the applicability of the developed tattoo sensor in liquid phase, the sensors were tested for DFP detection in real water samples by spiking 5 different concentrations (10, 20, 40, 80, 120 mM) in untreated lake and pond water samples. Fig. 3B (ii and iii) illustrates the favorable potentiometric response of tattoo sensors for the nerve agent simulant in lake (Lake Murray, San Diego, CA) and pond (San Diego) water, with well-defined changes in the potential signal upon increasing the DFP concentration. The lake water shows better response against pond water, which articulates that the tattoo sensor has more affinity to Lake Matrix due to the presence of less complex substances. Certainly, the obtained data showed significant promise of the new sensors for environmental field monitoring. The repeatability of the developed potentiometric biosensor represents another significant feature of wearable sensors. Moreover, it is also important to investigate whether the resulting signal drifts by time which would have a significant negative effect on the performed measurement. In general, it is imperative to achieve highly reproducible data points, which is of particular importance for continuous field monitoring of nerve agent threats, aimed at alerting for potential exposure. A fast and reversible response is required for wearable sensors to assess the accurate level of analyte. The reversibility of OPH-tattoo sensor was checked by measuring its response, using 7 different DFP concentrations (range: 5–100 mM) for more than 2000sec. Fig. 3C, i depicts the
responds instantaneously to these pH changes, yielding high linearity over the entire 2.2–8.2 pH range (slope: 65.30 mV/log10 [pH]). The sensor reaches a stable response in less than 20 s. The OPH-modified tattoo biosensors were evaluated for liquid-phase DFP detection by recording the pH change during the biocatalytic hydrolysis of the simulant nerve agent. Optimization the OPH loading was performed by changing the amount of OPH loading (20–60 ng) and monitoring the response to 80 mM DFP in liquid phase, with 60 ng offering the most favorable and stable response (Fig. S1). To facilitate the sensing step, the fabricated OPH-modified tattoo sensors were preconditioned for 2 min using 25 μL distilled water. The performance of the tattoo-based biosensor was assessed first towards screening the nerve agent threats in the liquid phase. Changes in the DFP concentration were used to evaluate the dynamic behavior of developed biosensor. Fig. 3B, i, illustrates the potential-time response upon increasing the DFP concentration over the 10–120 mM range. The potentiometric OP biosensor responds instantaneously to these DFP additions. The resulting calibration (shown in the inset) exhibits good linearity (R2 = 0.983). The estimated limit of quantification (LOQ) was 10 mM (n = 10), calculated in accordance with the IUPAC norms. Moreover, the reproducibility of biosensor was investigated by preparing five different tattoo-based sensors and testing them in similar manner for the detection of DFP in liquid phase. Calibration curves were performed over the 10 to 120 mM DFP concentration range. The obtained RSD of the resulting calibration curves was 4.8%, indicating a good reproducibility of the fabricated tattoo sensor. The DFP response was also assessed in the absence of OPH (control probe) on tattoo sensors (Fig. 3B, i, red curve, b). As expected from the absence of the enzyme (i.e., no release of protons), a negligible potential (i.e., pH) change is observed. This control experiment supports the crucial role of 969
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performance of tattoo sensor to check reversibility of the system in a wide range of DFP concentrations with a good trend of increase and decrease in OCV responses during the carry-over evaluation. Similarly, to examine the sensor performance with lower (40 mM) and higher (80 mM) dosage of DFP, carry-over evaluation was performed and repeatability was evaluated. Fig. 3C, ii illustrates a carry-over plot to showcase the good repeatability of OPH tattoo sensor in the liquid phase up to 9 cycles of low and high concentration (5 and 100 mM) over a prolonged (1600sec) period. The highly reproducible response, with no carry-over effects, signifies the potential for reliable on-body testing. 3.4. DFP detection in vapor phase Following the successful DFP detection in liquid phase, the wearable potentiometric tattoo sensor was tested for its ability to detect the OP threat in the vapor-phase. Such vapor detection was performed with utmost safety measures, specified in the experimental section 2.1, using the set-up presented in Fig. 1. The vapor phase DFP biosensing was facilitated by modifying the electrode surface with a PVA-acrylamide hydrogel, which allowed the vapors to absorb and diffuse towards the PANi-OPH sensing layer. The PVA-acrylamide hydrogel membrane was formed through crosslinking of polyacrylamide - polyvinyl alcohol with glutaraldehyde drop casted onto the carbon-based tattoo sensor and subsequently heated at 50 °C for 20 min. In terms of underlying mechanism, the acetyl bridges were developed among the pendant hydroxyl groups of the PVA chains and this generated the polyacrylamidepolyvinyl alcohol hydrogel. To mimic exposure to a G-series nerve agent vapor, such as sarin, a portable and hand-held nebulizer was used to generate the DFP vapor toward the wearable sensor placed on a mannequin arm (Fig. 1B). This DFP vapor detection was carried out by optimizing the vapor spraying. As illustrated in Fig. S3, different OP spraying/nebulizing times (15–60 s) were examined using 60 mM DFP at a fixed nebulizer-sensor distance. Based on these data, 15 s vapor spray duration was chosen to be the optimal time for further vapor phase detection experiments. No further change in the response was observed beyond 15 s. Upon detection of DFP, the obtained data were wirelessly transmitted to a smart device. Fig. 4A illustrates the experimental set-up consisting of the tattoo sensor integrated with potentiometric board placed on mannequin arm. DFP vapors were sprayed using handheld portable nebulizer. Fig. 4B, i and B, ii shows real time detection of increasing DFP vapor concentrations, along with the resulting calibration plot. The sensor responds rapidly to the DFP vapor, reaching steady-state response within 30 s. The response increases linearly with the vapor concentration over the entire concentration range (20–120 mM) of the nebulizer solution, with a LOQ of around 10 mM. A similar calibration experiment carried out with an ‘enzyme-free’ sensor displayed a negligible DFP response (red line of Fig. 4B, i and ii). Keeping in mind that the OPH is a class selective enzyme, hence, significant interfering effect could be expected from other organophosphate group analytes. In particular, the detection of these G-type nerve agents often must compete with a background level of OP pesticides [34]. MPOx was used here for testing the selectivity between the G-type nerve agent simulant, such as DFP and, common organophosphate pesticides (Fig. 4C). The selectivity experiments were carried out using 3 different mixed concentrations of 1:1 DFP + MPOx (40:40, 80:80 and 100:100 mM, DFP: MPOx). The response was compared to the signal of 3 different addition of MPOx solution (40, 80 and 100 mM). Surprisingly, no notable effect of MPOx interference is observed in comparison to the DFP response. As shown in Fig. 4C, i (black line), the tattoo sensor results in a well-defined DFP response (in the presence of similar level of MPOx) whereas MPOx alone yields a negligible potential response (compared to the DFP + MPOx response), illustrating the highly selective response of the sensor towards DFP. Such behavior is attributed to the significant difference in pKa values of the co-products of enzymatic reaction, HF (pKa ∿ 3) and p-nitrophenol (pKa ∿ 7) [35],
Fig. 4. Response of different DFP concentrations in vapor phase using the tattoo biosensor coated with PVA-acrylamide hydrogel (A) Illustration of the experimental set-up and electronic/potentiometric printed circuit board with an integrated tattoo that is attached to mannequin arm. DFP solution is sprayed using nebulizer. (B) Vapor phase DFP analysis. (i) Potentiometric response upon spraying DFP (20–120 mM) on the sensor (curve a) and similar real time response of DFP without OPH on the electrode surface (curve b). (B,ii) Calibration plot of the biosensor with (a) and without (b) OPH on the electrode surface. (C,i) Response to the mixed solution of DFP + MPOx (red) and only MPOx (black) (Cii) Linear points plotted based on the increase OCV values upon concentration increments of DFP + MPOx (40, 80 and 100 mM mixture) (black) and MPOx (red) (D) Carry-over evaluation of the potentiometric biosensor for 40 mM and 80 mM concentration range of DFP using PANi modified OPH sensor coated with PVA-acrylamide hydrogel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
which are released during the hydrolysis of DFP and MPOx respectively. This demonstrates the possibility for detecting organofluorophosphates in the environment where other non-fluoro class-homologues are present. Fig. 4C, ii shows the linear behavior of both the sensor, black line corresponds to DFP + MPOx response (both present in equal concentrations of 40, 80 and 100 mM) and red line shows linear response of MPOx. Similar to the liquid phase carry-over study, repeatability of the tattoo biosensor was performed to check the response of low and high dosage of DFP vapors. Fig. 4D illustrates a carry-over plot to assess the reversibility and repeatability of OPH tattoo sensor in vapor phase. It 970
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coupling the OPH biocatalytic recognition phase with a pH-responsive PANi layer and interfacing the resulting flexible potentiometric biosensor with a conformal wireless electronic interface. The skin-worn biosensor thus integrates all the required functionalities necessary for nerve agent screening in realistic field settings. Extensive in-vitro characterization of the new wearable OP potentiometric-tattoo biosensor system affirmed that it offers an attractive analytical performance, with a fast and stable response to the sarin analog, DFP. Such immediate response provides an instantaneous warning for a timely action. A single use of the tattoo sensor is desired considering this rapid warning goal and potential memory effect (associated with accumulation of reaction product within the hydrogel membrane). Possible changes in the sweat pH could be addressed using an enzyme-free PANi transducer. Besides its rapid warning capability, the wearable sensor can shed useful insight regarding the nature of a chemical attack. The new wearable biosensor addresses the existing gaps and major needs for protecting soldiers, civilians and farmers against unexpected exposure to chemical threats. Acknowledgments This work was supported by the Defense Threat Reduction Agency Joint Science and Technology Office for Chemical and Biological Defense (HDTRA1-16-1-0013). J.R.S. acknowledges the fellowships from CNPq (216981/2014-0). The authors thank Dr. Mustafa Musameh (CSIRO, Clayton, Australia) for providing the OPH enzyme. Fig. 5. Selectivity study: Response of the modified OPH/PANi based biosensor, coated with PVA-acrylamide hydrogel, to (a) methyl parathion (200 μM), (b) 200 μM MPOx, (c) 5% hydrogen peroxide (d) 100 ppm DNT (e) 60 mM DFP (f) 70% ethanol (g) acetone (h) ammonia (i) 70% methanol (j) formaldehyde and (k) toluene towards OP tattoo sensors, demonstrating the selectivity of the sensor towards DFP in vapor phase.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2018.07.001. References
can be seen from these data that there is an insignificant drift in the potentiometric biosensor response for the vapor up to 7 cycles (i.e. 900 s).
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3.5. Selectivity of potentiometric tattoo sensor for OP vapors The occurrence of other coexisting toxic chemical vapors in the environment might impact the selective detection of the target analyte, hence, it may compromise the accuracy of the new potentiometric wearable tattoo OP vapor sensor. Thus, an additional selectivity control study was performed using multiple potential interferences, such as ethanol, acetone, ammonia, methanol, MPOx, hydrogen peroxide, DNT, formaldehyde, isopropyl alcohol and toluene. These chemical vapors were selected as a control to study the DFP response among them, once they are commonly present in the environment [36–38]. Fig. 5 demonstrates the high selectivity of the new potentiometric OP-tattoo sensor toward the target nerve agent simulant (DFP) (e), along with the different potential interferences: (a) methyl parathion (200 μM) (B) 200 μM MPOx (c) 5% hydrogen peroxide (D) 100 ppm DNT (E) 60 mM DFP (f) 70% ethanol (g) acetone (h) ammonia (i) 70% methanol (j) formaldehyde and (k) toluene. These results illustrate that the presence of several other non-target vapors in the environment does not hinder the OP detection. Noteworthy, these tested chemical vapors do not show any drift or significant changes in the recorded signal, but the results were rather similar to the control signals except for the response of DFP shown in Fig. 5e. 4. Conclusions This work describes the performance of a fully integrated wireless potentiometric OP tattoo biosensor platform for real-time on-body monitoring of G-type nerve agent simulants in both liquid and vapor phases. Such effective on-the-spot detection of DFP has been realized by 971
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[17] G. Tan, M.Z.M. Nasir, A. Ambrosi, M. Pumera, 3D printed electrodes for detection of nitroaromatic explosives and nerve agents, Anal. Chem. 89 (2017) 8995–9001. [18] T.I. Kim, S.B. Maity, J. Bouffard, Y. Kim, Molecular rotors for the detection of chemical warfare agent simulants, Anal. Chem. 88 (2016) 9259–9263. [19] K. Smaali, D. Guerin, V. Passi, L. Ordronneau, A. Carella, T. Melin, et al., Physical study by surface characterizations of sarin sensor on the basis of chemically functionalized silicon nanoribbon field effect transistor, J. Phys. Chem. C 120 (2016) 11180–11191. [20] S. Cinti, C. Minotti, D. Moscone, G. Palleschi, F. Arduini, Fully integrated ready-touse paper-based electrochemical biosensor to detect nerve agents, Biosens. Bioelectron. 93 (2017) 46–51. [21] A.L. Simonian, J.K. Grimsley, A.W. Flounders, et al., Enzyme-based biosensor for the direct detection of fluorine-containing organophosphates, Anal. Chim. Acta 442 (2001) 15–23. [22] A.W. Flounders, A.K. Singh, J.V. Volponi, S.C. Carichner, et al., Development of sensors for direct detection of organophosphates. Part II: sol-gel modified field effect transistor with immobilized organophosphate hydrolase, Biosens. Bioelectron. 14 (1999) 715–722. [23] A.L. Simonian, B.D. diSioudi, J.R. Wild, An enzyme based biosensor for the direct determination of diisopropyl fluorophosphate, Anal. Chim. Acta 389 (1999) 189–196. [24] E.I. Rainina, E.N. Efremenco, S.D. Varfolomeyev, A.L. Simonian, J.R. Wild, The development of a new biosensor based on recombinant E.-coli for the direct detection of organophosphorus neurotoxins, Biosens. Bioelectron. 11 (1996) 991–1000. [25] A. Mulchandani, I. Kaneva, W. Chen, Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 2. Fiber optic microbial biosensor, Anal. Chem. 70 (1998) 5042–5046. [26] T.C. Marrs, Organophosphate poisoning, Pharmacol. Ther. 58 (1993) 51–66. [27] A. Shipp, G. Lawrence, R. Gentry, T. McDonald, H. Bartow, J. Bounds, N. Macdonald, H. Clewell, B. Allen, C. Van Landingham, Acrylamide: review of toxicity data and dose-response analyses for cancer and noncancer effects, Crit. Rev. Toxicol. 36 (2006) 481–608. [28] E.A. Kamoun, E.S. Kenawy, X. Chen, A review on polymeric hydrogel membranes for wound dressing applications: PVA-based hydrogel dressings, J. Adv. Res. 8 (2017) 217–233. [29] A.J. Bandodkar, W.Z. Jia, J. Wang, Tattoo-based wearable electrochemical devices:
a review, Electroanalysis 27 (2015) 562–572. [30] A.J. Bandodkar, V.W.S. Hung, W.Z. Jia, G. Valdes-Ramirez, et al., Tattoo-based potentiometric ion-selective sensors for epidermal pH monitoring, Analyst 138 (2013) 123–128. [31] J. Das, P. Sarkar, Enzymatic electrochemical biosensor for urea with a polyaniline grafted conducting hydrogel composite modified electrode, RSC Adv. 6 (2016) 92520–92533. [32] P. Mulchandani, A. Mulchandani, I. Kaneva, W. Chen, Biosensor for direct determination of organophosphate nerve agents. 1. Potentiometric enzyme electrode, Biosens. Bioelectron. 14 (1999) 77–85. [33] E.M. Genies, C. Tsintavis, Redox mechanism and electrochemical-behavior of polyaniline deposits, J. Electroanal. Chem. 195 (1985) 109–128. [34] R. Shunmugam, G.N. Tew, Chem. Eur. J. 14 (2008) 5409–5412. [35] K.C. Gross, P.G. Seybold, Substituent effects on the physical properties and pK(a) of phenol, Int. J. Quantum Chem. 85 (2001) 569–579. [36] P.Q. Hu, G.J. Du, W.J. Zhou, J.J. Cui, J.J. Lin, H. Liu, et al., Enhancement of ethanol vapor sensing of TiO2 nanobelts by surface engineering, ACS Appl. Mater. Interfaces 2 (2010) 3263–3269. [37] J.M. Sylvia, J.A. Janni, J.D. Klein, K.M. Spencer, Surface-enhanced Raman detection of 1,4-dinitrotoluene impurity vapor as a marker to locate landmines, Anal. Chem. 72 (2000) 5834–5840. [38] C. Wei, L.M. Dai, A. Roy, T.B. Tolle, Multifunctional chemical vapor sensors of aligned carbon nanotube and polymer composites, J. Am. Chem. Soc. 128 (2006) 1412–1413. Rupesh K. Mishra are post-doctoral fellows at UCSD (department of Nanoenigeering), Abbas Barfidokht are post-doctoral fellows at UCSD (department of Nanoenigeering). Aleksandar Karajic are post-doctoral fellows at UCSD (department of Nanoenigeering). Juliane R. Sempionatto is a graduate student at UCSD. Joshua Wang is an undergraduate student UCSD. Joseph Wang is Distinguished Professor in UCSD.
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Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Wearable potentiometric tattoo biosensor for on-body detection of G-type nerve agents simulants
T
Rupesh K. Mishra1, Abbas Barfidokht1, Aleksandar Karajic1, Juliane R. Sempionatto1, ⁎ Joshua Wang, Joseph Wang Department of NanoEngineering, University of California San Diego, La Jolla, CA 92093, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Wearable devices Biosensor Nerve agents Security Flexible sensors Polyaniline Chemical warfare threats
A new wearable potentiometric tattoo biosensor for real-time on-body monitoring of G-type nerve agents simulant is described. The skin-worn flexible biosensor responds rapidly and selectively to the fluorine-containing organophosphates (OP) nerve agent simulant diisopropyl fluorophosphate (DFP, a model OP analyte) in both liquid and vapor phases. The epidermal potentiometric OP biosensor relies on the pH-sensitive polyaniline (PANi) coating on a flexible printed transducer for monitoring the proton release during the enzymatic hydrolysis of DFP by enzyme organophosphate hydrolase (OPH). The sensing electrodes are screen printed on a temporary tattoo paper and are interfaced to a conformal electronic interface that provides wireless data transmission. The skin-worn OP potentiometric sensor can withstand severe mechanical strains without compromising its analytical performance. The biosensor displays a wide dynamic range, fast response and high selectivity towards DFP (including efficient discrimination against organophosphate pesticides), and good reproducibility. The attractive performance of the new wearable biosensor indicates considerable promise for onbody threat detection towards rapid warning regarding potential exposure to G-series nerve agents.
1. Introduction The growing concerns regarding chemical warfare threats have led to urgent demands for rapid and sensitive on-site detection of such agents. Among the major chemical threats, organophosphates (OP) nerve agents, particularly the G-series agents sarin and soman, are extremely toxic and fast-acting warfare agents owing to their irreversible inhibition of acetylcholinesterase (AChE) [1–3]. These concerns have led to the development of analytical methods for detecting chemical warfare agents (CWA), including powerful chromatographic, mass spectrometry and capillary electrophoretic methods [4]. However, these techniques are limited to bulky and costly instrumentation. Hence, developing reliable field-deployable sensing platforms for detecting OP neurotoxins is of tremendous importance towards protecting soldiers and civilians [1]. Such warning against CWA threats could greatly benefit from recent advances in wearable electrochemical sensors [5]. Over the past decade we have witnessed a dramatic growth in wearable sensors and biosensors that provide real-time (bio)chemical information [6,7]. While the majority of these new wearable chemical sensors have been focused on healthcare and fitness monitoring, recent
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1
Corresponding author. E-mail address: [email protected] (J. Wang). Authors with equal contribution.
https://doi.org/10.1016/j.snb.2018.07.001 Received 6 May 2018; Received in revised form 26 June 2018; Accepted 1 July 2018
Available online 02 July 2018 0925-4005/ © 2018 Elsevier B.V. All rights reserved.
efforts have led to on-body sensors for security applications [8]. These sensors can be integrated into diverse wearable platforms such as, rings [9], gloves [10], patch [11,12], balloons [13] or microneedles [14]. In addition to these wearable threat-detection biosensors, other miniaturized devices have been recently developed for detecting OP nerve agents, including wireless hazard badges [15], chemiresistor devices [16], 3D printed stainless steel sensors [17], fluorescent molecular rotors [18], silicon field effect transistor [19], and paper-based devices [20]. The majority of these recently developed wearable sensors for security applications have focused on liquid-phase measurements of explosives and OP nerve-agent/pesticides. Still, a major challenge is the detection of odorless and colorless vapor of OP agents, considering their particularly low vapor pressure [4]. A recent study demonstrated a wearable OPH-based sensor for detection of OP threats in the vaporphase through voltammetric monitoring of the nitro-phenol product [12]. It is also possible to use potentiometric sensors for detecting OP agents [21,22]. In principle, the OPH enzyme is able to cleave PeO, PeF, PeS, and PeCN bonds, resulting in hydrolyzed products able to change the local pH [21,23]. This mechanism has been exploited for developing classical potentiometric biosensors for detecting of OP
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container was sealed by a double-layer protected glove box. A circular hole was drilled in the wall of the polymeric container to place the Mesh Nebulizer (mini Air 360+ A, LFS, Shenzhen, China) that is used to generate the DFP vapors. The electronic board was connected to the sensors and wrapped in parafilm to avoid any contamination from DFP vapor. The recorded data were automatically transmitted to laptop via Bluetooth. Different DFP concentrations (1–120 mM) were prepared by diluting 0.1 M DFP aqueous stock solution to the final concentrations. Both liquid and vapor phase experiments were conducted separately under the fume hood inside the glove box. Safety note: Diisopropyl fluorophosphate (DFP) is an extremely toxic organophosphate pesticide [26]. Poisoning effects may be delayed for up to 12 h; extreme caution is advised. Experiments were thus performed in a sealed air bag-based glove box (4 gal-capacity), containing an airtight (1gal-capacity) polymer box inside. This rigid box had a circular hole to insert the inlet of the Nebulizer. The complete set up was placed inside the fume hood with continuous air flow. The bagbased glove box was double layered protected with an additional bagbased glove box to provide further protection from the OP simulant vapors nebulized inside the fume hood. Proper attention should be taken to the toxicity of the acrylamide component [27]. Although the tattoo sensor is not facing the skin, possible toxicity of the PVA-acrylamide hydrogel membrane must be considered carefully before onbody testing. It should be noted that the risk of using acrylamide is considerably decreased when it is polymerized to form the PVA gel. Some hydrogel membranes containing acrylamide, e.g., Geliperm®, are commercially used as wound dressings [28].
compounds liberating protons via detection of the pH change. They have relied on conventional pH electrodes, pH-sensitive field effect transistors (pH-FETs), or pH-sensitive fluorescent dyes [22,24,25], but not in connection to wearable sensing devices. Herein, we demonstrate the first example of a wearable potentiometric OP tattoo sensor for selective real-time screening of a model Gtype nerve agent simulant. The attractive performance of the new skinworn potentiometric sensor was demonstrated for liquid- and vaporphase detection of diisopropyl fluorophosphate (DFP), a fluorine-containing organophosphate with structural similarity to the chemical warfare agent’s Sarin and Soman. DFP has thus been widely used as a common simulant for G-series nerve agents [1]. On-body detection of chemical agents, such as DFP, is essential for providing rapid warning regarding personal exposure to G-series nerve agents towards timely countermeasures. The present biosensor relies on the enzymatic hydrolysis of DFP by the OPH enzyme that results in proton release [25]. The resulting pH change offers direct detection of DFP in the liquid and gas phases by the skin-worn potentiometric pH-sensing transducer. Such solid-state pH transducer relies on the attractive pH-sensitive conductivity of poly(aniline) (PANi). The epidermal sensor offers a selective response to G-series nerve agents even in the presence of common OP pesticide residues. Such epidermal OPH–pH biosensor has been printed onto a temporary tattoo paper and integrated to a miniaturized conformal wireless electronic interface (Fig. 1). The transducer surface was covered with a PVA-acrylamide hydrogel which ensures surface distribution of the target DFP vapors. The DFP interaction with the immobilized OPH results in a rapid pH-induced potentiometric response which is wirelessly transmitted to a mobile device. This wearable platform offers considerable promise for on-body sensing of chemical threats towards personal exposure warning in field settings, and for alerting farmers about exposure to pesticides.
3. Results and discussion 3.1. Design of the potentiometric tattoo sensor
2. Experimental
The development of the new epidermal OP tattoo biosensor builds on our early introduction of temporary transfer tattoo-based printed electrochemical sensors [29]. Fig. 1A shows the concept of the potentiometric epidermal tattoo sensor for monitoring the fluorine-containing nerve agent simulant, DFP, using a PANi-coated working electrode. The sensor integrates commercially-available temporary tattoo paper with inexpensive screen-printing technique. The new potentiometric tattoo sensor was designed in a “skull face’’ layout with one printed carbon ‘eye’, serving as a working electrode, and the second, Ag/AgCl ‘eye’ (Fig. 1A, a), as a reference electrode. The two-upper printed ‘bones’ were employed as connectors for attaching the sensor to
The reagents, sensor preparation and the wireless transceiver are described in the supplementary Information section. 2.1. Nerve agent detection in liquid and vapor phase Diisopropyl fluorophosphate is acutely potent neurotoxin. Hence, to safeguard, practical scenarios were mimicked in the lab fume hood by using double layered glove box. The tattoo sensors were transferred onto a mannequin arm and placed inside a polymeric container. The
Fig. 1. Tattoo biosensor for detecting nerve agents; (A) Illustration of the sensor embedded with electronic board showing the reagents layers on the working carbon electrode. Schematics of the potentiometric tattoo sensor working mechanism showing DFP hydrolysis in the vapor phase using OPH based electrode upon spraying of OP simulant (DFP) is also shown. During this step, protons are released and protonate the PANi layer. The data was transmitted wirelessly. The Ag/AgCl reference electrode (left side of the sensor) was modified with mixture of the polymeric PVB membrane containing NaCl. (B) 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 DFP vapors throughout the vapor study. (C) Sensor printing on the tattoo paper. (D) Sensor transfer to the skin through removal of the protective layer (bottom image). 967
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shown (g). A visual inspection of the epidermal sensor, after repetitive deformations exemplifies its ability to endure numerous extreme strains while remaining attached to the forearm reflecting the good skin contact. No observable structural damage to the tattoo electrodes has been observed after such repeated stress cycles. The influence of such strains upon the electrochemical behavior was examined by cyclic voltammetry of a ferricyanide marker. Fig. 2h displays the electrochemical behavior of the tattoo sensor after 60 strains (20 of each stress shown in d–f). No substantial changes are seen in the CV before (black curve) and after (red curve) the mechanical distortion, indicating that the sensor can maintain its performance in the face of complex mechanical deformations. While no DFP measurements were performed on the human skin, the mechanical strain experiments were demonstrated on human skin.
the miniaturized potentiometric PCB transducer (Fig. 1B). Fig. 1C illustrates the steps of the printing process on the tattoo paper by using Ag/AgCl printing first and subsequently, carbon ink for completing the “skull design”. The printed tattoo sensor preparation was followed by electropolymerization of PANi on the working electrode and by drop casting both Nafion/OPH enzyme layer and PVA-based hydrogel (Fig. 1A). The pH-sensing PANi-layer is suited for developing biocompatible epidermal tattoo-based potentiometric sensors [30]. The solid-state reference electrode was fabricated by drop casting the reference cocktail (mixture of 78.1 mg PVB and 50 mg NaCl in 1 mL of methanol) and polyurethane (PU; protective membrane) on the printed Ag/AgCl surface, respectively. Fig. 1A shows the mechanism involved in the potentiometric DFP detection by the epidermal tattoo sensor. Once the DFP vapor reaches the outer hydrogel layer of the biosensor, it absorbs and diffuses towards the Nafion/OPH layer where the enzymatic hydrolysis of DFP takes place. The enzymatically catalyzed hydrolysis of the fluorine-containing nerve agent results in the generation of protons, originating from the dissociation of generated HF and eP−OH group [23]. The released protons then diffuse towards pH sensing PANi layer and protonate its active sites [31]. Once the PANi protonation reaches the equilibrium state, a difference between the potential of the working and reference electrode is recorded. Since the concentration of released protons is proportional to the amount of the hydrolyzed chemical-threat substrate, the difference of potential signal offers rapid and effective quantification of DFP. The bio-catalytic recognition of the nerve agent target thus generates a potentiometric signal which is transmitted wirelessly to a mobile device, (Fig. 1B). Such monitoring of the OPH-catalyzed hydrolysis product of DFP offers the advantages of simple and fast measurements of OP nerve agents [32]. The placement of the tattoo biosensor on the skin and removal of protective layer are illustrated in Fig. 1D (upper) while the complete epidermal tattoo on the human subject is depicted in Fig. 1D (bottom).
3.3. Potentiometric bio-sensing of DFP in liquid phase The stoichiometric production of hydrogen ions, resulting from enzymatic hydrolysis of DFP by OPH enzyme, offers an opportunity to detect the corresponding threat substrate by the change in pH. Before testing the modified tattoo biosensor (OPH/PANi) for nerve agent simulant, the response of the new potentiometric epidermal tattoo sensor was evaluated employing standard McIlvaine’s buffer solution. The main advantage of polyaniline is its conductivity and ability to sense the changes in the pH based on the transition between emeraldine salt (ES) and emeraldine base (EB) [31]. Fig. 3A, i illustrates cyclic voltammograms recorded during the electropolymerization of PANi film. During the first cycle of electropolymerization, two peaks that correspond to the initial oxidation of aniline on a screen-printed carbon are observed at +0.7 and +0.9 V vs. Ag/AgCl (1 M NaCl) (not shown). Subsequently, peaks at +0.24, +0.52 and +0.77 V emerges during the consecutive scans showing the reversible performance of deposited PANi layer as it has been previously reported for PANi electropolymerization in acidic media [33]. The initial performance of the potentiometric tattoo sensor was studied by recording the Open Circuit Voltage (OCV) in McIlvaine’s buffer solutions with different pH levels between 8.2 to 2.2 using 1.0 pH value intervals. Aliquot of 100 μL of the buffer test solution was dispensed on the sensor’s surface and then OCV was recorded until a stable response was obtained, following by repeating the process with the next test solution (with distilled water rinsing between each test). Fig. 3A, ii displays the response of McIlvaine’s buffer obtained using PANi-modified tattoo sensor during these tests, along with the corresponding calibration plot (OCV vs pH; inset). This figure illustrates that the PANi-modified pH sensitive tattoo sensor
3.2. Mechanical resiliency study The new flexible threat-sensing system withstands mechanical deformations anticipated from the wearer’s movement. The impact of mechanical stress was investigated for assessing the resiliency of the wearable sensor against mechanical strains. As illustrated in Fig. 2(a–h), firstly, the tattoo was transferred to the human subject (a), subsequently, the protective layer was removed (b), then stress fatigue tests were performed by applying continuous 20 stretching (with alternate relaxing and stretching) (c), 20 bending (d), 20 twisting (e), indentation (f); the tattoo sensor after all mechanical strains is also
Fig. 2. Resiliency of the skin-worn OP tattoo sensor to mechanical strains. Images obtained after different repeated stress cycles. Transfer of the tattoo sensor onto the skin includes: (a) removal of the protective paper cover, (b) adhesion onto the skin. The strain test consists of 20 repeated (c) stretching, (d) bending (e) twisting cycles, and (f) indentation events of the skin (g) Image of tattoo sensor after 60 consecutive strain tests. (h) Cyclic voltammograms for 1 mM ferricyanide (in 0.1 M KCl) before (black plot) and after (red plot) applying the 60 strains of.d–f. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 968
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Fig. 3. Potentiometric pH sensitive biosensor for DFP detection in liquid phase using OPH Tattoo modified with PANi layer. (Ai) Electrodeposition of PANi in 1 M HCl solution containing 0.1 M aniline, by cycling the potential from −0.2 V to 1.0 V at a scan rate of 100 mVs−1. (ii) Real time calibration curve for pH sensor obtained in McIlvaine’s buffer at different pH values by using PANi modified tattoo sensor; the inset illustrates the linear response of the calibration curve. (Bi) Real time potentiometric response of DFP (10–120 mM) in the presence (black line, a) and absence (red line, b) of OPH on the sensor; inset shows the linear trend obtained from DFP response in the presence and absence of OPH on the surface of the transducer. (Bii) Detection of DFP (sarin simulant) in lake and (Biii) pond water samples; samples were spiked with different DFP concentrations (10, 20, 40, 80, 120 mM). (Ci) Carry-over evaluation of the OP biosensor from 5 mM to 100 mM DFP concentrations, and (Cii) for High and Low (40 mM and 80 mM) DFP concentrations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the immobilized OPH in the biosensor operation. Sensor stability is a major requirement for a successful wearable sensor. The storage stability of the OP tattoo sensors was evaluated by assessing the potentiometric behavior of a set of freshly prepared tattoo sensors over different time periods. Fig. S2 illustrates the stability performance of the sensor for up to 5 days without losing significant activity. The good stability signifies the judicious and systematic preparation of the sensors. To assess the applicability of the developed tattoo sensor in liquid phase, the sensors were tested for DFP detection in real water samples by spiking 5 different concentrations (10, 20, 40, 80, 120 mM) in untreated lake and pond water samples. Fig. 3B (ii and iii) illustrates the favorable potentiometric response of tattoo sensors for the nerve agent simulant in lake (Lake Murray, San Diego, CA) and pond (San Diego) water, with well-defined changes in the potential signal upon increasing the DFP concentration. The lake water shows better response against pond water, which articulates that the tattoo sensor has more affinity to Lake Matrix due to the presence of less complex substances. Certainly, the obtained data showed significant promise of the new sensors for environmental field monitoring. The repeatability of the developed potentiometric biosensor represents another significant feature of wearable sensors. Moreover, it is also important to investigate whether the resulting signal drifts by time which would have a significant negative effect on the performed measurement. In general, it is imperative to achieve highly reproducible data points, which is of particular importance for continuous field monitoring of nerve agent threats, aimed at alerting for potential exposure. A fast and reversible response is required for wearable sensors to assess the accurate level of analyte. The reversibility of OPH-tattoo sensor was checked by measuring its response, using 7 different DFP concentrations (range: 5–100 mM) for more than 2000sec. Fig. 3C, i depicts the
responds instantaneously to these pH changes, yielding high linearity over the entire 2.2–8.2 pH range (slope: 65.30 mV/log10 [pH]). The sensor reaches a stable response in less than 20 s. The OPH-modified tattoo biosensors were evaluated for liquid-phase DFP detection by recording the pH change during the biocatalytic hydrolysis of the simulant nerve agent. Optimization the OPH loading was performed by changing the amount of OPH loading (20–60 ng) and monitoring the response to 80 mM DFP in liquid phase, with 60 ng offering the most favorable and stable response (Fig. S1). To facilitate the sensing step, the fabricated OPH-modified tattoo sensors were preconditioned for 2 min using 25 μL distilled water. The performance of the tattoo-based biosensor was assessed first towards screening the nerve agent threats in the liquid phase. Changes in the DFP concentration were used to evaluate the dynamic behavior of developed biosensor. Fig. 3B, i, illustrates the potential-time response upon increasing the DFP concentration over the 10–120 mM range. The potentiometric OP biosensor responds instantaneously to these DFP additions. The resulting calibration (shown in the inset) exhibits good linearity (R2 = 0.983). The estimated limit of quantification (LOQ) was 10 mM (n = 10), calculated in accordance with the IUPAC norms. Moreover, the reproducibility of biosensor was investigated by preparing five different tattoo-based sensors and testing them in similar manner for the detection of DFP in liquid phase. Calibration curves were performed over the 10 to 120 mM DFP concentration range. The obtained RSD of the resulting calibration curves was 4.8%, indicating a good reproducibility of the fabricated tattoo sensor. The DFP response was also assessed in the absence of OPH (control probe) on tattoo sensors (Fig. 3B, i, red curve, b). As expected from the absence of the enzyme (i.e., no release of protons), a negligible potential (i.e., pH) change is observed. This control experiment supports the crucial role of 969
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performance of tattoo sensor to check reversibility of the system in a wide range of DFP concentrations with a good trend of increase and decrease in OCV responses during the carry-over evaluation. Similarly, to examine the sensor performance with lower (40 mM) and higher (80 mM) dosage of DFP, carry-over evaluation was performed and repeatability was evaluated. Fig. 3C, ii illustrates a carry-over plot to showcase the good repeatability of OPH tattoo sensor in the liquid phase up to 9 cycles of low and high concentration (5 and 100 mM) over a prolonged (1600sec) period. The highly reproducible response, with no carry-over effects, signifies the potential for reliable on-body testing. 3.4. DFP detection in vapor phase Following the successful DFP detection in liquid phase, the wearable potentiometric tattoo sensor was tested for its ability to detect the OP threat in the vapor-phase. Such vapor detection was performed with utmost safety measures, specified in the experimental section 2.1, using the set-up presented in Fig. 1. The vapor phase DFP biosensing was facilitated by modifying the electrode surface with a PVA-acrylamide hydrogel, which allowed the vapors to absorb and diffuse towards the PANi-OPH sensing layer. The PVA-acrylamide hydrogel membrane was formed through crosslinking of polyacrylamide - polyvinyl alcohol with glutaraldehyde drop casted onto the carbon-based tattoo sensor and subsequently heated at 50 °C for 20 min. In terms of underlying mechanism, the acetyl bridges were developed among the pendant hydroxyl groups of the PVA chains and this generated the polyacrylamidepolyvinyl alcohol hydrogel. To mimic exposure to a G-series nerve agent vapor, such as sarin, a portable and hand-held nebulizer was used to generate the DFP vapor toward the wearable sensor placed on a mannequin arm (Fig. 1B). This DFP vapor detection was carried out by optimizing the vapor spraying. As illustrated in Fig. S3, different OP spraying/nebulizing times (15–60 s) were examined using 60 mM DFP at a fixed nebulizer-sensor distance. Based on these data, 15 s vapor spray duration was chosen to be the optimal time for further vapor phase detection experiments. No further change in the response was observed beyond 15 s. Upon detection of DFP, the obtained data were wirelessly transmitted to a smart device. Fig. 4A illustrates the experimental set-up consisting of the tattoo sensor integrated with potentiometric board placed on mannequin arm. DFP vapors were sprayed using handheld portable nebulizer. Fig. 4B, i and B, ii shows real time detection of increasing DFP vapor concentrations, along with the resulting calibration plot. The sensor responds rapidly to the DFP vapor, reaching steady-state response within 30 s. The response increases linearly with the vapor concentration over the entire concentration range (20–120 mM) of the nebulizer solution, with a LOQ of around 10 mM. A similar calibration experiment carried out with an ‘enzyme-free’ sensor displayed a negligible DFP response (red line of Fig. 4B, i and ii). Keeping in mind that the OPH is a class selective enzyme, hence, significant interfering effect could be expected from other organophosphate group analytes. In particular, the detection of these G-type nerve agents often must compete with a background level of OP pesticides [34]. MPOx was used here for testing the selectivity between the G-type nerve agent simulant, such as DFP and, common organophosphate pesticides (Fig. 4C). The selectivity experiments were carried out using 3 different mixed concentrations of 1:1 DFP + MPOx (40:40, 80:80 and 100:100 mM, DFP: MPOx). The response was compared to the signal of 3 different addition of MPOx solution (40, 80 and 100 mM). Surprisingly, no notable effect of MPOx interference is observed in comparison to the DFP response. As shown in Fig. 4C, i (black line), the tattoo sensor results in a well-defined DFP response (in the presence of similar level of MPOx) whereas MPOx alone yields a negligible potential response (compared to the DFP + MPOx response), illustrating the highly selective response of the sensor towards DFP. Such behavior is attributed to the significant difference in pKa values of the co-products of enzymatic reaction, HF (pKa ∿ 3) and p-nitrophenol (pKa ∿ 7) [35],
Fig. 4. Response of different DFP concentrations in vapor phase using the tattoo biosensor coated with PVA-acrylamide hydrogel (A) Illustration of the experimental set-up and electronic/potentiometric printed circuit board with an integrated tattoo that is attached to mannequin arm. DFP solution is sprayed using nebulizer. (B) Vapor phase DFP analysis. (i) Potentiometric response upon spraying DFP (20–120 mM) on the sensor (curve a) and similar real time response of DFP without OPH on the electrode surface (curve b). (B,ii) Calibration plot of the biosensor with (a) and without (b) OPH on the electrode surface. (C,i) Response to the mixed solution of DFP + MPOx (red) and only MPOx (black) (Cii) Linear points plotted based on the increase OCV values upon concentration increments of DFP + MPOx (40, 80 and 100 mM mixture) (black) and MPOx (red) (D) Carry-over evaluation of the potentiometric biosensor for 40 mM and 80 mM concentration range of DFP using PANi modified OPH sensor coated with PVA-acrylamide hydrogel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
which are released during the hydrolysis of DFP and MPOx respectively. This demonstrates the possibility for detecting organofluorophosphates in the environment where other non-fluoro class-homologues are present. Fig. 4C, ii shows the linear behavior of both the sensor, black line corresponds to DFP + MPOx response (both present in equal concentrations of 40, 80 and 100 mM) and red line shows linear response of MPOx. Similar to the liquid phase carry-over study, repeatability of the tattoo biosensor was performed to check the response of low and high dosage of DFP vapors. Fig. 4D illustrates a carry-over plot to assess the reversibility and repeatability of OPH tattoo sensor in vapor phase. It 970
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coupling the OPH biocatalytic recognition phase with a pH-responsive PANi layer and interfacing the resulting flexible potentiometric biosensor with a conformal wireless electronic interface. The skin-worn biosensor thus integrates all the required functionalities necessary for nerve agent screening in realistic field settings. Extensive in-vitro characterization of the new wearable OP potentiometric-tattoo biosensor system affirmed that it offers an attractive analytical performance, with a fast and stable response to the sarin analog, DFP. Such immediate response provides an instantaneous warning for a timely action. A single use of the tattoo sensor is desired considering this rapid warning goal and potential memory effect (associated with accumulation of reaction product within the hydrogel membrane). Possible changes in the sweat pH could be addressed using an enzyme-free PANi transducer. Besides its rapid warning capability, the wearable sensor can shed useful insight regarding the nature of a chemical attack. The new wearable biosensor addresses the existing gaps and major needs for protecting soldiers, civilians and farmers against unexpected exposure to chemical threats. Acknowledgments This work was supported by the Defense Threat Reduction Agency Joint Science and Technology Office for Chemical and Biological Defense (HDTRA1-16-1-0013). J.R.S. acknowledges the fellowships from CNPq (216981/2014-0). The authors thank Dr. Mustafa Musameh (CSIRO, Clayton, Australia) for providing the OPH enzyme. Fig. 5. Selectivity study: Response of the modified OPH/PANi based biosensor, coated with PVA-acrylamide hydrogel, to (a) methyl parathion (200 μM), (b) 200 μM MPOx, (c) 5% hydrogen peroxide (d) 100 ppm DNT (e) 60 mM DFP (f) 70% ethanol (g) acetone (h) ammonia (i) 70% methanol (j) formaldehyde and (k) toluene towards OP tattoo sensors, demonstrating the selectivity of the sensor towards DFP in vapor phase.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.snb.2018.07.001. References
can be seen from these data that there is an insignificant drift in the potentiometric biosensor response for the vapor up to 7 cycles (i.e. 900 s).
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3.5. Selectivity of potentiometric tattoo sensor for OP vapors The occurrence of other coexisting toxic chemical vapors in the environment might impact the selective detection of the target analyte, hence, it may compromise the accuracy of the new potentiometric wearable tattoo OP vapor sensor. Thus, an additional selectivity control study was performed using multiple potential interferences, such as ethanol, acetone, ammonia, methanol, MPOx, hydrogen peroxide, DNT, formaldehyde, isopropyl alcohol and toluene. These chemical vapors were selected as a control to study the DFP response among them, once they are commonly present in the environment [36–38]. Fig. 5 demonstrates the high selectivity of the new potentiometric OP-tattoo sensor toward the target nerve agent simulant (DFP) (e), along with the different potential interferences: (a) methyl parathion (200 μM) (B) 200 μM MPOx (c) 5% hydrogen peroxide (D) 100 ppm DNT (E) 60 mM DFP (f) 70% ethanol (g) acetone (h) ammonia (i) 70% methanol (j) formaldehyde and (k) toluene. These results illustrate that the presence of several other non-target vapors in the environment does not hinder the OP detection. Noteworthy, these tested chemical vapors do not show any drift or significant changes in the recorded signal, but the results were rather similar to the control signals except for the response of DFP shown in Fig. 5e. 4. Conclusions This work describes the performance of a fully integrated wireless potentiometric OP tattoo biosensor platform for real-time on-body monitoring of G-type nerve agent simulants in both liquid and vapor phases. Such effective on-the-spot detection of DFP has been realized by 971
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