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Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Early detection and monitoring of chronic wounds using low-cost,omniphobic paper-based smart bandages

Aniket Pala, Debkalpa Goswamia, Hugo E. Cuellara, Beatriz Castrob, Shihuan Kuangb,Ramses V. Martineza,c,⁎

a School of Industrial Engineering, Purdue University, 315 N. Grant Street, West Lafayette, IN 47907, USAbDepartment of Animal Sciences, Purdue University, 270 S. Russell St, West Lafayette, IN 47907, USAcWeldon School of Biomedical Engineering, Purdue University, 206 S. Martin Jischke Drive, West Lafayette, IN 47907, USA

A R T I C L E I N F O

Keywords:Smart bandagesChronic wound monitoringPressure ulcersWearable sensorpH sensorUric acid sensor

A B S T R A C T

The growing socio-economic burden of chronic skin wounds requires the development of new automated andnon-invasive analytical systems capable of wirelessly monitoring wound status. This work describes the low-costfabrication of single-use, omniphobic paper-based smart bandages (OPSBs) designed to monitor the status ofopen chronic wounds and to detect the formation of pressure ulcers. OPSBs are lightweight, flexible, breathable,easy to apply, and disposable by burning. A reusable wearable potentiostat was fabricated to interface with theOPSB simply by attaching it to the back of the bandage. The wearable potentiostat and the OPSB can be used tosimultaneously quantify pH and uric acid levels at the wound site, and wirelessly report wound status to the useror medical personnel. Additionally, the wearable potentiostat and the OPSBs can be used to detect, in an in-vivomouse model, the formation of pressure ulcers even before the pressure-induced tissue damage becomes visible,using impedance spectroscopy. Our results demonstrate the feasibility of using inexpensive single-use OPSBs anda reusable, wearable potentiostat that can be easily sterilized and attached to a new OPSB during the dressingchange, to provide long term wound progression data to guide treatment decisions.

1. Introduction

Chronic wounds, where full regeneration of the damaged tissue doesnot complete in three months, are a worldwide health problem thatcauses a significant burden to healthcare systems; both in terms of thenumber of patients affected and the expenses derived from their pre-vention and treatment (Posnett and Franks, 2010; Sen et al., 2009). Theneed to reduce the burden of chronic wounds on patient's quality of lifeand national health budgets has led to the development of advancedwound care technologies for automatic monitoring of wound status(Finlay, 2016). These “smart bandages” monitor wound biomarkersusing sensors fabricated on flexible substrates in order to reduce thenumber of dressing changes and minimize the stress and pain sufferedby the patient (Mehmood et al., 2014; Weber et al., 2010). Effectivesmart bandages should be mechanically flexible, breathable, easy toapply, and capable of reporting quantitative information about thewound status in real time to guide treatment decisions (Sen et al.,2009). Although a variety of smart bandages have been proposed tomonitor physical and chemical parameters important in wound healing,most of these devices often require expensive and relatively

cumbersome equipment, which limits the mobility of the patients andmakes the dressings uncomfortable to wear (Jankowska et al., 2017;Phair et al., 2014; Schreml et al., 2014; Sharp and Davis, 2008).Moreover, the need of trained personnel to apply the smart dressingsand to interpret the results limits the implementation of these devicesoutside clinical settings. Since it is recommended to change dressingsfrequently (at least once per day (Sood et al., 2014)) smart bandagesneed to be low cost and disposable for single-use applications. There-fore, a low cost strategy to fabricate sensitive and easy to use smartbandages, so that they can be used as single use devices by minimallytrained individuals, would be desirable to improve chronic woundhealing outcomes; particularly in resource limited and home environ-ments. Here, we propose to incorporate low-cost electrochemical andimpedance sensors, fabricated using omniphobic (hydrophobic andoleophobic) paper, into commercially available bandages to monitorthe status of open chronic wounds and pressure ulcers. The simple in-terfacing between these single-use smart bandages with a reusable,wearable measuring system facilitates their use as a non-invasivewireless platform for real-time monitoring of wound status.

Recent advances in electrochemical sensors using flexible electronic

https://doi.org/10.1016/j.bios.2018.06.060Received 3 April 2018; Received in revised form 6 June 2018; Accepted 27 June 2018

⁎ Corresponding author at: School of Industrial Engineering, Purdue University, 315 N. Grant Street, West Lafayette, IN 47907, USA.E-mail address: rmartinez@purdue.edu (R.V. Martinez).

Biosensors and Bioelectronics 117 (2018) 696–705

Available online 05 July 20180956-5663/ © 2018 Elsevier B.V. All rights reserved.

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platforms have provoked the emergence of several advanced dressingsthat quantitatively report wound status by monitoring physiologicalindicators such as temperature (Hattori et al., 2014), moisture (McCollet al., 2007), partial pressure of oxygen (Li et al., 2014; Schreml et al.,2014), concentration of biomarkers (Fernandez et al., 2014; McListeret al., 2017; Schneider et al., 2007), and bacterial load (Farrow et al.,2012; Zhou et al., 2010). The use of flexible substrates allows them toconformably cover the wound without damaging the repairing tissue.Some of these technologies demonstrated the ability to transfer thesensor reading wirelessly to an external device (Farooqui and Shamim,2016; Kassal et al., 2017). Despite the high sensitivity of these smartbandages, the integration of a power source and the electronic circuitrynecessary to acquire and transmit measurement data complicates theirdesign and fabrication, increasing their cost and hindering their im-plementation as disposable, single-use solutions (Liu et al., 2017).

Among the different biomarkers present in wound exudate, uric acid(UA) and pH are recognized to play a pivotal role in the biochemicalevents associated with wound healing (McLister et al., 2016). Thecontinuous monitoring of UA has demonstrated to serve as an accurateindicator of S. aureus or P. aeruginosa colonization in open woundssince bacteria decrease the concentration of UA within wound exudateby metabolization (Ochoa et al., 2014; Sharp and Davis, 2008). Severalbandage-based electrochemical sensors have used voltammetry andamperometry for the non-enzymatic detection of UA in wounds (Kassalet al., 2015; Liu et al., 2013; Phair et al., 2014). Unfortunately, vol-tammetry-based approaches to measure UA often require the applica-tion of a relatively large potential on the working electrode, whichcauses interferences from other metabolites and easily oxidizing speciesin the wound exudate (Dargaville et al., 2013). The low voltages typi-cally needed to monitor UA using amperometry significantly decreaseinterferences; however, electrode biofouling has proved to decrease thedetection accuracy of UA in wound exudate or blood after multiplemeasurements (Sharp et al., 2008).

Elevated pH values of the wound exudate can also be used toidentify bacterial infections, since the alkaline byproducts of bacterialproliferation raise the pH out of the narrow acidic range (5.5–6.5) ofnon-infected open wounds (Ochoa et al., 2014; Rahimi et al., 2016).While commercial pH meters (glass probes) have demonstrated theadequate evaluation of uniform acute wounds, most recent pH sensingplatforms for wound monitoring rely on flexible electrodes coated witha pH-sensitive layer (mainly metal oxides (Korostynska et al., 2008) orconductive polymers (Ferrer-Anglada et al., 2006; Guinovart et al.,2014; Rahimi et al., 2017)). Compliant sensors with metal oxide pH-sensitive layers (e.g., SnO2, RuO2) exhibit good thermal stability, re-sponse times of only a few seconds due to their rapid absorption ofhydrogen ions from the wound exudate, and high sensitivities(≈−80mV/pH) (Huang et al., 2011). Unfortunately, metal-oxide-based pH sensors require frequent calibrations due to drift and have notbeen widely adopted due to the high cost of their fabrication materials(Kurzweil, 2009). The use of polymeric pH-sensitive layers (e.g., poly-pyrrole, polyaniline), which are capable of measuring pH using theprotonation and deprotonation of the nitrogen atoms in their structures,enabled the low-cost fabrication of highly sensitive (≈−1300mV/pH),flexible, and stable pH sensors by electropolymerization or drop-casting(Rahimi et al., 2017, 2016). While several sensors for UA or pH mon-itoring have been demonstrated using wireless platforms, the com-plexity of their fabrication process, the cost of the materials, and theiroften uncomfortable design make them unsuitable for chronic woundmonitoring (Farooqui and Shamim, 2016; Liu et al., 2017).

Patients with limited mobility are prone to develop pressure ulcerswhen a part of their body holds sustained pressure for a prolongedperiod. Elderly (Gist et al., 2009), obese (Sen et al., 2009), and diabetic(Blakytny and Jude, 2006) patients are more likely to develop pressureulcers, especially while recovering from surgical operations. Pressureulcers pose the additional problem of being difficult to predict, since theamount of pressure that causes them varies widely between patients

(Ayello and Lyder, 2008; Sen et al., 2009; Wong, 2011). Several tech-niques have been proposed for the early detection and monitoring ofpressure ulcer development using ultrasounds (Aoi et al., 2009; Kimet al., 2008), digital photography (Chang et al., 2017; Lima et al.,2018), impedance spectroscopy (Swisher et al., 2015), or measurementsof the partial pressure of carbon dioxide of the damaged tissue(Mirtaheri et al., 2015). To date, however, there are no wearablewireless devices capable of detecting and continuously monitoring thehealing process of pressure ulcers.

Our previous work in point-of-care diagnostics demonstrated thelow-cost fabrication of microfluidic devices (Glavan et al., 2013) andself-powered electrochemical sensors using hydrophobic paper (Palet al., 2017). Here, we present omniphobic paper-based smart bandages(OPSBs), a low cost platform capable of measuring multiple parametersto perform real-time monitoring of all kinds of chronic wounds (bothopen wounds and pressure ulcers). OPSBs, in combination with awireless wearable potentiostat, can provide simultaneous quantitativemeasurements of pH and uric acid in open wounds, and assess tissuedamage in closed chronic wounds like pressure ulcers. OPSBs also offerseveral advantages as follows: (i) They are flexible, lightweight,breathable, easy to apply and dispose, and suitable for single-use ap-plications; (ii) their manufacturing is simple, inexpensive, and compa-tible with mass-scale production techniques, such as spray deposition orroll-to-roll printing; (iii) they can be used to monitor both open woundsand pressure ulcers; and (iv) their wireless measurement and commu-nication module is wearable and reusable, enabling non-invasive re-mote monitoring of wound status.

2. Materials and methods

2.1. Chemicals and instruments

We purchased Whatman #1 paper from GE Healthcare Inc.(Pittsburgh, PA) and two conductive inks, Ag/AgCl (AGCL-675) andcarbon (C-200), from Applied Ink Solutions (Westborough, MA).Potassium ferricyanide, potassium ferrocyanide, uric acid, uricase(from Candida sp., 4.1 U/mg), polyaniline emeraldine base (PANi-EB,Mw=50 kDa), disodium phosphate (sodium hydrogen phosphate), ci-tric acid (2-hydroxypropane-1,2,3-tricarboxylic acid), and RFSiCl3(CF3(CF2)5(CH)2SiCl3, trichloro-(1H,1H,2H,2H-perfluorooctyl)silane)were purchased from Sigma Aldrich Inc. (St. Louis, MO). We used acommercial, benchtop potentiostat (Reference 3000; GamryInstruments, Warminster, PA) to test the performance of the electro-chemical sensor and the wearable potentiostat. We purchased BAND-AID® adhesive bandages from Johnson & Johnson Consumer Inc. (NewBrunswick, NJ).

2.2. Fabrication of the wearable potentiostat

We fabricated a rechargeable, wearable potentiostat using a low-power programmable front end for electrochemical sensing applications(LMP91000, Texas Inst. Inc.) and a high precision impedance analyzer(AD5933, Analog Devices Inc). The wearable potentiostat is powered bya rechargeable battery (LIR2032, Duracell Inc.), and controlled by anopen-source microcontroller prototyping platform (Arduino Nano v3.0,Arduino Inc.) (code provided in Supplementary information). An RFtransceiver IC (nRF24L01, Nordic Semiconductors Inc.) inside thewearable potentiostat performs for wireless communication through the2.4 GHz ISM band. We sterilized the wearable potentiostat by spraying70% ethanol before attaching it to a new OPSB. After the ethanol dried,we used a laser cut ring of double-sided adhesive tape (410M, 3M Inc.)to attach the wearable potentiostat to the bandage and provide a stableelectrical connection with the paper-based sensors embedded in theOPSB.

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2.3. Fabrication of omniphobic paper-based uric acid sensors to monitoropen chronic wounds

We rendered Whatman #1 paper omniphobic by spraying it with a2% solution of fluorinated alkyltrichlorosilane (RFSiCl3) in iso-propylalcohol (IPA) and drying it in a desiccator at 36 Torr for 20min (Glavanet al., 2013). We stencil printed three flexible electrodes on the omni-phobic paper using conductive inks: working (WE) and counter (CE)electrodes with carbon ink, reference electrode (RE) and contact padswith Ag/AgCl ink (Fig. S1). We dried the conductive inks in a desiccatorat 36 Torr for 30min, producing electrodes with highly reproducibleconductivities (Fig. S2). Prior to mounting the paper-based UA sensorson the adhesive bandages, we drop cast 5 μL of a uricase solution on theelectrochemical test zone and allowed it to dry at room temperature.We used uricase (from Candida sp.) as it has been shown to have ex-cellent selectivity towards UA (Erden and Kiliç, 2013; Liu et al., 2013;Mostafalu et al., 2015; Zhang et al., 2004). The presence of potentialinterferants available in wound exudate—such as creatinine, glucose,lactate, or ascorbic acid—has also been demonstrated to have no sig-nificant effect on the selectivity of uricase (Kassal et al., 2015;Mostafalu et al., 2015; Piermarini et al., 2013). We prepared the uricasesolution by mixing 3 μg/mL uricase with a 100mM solution of po-tassium ferricyanide in 1:1 ratio. All solutions were made in phosphatebuffered saline (PBS, 1×, pH 7.4). To calibrate the wearable

potentiostat, we used different concentrations (0.2–1mM in steps of0.2 mM) of UA in PBS. We pipetted 5 μL of the uric acid solutions overthe uricase modified electrodes and performed chronoamperometricassays using a 300mV step potential (with respect to the RE) at asampling rate of 10 Hz. The use of such a low working potential ensuresminimal interference from other easily oxidized species in the woundexudate. Integrating the measured current with respect to time enabledthe calculation of the net charge exchanged during the redox reactionby chronocoulometry (Eq. S2).

2.4. Fabrication of omniphobic paper-based pH sensors to monitor openchronic wounds

We printed the electrodes of the pH sensors using Ag/AgCl ink. Weprepared a pH-responsive polymeric composite by mixing 150mg ofPANi-EB with 250mg of silver microflakes (particle size 2–5 µm,Inframat® Advanced Materials™ LLC) (Fig. S5a, b) in 5mL of IPA. Themixture was sonicated for 1 h to create a uniform suspension. We pi-petted 10 μL of the Ag/PANi-EB solution between the electrodes of thepH sensor and dried it at 60 °C for 30mins to create a thin solid film ofAg/PANi-EB composite (blue colored). We exposed the Ag/PANi-EBcomposite to hydrochloric acid (HCl) vapors in a desiccator at 36 Torrfor 30min. The H+ ions from the HCl vapors transform the PANi-EBpart of the composite into its emeraldine salt (ES) form (green colored),

Fig. 1. OPSB fabrication and assembly process. (a)Schematic diagram of the fabrication of OPSBs: 1)Whatman #1 paper is rendered omniphobic by spraying a2% solution of RFSiCl3 in IPA; 2) stencil printing is used topattern flexible conductive electrodes using carbon andAg/AgCl inks; 3) openings are laser cut on the adhesivelayer of the bandage to interface the wearable potentiostatwith the paper-based sensors in the OPSB. OPSBs are as-sembled by placing the paper-based sensors between theadhesive layer and the absorbent pad of the commercialbandages. (b) OPSBs used to monitor uric acid and pHlevels in open wounds. (c) OPSBs used for the early de-tection of pressure ulcers. (d, e) Interfacing of the wear-able potentiostat with OSPBs for monitoring open woundsand detecting pressure ulcers, respectively. (f) Packagingof the electronics in the rechargeable, wearable potentio-stat.

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which exhibits a higher conductivity (Fig. S3, S5c, d). We rinsed theAg/PANi-ES pH sensors with deionized water and dried them in a ni-trogen stream before their embedding into a commercial bandage.

We prepared pH buffer solutions (McIlvaine) across the clinicalrange (Ochoa et al., 2014; Rahimi et al., 2016) of open wound exudate(5.5–8.5) to calibrate the pH sensors. McIlvaine buffers were preparedby mixing 0.2M solution of disodium phosphate and a 0.1 M solution ofcitric acid in different ratios (Pearse, 1968) (Table S1). We verified thepH of all the resulting solutions using a digital pH meter (Model IQ125,IQ Scientific Instruments, USA). We pipetted 10 μL of the pH buffers onthe Ag/PANi-ES composite and performed impedance spectroscopyacross the electrodes by applying sinusoidal signals with an amplitudeof 100mV and frequencies ranging 10 Hz–100 kHz to calibrate themeasured impedance with pH.

2.5. Fabrication of omniphobic paper-based impedance sensor array tomonitor pressure ulcers

We printed seven equidistant electrodes in a hexagonal array usingAg/AgCl ink on omniphobic Whatman #1 paper to measure, in vivo,tissue impedance across pressure ulcers models induced on mice (Fig.S1). To improve the electrical contact between the electrodes and theskin of the mice, we selectively coated the electrodes with a conductivehydrogel (SPECTRA® 360, Parker Laboratories Inc.) using a stencilmask. The omniphobic paper substrate impeded the spreading of theconductive hydrogel over the paper, and avoided short circuits amongthe electrodes. After placing the electrode array on the shaved skin ofthe mouse and ensuring its uniform contact, impedance data was re-corded from each pair of nearest neighbor electrodes. The wearablepotentiostat enables the detection of tissue damage using a AD5933impedance analyzer chip and transmits the results to the user via an RFtransceiver module. The AD5933 chip applied signals with an AC

voltage of 0.97 V, DC bias of 0.76 V, and frequencies ranging 1–100 kHzto perform impedance spectroscopy. The wearable potentiostat readsthe response signal, and calculates the magnitude and phase of theimpedance of the underlying tissue.

2.6. Early in vivo detection of pressure ulcers

We used ten laboratory mice (C57B6J, 8–15 weeks old, male) withmixed backgrounds to detect pressure-induced tissue damage in vivo. Aketamine-xylazine cocktail (0.1 g per kg of body weight). was used toanaesthetize the mice. We used two disc magnets (D601, www.kjmagnetics.com; NdFeB, 10mm diameter, 2 mm thickness) to con-trollably create a pressure ulcer model on mice (Swisher et al., 2015).Prior to the application of the magnets, hair was removed from the backof the mice using depilatory cream (Nair) and then the area was washedwith mild detergent (Dawn). The shaved skin of the mice was gentlytented up and placed between the two disc magnets (Fig. S6), whichapplied a ~ 6.7 kPa pressure during the ischaemia cycle. The magnetsdid not interfere with the normal activity of the mice after they re-covered from the anesthesia. The magnets were kept in place for 1 or3 h to create different degrees of tissue damage. Each mouse receivedonly one pressure-induced wound, and impedance measurements of thedamaged tissue were collected for three days after the ischaemia cycle.During the measurements, the wearable potentiostat placed over theanesthetized mouse performed impedance spectroscopy across eachpair of nearest neighbor electrodes in the array (Fig. S1). After samplingall the electrodes and transmitting the results wirelessly to a laptop, amap of the impedance of the tissue was constructed and used to assessthe healing process of tissue damaged by 1 h- and 3 h-long ischaemicevents. We drew registration marks onto the skin of the mice to locatethe different features of the pressure ulcer with respect to the positionof the electrodes. We used the same OPSB to collect impedance

Fig. 2. Schematics of the electronics in thewearable potentiostat used for wireless chronicwound monitoring. The LMP91000 andAD5933 chips performed electrochemicalmeasurements and impedance spectroscopy,respectively. Both chips are controlled by amicrocontroller and powered by a recharge-able 3.6 V battery. Test results are wirelesslytransmitted by a radio frequency communica-tion module.

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measurements, once per day, over three consecutive days. All proce-dures involving mice were performed in accordance with Purdue Uni-versity's Animal Care and Use Committee.

3. Results and discussion

3.1. Design and assembly of OPSBs

Fig. 1a depicts the fabrication steps followed to make OPSBs. Wefabricated flexible electrodes by screen printing conductive inks onpaper, which was previously rendered omniphobic by spraying a so-lution of RFSiCl3 in IPA. The silane, while chemically modifying thecellulose fibers, does not block the pores of the paper, preserving itsbreathability to ensure oxygenation of the wound (Fig. S3, Movie S1).These paper-based sensors are then embedded into commercial ban-dages to create the OPSBs (Fig. 1a–c), without compromising the flex-ibility of the bandage. We laser cut openings on the adhesive bandageand folded the contact pads of the sensors to allow them to interfacewith the wearable potentiostat (Fig. 1a). The absorbent pad of thebandage transfers the wound exudate to the surface of the paper-basedsensors. We fabricated two sets of OPSBs: one with sensors capable of

monitoring both UA and pH levels in the exudate of open wounds(Fig. 1b), another with an electrode array capable of detecting pressureulcers (Fig. 1c). After the bandage is applied on top of the wound, asingle-use, double-sided adhesive layer sticks the wearable potentiostatto the OPSB, making it easy to apply, and securing the electric contactswith the paper-based sensors. To change these smart dressings, wesimply remove the OPSB (while still attached to the wearable po-tentiostat) from the skin of the user and then peel the wearable po-tentiostat from the adhesive layer.

Supplementary material related to this article can be found online atdoi:10.1016/j.bios.2018.06.060.

3.2. Characterization of the wearable wireless potentiostat

We developed a lightweight (~ 8 g) and low-cost (~ $18, Table S4)wearable potentiostat, capable of performing 3-electrode electro-chemical measurements and impedance spectroscopy (Fig. 2). We de-signed the housing of the potentiostat as a “smiley face” to promotepatient adoption of the device (Fig. 1d–f). The wireless communicationmodule integrated in the wearable potentiostat transmits the mea-surements so that the results can be stored and displayed on the user's

Fig. 3. Characterization of the electrochemical performance of the flexible electrodes stencil-printed on omniphobic paper substrates. (a) Cyclic voltammogramsrecorded with the wearable potentiostat for various concentrations of potassium ferricyanide in PBS at a scan rate of 10mV/s. The insets show the static contact angle(147°) of water on omniphobic Whatman #1 paper and the electrodes used. (b) Dependence of the anodic and the cathodic peak currents on the concentration ofpotassium ferricyanide (average RSD=8% and 12% respectively). (c) Chronoamperometric current response measured by applying a constant potential of 300mVwith respect to the RE, as increasing concentrations of potassium ferricyanide in PBS (0.1–1mM, in steps of 0.1 mM) are added at 2min intervals. (d) Lineardependence of the exchanged charge on the square root of time for the different concentrations of potassium ferricyanide, as expected from the Cottrell equation (Eq.S1).

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phone (Pal et al., 2017) or laptop and then transferred from on-site toexperts, over the web, to facilitate remote consultation. Additionally,the wireless card in the wearable potentiostat enables the wireless re-configuration of the LMP91000 and the AD5933 chips through themicrocontroller, making it possible to choose between impedancespectroscopy, chronoamperometry, and chronocoulometry, as well as toselect the proper scanning parameters, for a fully automated wirelessmonitoring of wound status. A 3.6 V rechargeable battery powers themicrocontroller and the low-power chemical sensors using low supplycurrents (< 15 μA), maximizing battery life (over 7 days, measuringonce every two hours) and making the device safe for the patient. WhileOPSBs can be easily disposed by incineration (Fig. S7), the wearablepotentiostat can be reused, after its sterilization and recharging by at-taching it to a new OPSB.

To characterize the electrochemical performance of the OPSBs andthe wearable potentiostat, we performed cyclic voltammetry (CV) withseveral solutions of ferricyanide/ferrocyanide, one of the electroactivesystems most commonly used for testing electrochemical electrodes(Hamedi et al., 2016; Nemiroski et al., 2014; Pal et al., 2017). Fig. 3ashows the voltammograms of several concentrations (0.5, 1.0, 1.5, 2.0,2.5, and 5.0 mM) of potassium ferricyanide in PBS, recorded with thewearable potentiostat while applying a linear voltage sweep from −0.4

to 0.6 V at 10mV/s scan rate. The linear relationship between theanodic and cathodic peak currents and the square root of the scan rates(R̅2 =0.993 and 0.997 respectively, Fig. 3b) demonstrates that theOPSBs and the wearable potentiostat are suitable to perform electro-chemical analysis (Glavan et al., 2016; Hamedi et al., 2016; Pal et al.,2017). Fig. 3c shows the chronoamperometric current response forgradually increasing potassium ferricyanide concentrations. The linearrelationship between the net exchanged charge and the square root oftime (Fig. 3d) verifies that the reaction is diffusion controlled andgoverned by the Cottrell equation (Eq. S1) (Glavan et al., 2016).

3.3. Real-time monitoring of uric acid using OPSBs

The electrochemical measurement of UA levels in open wounds canbe used to monitor bacterial infection (Ochoa et al., 2014; Sharp andDavis, 2008). We used the OPSBs and the wearable potentiostat towirelessly measure different concentrations of UA in a wound exudatemodel (Fig. 4). The UA is oxidized to allantoin by the uricase in theOPSB, while potassium ferricyanide is reduced to potassium ferrocya-nide (Eq. S3, (Pal et al., 2017)). Using chronoamperometry, we cali-brated the linear relationship between the oxidation current and theconcentration of UA (Fig. 4b, c), according to the Cottrell equation (Eq.

Fig. 4. Real-time monitoring of UA using OPSBs and a wearable potentiostat. (a) Picture of the wearable potentiostat interfacing a functional OPSB. The wearablepotentiostat can be easily adhered on top of the smart bandage and removed, sterilized, and reused on different bandages. (b) Chronoamperograms measured withthe wearable potentiostat for different concentrations of UA in the wound model. (c) Calibration plots of the current as a function of the concentration of UA atdifferent reading times: 5 s (R̅2 =0.996, average RSD=10.8%), 11.5 s (R̅2 =0.997, average RSD=9.6%), 20 s (R̅2 =0.998, average RSD=8.9%), and 30 s(R̅2 =0.998, average RSD=10.1%). (d) Linear dependence of the total charge measured by chronocoulometry on the concentration of UA at different reading times:5 s (R̅2 =0.957), 11.5 s (R̅2 =0.995), 20 s (R̅2 =0.998), and 30 s (R̅2 =0.999).

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S1). Fig. 4d shows the performance of the wearable potentiostat and theOPSBs to acquire and wirelessly transmit chronocoulometry measure-ments of UA. We noticed that using chronocoulometry provides morereliable quantitative measurements of the concentration of UA whencompared to chronoamperometry, as any random experimental noise isaveraged out by integrating the oxidation current over time (Eq. S2).OPSBs, calibrated using the curve shown in Fig. 4d, exhibit a limit ofdetection for UA of 0.2mM using the wearable potentiostat in chron-ocoulometry configuration, demonstrating the accurate sensing of thesesmart bandages over the clinically relevant range for open chronicwounds (0.22–0.75mM, (Kassal et al., 2015)).

3.4. Determination of pH levels using OPSBs

The wearable potentiostat can also be wirelessly configured to op-erate as a resistive sensor to accurately quantify pH levels of a woundexudate model (Fig. 5). To measure pH, we use OPSBs with a pair ofAg/AgCl parallel electrodes printed on omniphobic paper and separatedby a layer of Ag/PANi-ES composite (Fig. 5a, b, S4). PANi-ES graduallytransforms into PANi-EB (more resistive) when exposed to alkaline pHenvironments (Rahimi et al., 2017). Silver microflakes decrease theresistivity of PANi, facilitating the accurate determination of the pH ofthe wound exudate by applying low voltages (100mV). We measuredthe impedance across the electrodes of the pH sensor over frequenciesranging 10 Hz–100 kHz, and observed no frequency dependence of theimpedance up to 10 kHz (Fig. 5c). We chose a frequency of 100 Hz tocalibrate the relationship between pH and impedance for our omni-phobic, paper-based sensors (Fig. 5d). The PANi in the OPSB undergoesa reversible chemical transformation between PANi ES and PANi EB(Fig. S5) depending on the concentration of H+ ions of its surroundings.The impedance measured by the pH sensor correlates linearly with theconcentration of H+ (and thus exponentially with the pH) in the woundexudate model. After the OPSBs and the wearable potentiostat are ca-librated using the curve shown in Fig. 5d, they can be used to accurately

quantify pH levels across the clinically relevant pH range for openchronic wounds (5.5–8.5, (Ochoa et al., 2014)).

3.5. Early in-vivo detection and monitoring of pressure ulcers using OPSBs

While open chronic wounds are relatively simple to identify andmonitor by analyzing wound exudate, the early identification of theformation of subcutaneous (closed) wounds is challenging because bythe time the wound becomes visible on the skin, the tissue damageunderneath is often already severe (Ayello and Lyder, 2008; Wong,2011). Fig. 6 shows the early in-vivo detection and monitoring ofpressure ulcers on a mouse model using OPSBs. Cell damage or deathcauses the breakage of the cellular membrane, allowing the ion-richcytoplasm of the cell to merge with the extracellular fluid. This makesthe damaged tissue more conductive and less capacitive compared tohealthy tissue (Swisher et al., 2015). Fig. 6a, b show that a 3 hischaemic event decreases the magnitude of the impedance of the da-maged tissue more than a 1 h event, according to the amount of damageinduced to the tissue. Similarly, the phase of the impedance increaseswith the amount of damage caused by the ischaemic event. Fig. 6c, dshow the estimated transfer functions (ETF) used to mathematicallydescribe the impedance behavior of the in vivo model as a function offrequency. We found that OPSBs provided a maximum contrast be-tween the impedances of the healthy and damaged tissue over the40–60 kHz frequency range. A central frequency of 50 kHz was chosento create surface maps of impedance to detect damaged tissue under thebandage (Fig. 6e, f). The combination of magnitude and phase datamakes the detection of pressure ulcers more reliable than detectionmethods using only one parameter (Swisher et al., 2015). Using bothmagnitude and phase also minimizes the effect of experimental noiseand random variations in animal characteristics (age, skin roughness,weight, etc.). 1 h ischaemic events cause an immediate damage of theunderlying tissue that is partially recovered after 48 h and totally re-stored after 72 h (Fig. 6e). 3 h ischaemic events cause a damage of the

Fig. 5. Monitoring pH using OPSBs with Ag/PANi composite electrodes. (a) Schematic diagram describing the fabrication of the pH sensors: (1) PANi-EB is added to asuspension of silver microflakes in IPA and sonicated. (2) The resulting solution is drop cast between the stencil-printed electrodes and dried to create a Ag/PANi-EBlayer. (3) The Ag/PANi-EB composite is doped with H+ ions from HCl vapors, converting the PANi-EB to PANi-ES. (b) SEM image of a Ag/PANi-ES composite layeron omniphobic paper. The inset shows a high-resolution, false-colored SEM image of the Ag/PANi-ES composite with PANi-ES in green and Ag microflakes in blue(scale bar is 10 µm). (c) Bode diagram of the magnitude of the impedance for wound exudate models with different pH values. (d) Calibration of magnitude of theimpedance, measured at 100 Hz, as a function of the pH (R̅2 =0.998).

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Fig. 6. Early in-vivo detection and monitoring of pressure-induced tissue damage using OPSBs. (a, b) Bode diagrams of the magnitude and phase of the impedancemeasured across pressure ulcers models induced on a mouse by 1 h (a) and 3 h (b) ischaemia cycles. “Healthy” indicates the control measurements taken beforeapplying pressure to the tissue with two magnets. “0 h” corresponds to measurements taken immediately after the ischaemia cycle. “48 h” and “72 h” corresponds tomeasurements taken two and three days after the ischaemia cycle, respectively. (c, d) Dependence on frequency of the magnitude and phase of the impedanceobtained from a representative pair of electrodes after 1 h (c) and 3 h (d) ischaemia cycles. The markers denote the experimentally measured data, while the solidlines and shaded regions show the ETF and the 95% fit confidence interval respectively. (e, f) Surface maps of the magnitude and phase of the impedance measuredacross the tissue damaged by 1 h (e) and 3 h (f) ischaemia cycles. Images of the damaged region are provided at the right of each map. The dashed circle correspondsto the size of the magnets and indicates where the pressure was applied.

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underlying tissue that cannot complete its healing even after 72 h(Fig. 6f). These impedance maps demonstrate that the OPSBs and thewearable potentiostat can be used to non-invasively detect pressure-induced tissue damage, even when such damage cannot be visuallyidentified (Fig. 6e, f). These results demonstrate the feasibility of usingimpedance measurements obtained with paper-based wireless smartbandages to detect pressure-induced tissue damage at an early stageand to monitor its healing process across multiple animals.

4. Conclusions

This work reports a simple, low-cost, and non-invasive strategy tomonitor open wound status wirelessly, using OPSBs. This work alsoprovides the first demonstration of in-vivo early detection and mon-itoring of pressure ulcers using wireless smart bandages. OPSBs havefive significant advantages over previously reported smart bandages: (i)They are lightweight, inexpensive to manufacture, easy to apply, anddisposable by burning; (ii) a single OPSB can simultaneously quantifypH and UA at the wound site; (iii) they enable the early detection ofpressure ulcers by providing a surface map of the location and severityof the tissue damage; (iv) the use of omniphobic paper as a flexiblesubstrate facilitates oxygenation of the wound by preserving the gaspermeability of the bandage, and enables accurate wound monitoringfor up to three days without significant change in performance.Replacing OPSBs enables wound monitoring over longer periods oftime; (v) the wearable potentiostat wirelessly reports quantitative in-formation about the status of the wound, which can be used to informthe patient and remote medical staff about the need to change thebandage, disinfect the wound, or apply preventive treatment. TheOPSBs and the wearable potentiostat described here, at their presentlevel of development, also have two limitations: (i) The accuracy of themeasurements on open wounds performed with Ag/AgCl referenceelectrodes depends on the chloride concentration, which, while it isexpected to be tightly regulated within the blood, might vary over theinjury depending on the wound dynamics; (ii) to measure the in-vivoimpedance of pressure ulcers, OPSBs use a two-point probe configura-tion, rather than the theoretically more accurate, four-point probeconfiguration. This approach is taken due to the lack of homogeneity inthe impedance of the skin and the non-ideal connections between theelectrodes and the user; an inherent complication while measuringimpedance on complex biological tissues. The strategy to integrateomniphobic paper-based sensors in commercially available dressings is,however, versatile, applicable to other biosensors and, with furtherdevelopment, will be able to expand the sensing repertoire of currentsmart bandages to monitor the healing process of chronic wounds.

Acknowledgements

The authors gratefully acknowledge start-up funding from PurdueUniversity. The authors also acknowledge the funding from Procter &Gamble (Grant number 209621). D. G. thanks the Ross Fellowshipprogram at Purdue University for providing partial support of his work.B. C. gratefully acknowledges support from the Alfonso MartinEscudero Foundation.

Appendix A. Supporting information

Supplementary data associated with this article can be found in theonline version at doi:10.1016/j.bios.2018.06.060.

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