Introduction

The largest organ of the human body, skin, acts as the primary defense against environmental insults—thus its integrity is of great importance to human health. When the skin becomes compromised, wound healing proceeds through four phases (bleeding and hemostasis; inflammation; cell proliferation; and remodeling) in a coordinated and timely manner1,2,3,4,5,6,7,8. A component of the wound healing process occurs during the inflammatory phase where reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are generated which target potentially pathogenic microorganisms9,10.

Chronic wounds, affecting ~2% of the United States population annually, arise when wound healing fails to efficiently complete11. Chronic wounds can affect healthy individuals but disproportionally impact those with reduced skin barrier function (e.g., aged skin, diabetes mellitus)11. A prolonged state of tissue disrepair can be further complicated with infection, particularly by microorganisms that can overcome traditional antibiotic therapy through genetically acquired resistance and/or biofilm-associated tolerance. Antibiotic alternatives, such as débridement or topical wound dressings (e.g., hydrogels, silver-based, sponges) can be used12, but many chronic wounds require prolonged treatment and, in some cases, result in permanently compromised functionality (e.g., limb amputation in the case of diabetic foot ulcers).

H2O2 is a biocide naturally generated during the wound healing process and has broad-spectrum activity and multiple mechanisms of action. For example, myeloperoxidase can bind H2O2 as a substrate which ultimately damages amino acid side chains and DNA; H2O2 also directly participates in Fenton chemistry which can oxidize lipids, proteins, and nucleic acids7,13,14,15,16. Therefore, implementing H2O2 as a wound infection prevention and treatment strateg, combined with a wound healing facilitation strategy, requires controlled, precise delivery6,17,18. Bulk application of H2O2 during wound débridement is done to overcome fast biocide degradation, but high initial H2O2 concentrations may be harmful to tissues. Considering this, there is a need for technologies that provide H2O2 at non-toxic concentrations for prolonged periods. Our group has developed a H2O2 producing electrochemical bandage (e-bandage), which generates continuous micromolar concentrations of H2O2 over extended periods of time19,20,21,22,23,24.

The 1.77 cm2 e-bandage is composed of carbon fabric layers separated by cotton fabric, with a silver/silver chloride (Ag/AgCl) wire serving as a quasi-reference electrode placed between cotton fabric layers. The first layer of carbon fabric (here termed working electrode) is covered by hydrogel and contacts the wounded skin surface, generating H2O2 by oxygen reduction (O2 + 2H+ + 2e− → H2O2) when polarized to −0.6 VAg/AgCl. O2 is delivered to the working electrode surface from air. A second layer of carbon fabric (here termed counter electrode) has positive polarity which allows generation of hydrogen ions (H+) and electrons (e−). Sodium chloride (NaCl) containing hydrogel embedded in the e-bandage serves as an electrolyte supporting electrochemical reactions with NaCl while enhancing ionic conductivity of the hydrogel. The ability of an e-bandage to generate H2O2 for a given period is controlled by a lightweight micropotentiostat19,20,21,22,23,24. The 1.77 cm2 e-bandage has demonstrated in vitro, ex vivo, and in vivo (in mice) efficacy for infection prevention and/or treatment against bacterial and fungal biofilms (single-, dual-, or tri-species)19,20,21,22,23,24. The current study advances this technology closer to the clinic by analyzing (1) shelf-life efficacy of ready-to-use 1.77 cm2 e-bandages; and (2) 1.77 cm2 e-bandage safety on the skin of healthy adults for 24 h with either no H2O2 being produced or H2O2 produced for 3, 6, 12, or 24 h. Results indicate retention of biocidal efficacy of ready-to-use 1.77 cm2 e-bandages for at least one month and demonstrate tolerability of 1.77 cm2 e-bandages in humans.

Materials and methods

The study was designed and conducted under the oversight of Dr. Robin Patel; neither the specified funding source nor patients nor the public were involved in the process. The study was approved by the Mayo Clinic Institutional Review Board (IRB; protocol #23–003805). This trial was registered with ClinicalTrials.gov under the identifying number NCT05940207 (https://clinicaltrials.gov/study/NCT05940207?term=23-003805&rank=1) on 11/07/2023 (first posted date), where the full trial protocol is publicly available. All experiments involving humans were performed in accordance with institutional and federal guidelines. De-identified study subject information, participant group assignment, and raw data are provided in Supplementary Data S1. This study was a dose-escalation trial with sequential cohort design investigating safety of a 1.77 cm2 H2O2-generating e-bandage,19,20,21,22,23,24 polarized for 0, 3, 6, 12, and 24 h on healthy human skin.

Device construction, sterilization, and packaging

The e-bandage has been previously described19,20,21,22,23,24. Ready-to-use 1.77 cm2 e-bandages were assembled and sterilized in Dr. Haluk Beyenal’s laboratory at Washington State University before being shipped to Mayo Clinic at Rochester, MN. The e-bandage has a working electrode made of carbon fabric (Panex, fuelcellstore.com) and a counter electrode made of carbon fabric with a porous carbon layer on both sides (Elat®, fuelcellstore.com) whose surface measured 1.77 cm2. Working and counter electrodes were separated by two layers of cotton fabric (Kona®, michaels.com, catalog number 10736967)—embedded within these cotton fabric layers wass a AgCl coated Ag wire (ThermoScientific, catalog number 041455.H4) that served as a quasi-reference electrode. The exposed portion of the Ag/AgCl wire was protected with PTFE tubing to prevent mechanical failure (Component Supply Company, componentsupplycompany.com, catalog #STT-26-C). An additional layer of cotton fabric was placed over the counter electrode to enhance moisture retention. e-Bandage layers were secured by applying a light layer of silicon adhesive (GE, Amazon.com, catalog #GE312A) to the outer edges. Electrical connections to the working and counter electrodes were established by pressing titanium (Ti) wires (TEMCo, Amazon.com, catalog #RW0524) onto the carbon fabric using nylon sew on caps (Dritz, Spartanburg, SC, item#85), creating a link between the e-bandage and micropotentiostat. To bolster attachment of the titanium and Ag/AgCl wires, a 5-minute epoxy resin (Permatex, Amazon.com, model #84101) was applied to the snaps and the edge of the cotton fabric where it met the PTFE tubing. Then, the e-bandage was left to dry for 24 h to allow the silicone and epoxy to cure.

Once built, e-bandages were placed in Mylar foil pouches (QQ Studio, qq-studio.com, catalog #1101–081216) with the e-bandage-micropotentiostat connection port near the closed end of the pouch. The pouch was sealed just below the port that connects to the e-bandage and silicone applied to any unsealed openings to prevent hydrogel leakage into the port (to avoid corrosion of this component). Once the silicone was cured for 24 h, 5 mL of 3 M Tegaderm hydrogel (Solventum; 3M ID: 7100246374) containing 0.9% NaCl was dispensed into the pouch such that hydrogel covered the front and back of the e-bandage. Packaged e-bandages were wrapped with a layer of paper towel and aluminum foil and sterilized at 121 °C for 30 min. After autoclaving, the open end of the pouch was sealed thrice using a heat impulse sealer. The e-bandage and its sterilization process are shown in Supplementary Figure S1a. Note that for the first three participants (i.e., HP19, HP4, HP1), the e-bandage had excess Ti wire material protruding from the snaps; these were removed for the remaining participants as they led to a slight abrasion to the skin of HP1—the modification is shown in Supplementary Figure S2.

The programmable wearable micropotentiostat design and functionality has been described20,24. Here, the micropotentiostat was programmed to maintain operational potential of the working electrode at −0.6 VAg/AgCl for 0, 3, 6, 12, or 24 h, starting immediately after the device was switched on. After polarization ended, the micropotentiostat was programmed to not apply operational potential to e-bandage. In other words, placing the switch in the ON position enabled the programmed sequence to operate. If the micropotentiostat was programmed for 0 h, no H₂O₂ would be generated (but the micropotentiostat would be turned ON). Conversely, if it was programmed to generate H₂O₂ for 3 out of 24 h, then upon turning the switch ON, H₂O₂ was generated during the first 3 h, after which polarization ceased for the remaining 21 h. These operations were controlled by a microprocessor on the micropotentiostat. Thus, micropotentiostats were designed to drive electrochemical generation of H2O2 at the e-bandage working electrode surface immediately after being turned on and to stop electrochemical H2O2 generation after the specified time (i.e., 0, 3, 6, 12, or 24 h). Power was supplied to the micropotentiostat with a Medcell® 2032 lithium coin battery (Medline). To protect both participants’ skin from micropotentiostat-induced skin irritation and micropotentiostats from water contact, micropotentiostats were enclosed in a 3D printed case made of a medical grade polycarbonate filament (Lattice Services, Loos, France, Reference: IFU-BOB-PC-0002).

To sterilize micropotentiostats and their cases (Supplementary Figure S1b), components were placed on aluminum foil, the case was opened, and clear tape was attached with a small part of the tape extending out, acting as an “arm”. The micropotentiostat was placed directly on the foil with no tape. Both the micropotentiostat and case were sterilized using ultraviolet (UV) light for 10 min before being flipped (the case was flipped using the tape “arm”, while the micropotentiostat was flipped using sterile tweezers) and UV sterilized for another 10 min. Then, the micropotentiostat was enclosed in the case (using a sterile tweezer) and the case transferred to a Mylar foil pouch that was closed and labeled. Once e-bandages and micropotentiostats were sterilized, packaged, and labeled (using an IRB-approved labeling format), they were sent to Mayo Clinic for use in the shelf life study and safety trials.

Shelf-life study

An outline of the shelf-life study method is provided in Supplementary Figure S3a. A day-old polycarbonate biofilm method, akin to established in vitro biofilm protocols25,26, was utilized. Briefly, a methicillin-resistant Staphylococcus aureus (MRSA) IDRL-6169 colony was inoculated into 2 mL tryptic soy broth (TSB) and allowed to grow to a density of 0.5 McFarland at 37 °C. Then, 2.5 µL of this 0.5 McFarland culture was spotted onto UV sterilized membranes (Millipore Sigma Catalog Number: WHA10417001) that had been placed on a tryptic soy agar (TSA) plate; after the cell suspension dried on the membrane, the plate was incubated at 37 °C for 24 h. After 24 h, the membrane biofilm was transferred to a new TSA plate. Then, the ready-to-use e-bandage was connected to a battery-loaded micropotentiostat (programmed for 24 h polarization for H2O2 generation) and the working electrode portion of the e-bandage positioned on top of the biofilm while the micropotentiostat rested on the Tegaderm (1¾ in x 1¾ in; manufacturer number 1622W; to avoid micropotentiostat corrosion from agar-derived moisture). Tegaderm (1¾ in x 1¾ in) was placed on top of the e-bandage to retain moisture and sterility. Finally, the micropotentiostat switch was turned ON to initiate polarization. Lids were secured onto the Petri dish using parafilm, and plates incubated at 37 °C for 24 h.

The next day, micropotentiostat switches were turned OFF before removing the Tegaderm™. e-Bandages were removed and membranes individually placed into Falcon® tubes. Residual biofilm was removed from the e-bandage working electrode with 5 mL saline before transferring the saline to the Falcon® tube containing the membrane. The Falcon® tube was then vortexed at maximum speed for 30 s on a Vortex Genie2 (Scientific Industries) before being sonicated at 0.22 ± 0.4 W/cm2, 40 kHz for 5 min (model T400-1, Zenith Ultrasonics Inc). Cells were centrifuged at 5,000 rpm for 10 min. The supernatant was removed, and the cell pellet resuspended in 1 mL saline. The cell resuspension was serially diluted 1:10 in saline and 20 µL of each dilution was spot plated onto TSA. Plates were incubated at 37 °C for 24 h before bacterial enumeration using the following equation: \(\:\frac{CFU}{cm2}=\:\frac{\left(CFU\right)\left(cell\:pellet\:resuspension\:volume\right)}{\left(volume\:plated\right)\left(membrane\:surface\:area\right)}\). In the equation, colony forming units (CFU) is calculated by multiplying the number of colonies counted by the \(\:{10}^{dilution\:factor}\) and the membrane surface area calculated by multiplying the squared membrane radius by ∏. The limit of detection for this assay was 1.01 log10 colony forming units per centimeter squared (CFU/cm2).

Device application to study subject forearm

The study population was drawn from those responding to IRB-approved advertisements posted by Mayo Clinic. Inclusion criteria were as follows: an individual had to be 18 years or older (no exclusions based on race or gender), be in good health and have intact skin on forearms, have no history of Tegaderm intolerance, and be able to provide appropriate consent. Exclusion criteria were as follows: an individual considered part of a vulnerable population (e.g., minor, pregnant, and/or having skin disease), having non-intact skin on forearms and/or having a history of intolerance to Tegaderm™. The first twenty study participants who responded to IRB approved recruitment advertising and fit these criteria were asked for informed consent before participation in the study, with no assent being used. The initial twenty individuals recruited responded to Mayo Clinic advertisements in less than 24 h after the post went up; thereafter, people recruited to replace those who withdrew from the study were recruited, as needed. Those who responded to the advertisement first and met eligibility criteria (outlined above) were asked for informed consent. If they consented, study participants were placed into one of five study groups (i.e., four participants per group) based on their availability. In other words, whoever could sign up earliest was placed into the ongoing study group. The enrollment, consenting, and group assignment activities were completed by the study coordinator, Ms. Susan Holtegaard. In the “De-identified data” tab of Supplementary Data S1, “excluded” individuals recruited by Ms. Holtegaard who withdrew from the study before they could be scheduled for participation in the study (i.e., all participants who withdrew did so before the initiation of visit 1).

Study groups included Group 1: no e-bandage polarization for the complete 24 h of wear; Group 2: e-bandage polarization at −0.6 VAg/AgCl for the first 3 h of wear followed by 21 h of non-polarization; Group 3: e-bandage polarization for the first 6 h of wear followed by 18 h of non-polarization; Group 4: e-bandage polarization for the first 12 h of wear followed by 12 h of non-polarization; Group 5: e-bandage polarization for the complete 24 h of wear. Participants were not informed of their assigned group until after all three visits were completed, at which time that information was provided upon participant request. All study personnel were aware of the groups each participant belonged to.

After consent, each participant completed visits 1 and 2 at the Mayo Clinic Clinical Research and Trials Unit (CRTU), with the third visit completed via telephone. During visit 1, the participant chose which forearm the device (note that the term “device” refers to an e-bandage connected to its partner micropotentiostat) would be secured to. Overall tasks completed at each visit are outlined in Supplementary Figure S3b. The device was secured to the forearm as follows. 1) a layer of Tegaderm (1¾ in x 1¾ in) was secured to the forearm; 2) the case-enclosed micropotentiostat was removed from the Mylar pouch and a Medcell® 2032 lithium coin battery (Medline) inserted; 3) the sterile e-bandage was removed from its Mylar pouch and connected to the micropotentiostat; 4) the device was placed on the forearm with the micropotentiostat resting on the Tegaderm; 5) Tegaderm (1¾ in x 1¾ in) was placed over the e-bandage with a larger piece of Tegaderm (4 in x 4 ¾ in; manufacturer number 1626W) cut in half and placed on top; 6) the micropotentiostat ON/OFF toggle was switched to the ON position; and 7) waterproof skin tape was placed over the case-enclosed micropotentiostat. Photographs of the forearm were taken using an Apple iPad 10th generation (OS 18.1.1) before and after device placement. Each participant was given a box of saran wrap to cover the arm in the event of planned exposure to large amounts of water (e.g., showering).

Twenty-four hours later, each study subject returned to CRTU to complete visit 2. The e-bandage was removed, with photographs of the forearm taken using an Apple iPad 10th generation (OS 18.1.1) before and after device removal. Following device removal, study subjects were asked to verbally report reactions—using a structured grading scale of 1 (none), 2 (mild), 3 (moderate), or 4 (severe)—to device-related discomfort, swelling, local and systemic allergic reaction, skin discoloration, and “other” self-described outcomes. The study operator then recorded study participant responses (Supplementary Data S1). A flow diagram of visit 2 proceedings can be found in Supplementary Figure S4. The grading scale was developed internally and approved by the IRB; the same set of outcome-related questions was asked of each participant (Supplementary Figure S4).

Visit 3 occurred six days following visit 2 as a phone call follow-up where the participant was asked to report and grade any reactions, as described above and in Supplementary Figure S4. After all four study participants in each group had completed their visits, the group’s collective data was assessed to determine if the study should progress to the next level (i.e., increased e-bandage polarization time). A single operator interfaced with participants at all visits to minimize variability and ensure consistency of data collection and interpretation.

Four individuals per group allowed the ability to define several criteria for stopping points. The study would not move on to the next group if two or more participants described a Grade 3 or greater reaction for any category (e.g., discomfort, swelling) or if two or more devices fell off prematurely before removal by the investigators at visit 2.

Statistics

For the bench shelf-life study, normal distribution was first evaluated via Shapiro-Wilks test. Welch’s t-test was used for comparisons with normal distribution in compared groups; the Mann-Whitney U-test was used when one or both compared groups had a non-normal distribution. Several analytical pipelines were implemented to analyze study participant demographic information. For continuous data (i.e., age), the Kruskal-Wallis test was used to evaluate the presence of differences across study groups with an epsilon squared (ε2) value estimating the proportion of variability in age that could be explained by group membership. For categorical data (i.e., race, ethnicity, sex), the Fisher’s-exact test was used to assess for statistical differences across study groups to accommodate for small group sizes. To enhance interpretation of categorical demographics, variables were summarized using percent spread, calculated as the proportion of participants per group within each demographic category. p-values < 0.05 were considered statistically significant. For purely observational outcomes (i.e., photographs), no statistics were applied; thus, any missing data in these areas (e.g., failure to photograph a study subject’s arm after e-bandage placement), did not affect any analytical outcome.

Data visualization and statistical analysis software

Graphs reporting the shelf-life study (and to conduct the corresponding statistical analysis) as well as subjects’ reactions to the devices were generated using GraphPad Prism software version 10.1.1(270). Patient demographic statistics were evaluated in RStudio using base R version 4.5.0 along with effectsize 1.0.1 and rstatix 0.7.2.

Results

Ready-to-use e-bandages retain in vitro efficacy against MRSA biofilms for at least four weeks

The shelf-life study (Fig. 1, top) was performedd to determine whether shipping from Washington State University to Mayo Clinic and four weeks of storage would impact e-bandage activity. Ready-to-use e-bandages and micropotentiostats were made at Washington State University (Supplementary Figure S1) and shipped to Mayo Clinic, Rochester. The shelf-life study was done in parallel at both institutions over the course of four weeks, being initiated upon the receipt of device components at Mayo Clinic (i.e., week 0) and repeated two and four weeks later at both locations. As shown in Fig. 2, the e-bandages decreased the burden of in vitro MRSA IDRL-6169 biofilms. At Mayo Clinic, the average untreated MRSA IDRL-6169 burden at weeks 0, 2, and 4 was 8.82, 9.08, and 8.87 log10 CFU/cm2, respectively. This compares to an average of 8.00, 7.79, and 8.07 log10 CFU/cm2 for biofilms treated with e-bandages for 24 h at weeks 0, 2, and 4 (Fig. 2a). The shelf-life study conducted at Washington State University yielded similar results, with weeks 0, 2, and 4 untreated biofilms producing an average of 9.05, 9.39, and 9.42 log10 CFU/cm2, respectively, whereas e-bandage treated MRSA IDRL-6169 biofilms had an average of 8.32, 8.67, and 8.39 log10 CFU/cm2 (Fig. 2b). The drop in CFU/cm2 aligns with previous observations where e-bandages decreased microbial load in vitro (using the same polycarbonate membrane biofilm method above) as well as ex vivo and in vivo (murine wounds) reduction of microbial burden19,20,21,22,23. Accordingly, these data indicate that the e-bandages retained efficacy over the duration of four weeks, a property not impacted by location or shipping. Ready-to-use e-bandages for the Phase 0/1 trial described below were used within four weeks after being received by Mayo Clinic.

Fig. 1
figure 1

General timeline of shelf-life (light grey) and Phase 0/1 (dark grey) studies. Times needed to complete individual steps, once initiated, are indicated above the timeline, denoted by hourglasses. Additional timeline information is provided in parenthesis within each step. Abbreviations: Washington State University (WSU). Image created with Biorender.com.

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Fig. 2
figure 2

Shelf-life study of e-bandages and micropotentiostats. e-Bandages and micropotentiostats were made at Washington State University and shipped overnight to Mayo Clinic. The shelf-life study was done in parallel at both institutions over four weeks. Experiments were initiated upon receipt of device components at Mayo Clinic (week 0) and repeated two and four weeks later at Mayo Clinic (a) and Washington State University (b). Average initial log10 CFU/cm2 for weeks 0, 2, and 4 timepoints at Mayo Clinic were 8.65, 8.71, and 8.67, respectively; those at Washington State University were 9.02, 8.91, and 8.91, respectively. Statistics: Normal distribution was first evaluated via Shapiro-Wilks. Welch’s t-test was used for comparisons with normal distribution between groups; Mann-Whitney U-test was implemented once for week 4 Mayo Clinic data given the non-normal distribution in the treatment group. p-values < 0.05 were considered significant. The limit of detection was 1.01 log10 CFU/cm2.

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Inactive e-bandages do not adversely affect the skin of health study participants

A summary of the human study is described in Fig. 3, with a detailed study timeline of device usage portrayed in the bottom panel of Fig. 1. Four healthy adults with intact forearm skin had non-polarized e-bandages connected to micropotentiostats (herein referred to as “devices”) secured to their forearm skin—meaning that the micropotentiostat was not programmed to generate any H2O2, even when the switch was placed in the ON position. The first two participants (HP19 and HP4) experienced no device-related discomfort, redness, swelling, local or systemic allergic reaction, or skin discoloration (Fig. 4a). The third participant, HP1, experienced Grade 2 (i.e., minor) discomfort from a slight skin abrasion induced by excess Ti wire protruding from the nylon snaps of the e-bandage (Supplementary Figure S2). The protruding wire was removed for devices used in all remaining study participants, including the last participant in the inactive e-bandage group, HP14. At visit 2, HP14 reported Grade 2 discomfort due to a mild itching across the entire Tegaderm-covered area. During the phone call follow-up (i.e., visit 3), this participant additionally reported Grade 2 redness (described as small red bumps) and specified that the bumps were not solely localized to where the e-bandage had been placed. Red bumps resolved within a day after e-bandage removal. Representative photographs for the non-polar group device placement and removal are reported in Fig. 4b and c, respectively.

Fig. 3
figure 3

 Modified from the updated CONSORT 2025 extension guidelines for reporting randomized pilot and feasibility studies29. Image created with Biorender.com.

Flow diagram of participant enrollment, exclusion, and distribution to study groups. Overarching progress through the phases of this nonrandomized, step-up trial (enrollment, device allocation, follow-up, and data analysis) is shown. Study participants were allocated in order of schedule availability; each group completed all steps from device placement to analysis for all participants in the group before determining whether to advance to the next group.

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Fig. 4
figure 4

Evaluation of non-polarized devices. (a) Results of participant-described grading for device-related reactions to e-bandages. For each self-reported, graded reaction, study participants are represented by the following symbols: open circle (HP19), open square (HP4), open right-side-up triangle (HP1), and open upside-down triangle (HP14). (b-c) Representative photographs of participants’ skin before and after device application (b) and device removal (c); photographs are from HP1. (c) “Cleaned” means excess hydrogel was removed to determine if the skin had been discolored; the black circle shows where a protruding Ti wire slightly abraded the skin.

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e-Bandages polarized for increasing periods of time do not adversely affect healthy skin

For the remaining groups, the micropotentiostat was programmed to be polarized at −0.6 VAg/AgCl for 3, 6, 12, or 24 h (Figs. 5, 6 and 7, and 8, respectively). For the group exposed to 3 h of polarization, two study participants (HP20 and HP13) described Grade 2 discomfort due to minor itching across the Tegaderm-covered area; given the reoccurrence of this type of discomfort, all reports of non-device related discomfort were reported in the “other” category in the remaining study groups. A third participant, HP11, described Grade 2 discomfort at the second visit due to minor itching localized at the device working electrode. All reported Grade 2 discomforts for this group were resolved by the phone call follow-up (Fig. 5a). Notably, upon removing the e-bandage for these four participants, the hydrogel appeared discolored (Fig. 5b)—similar to what was previously observed in mice23. The skin itself was not discolored (Fig. 5c; a study-defined stopping point), however; therefore, the next polarization scheme was investigated.

Fig. 5
figure 5

Polarization for 3 of 24 h. (a) Results of participant-described grading of device-related reactions to e-bandages. For each self-reported, graded reaction, study participants are represented by the following symbols: open circle (HP12), open square (HP11), open right-side-up triangle (HP13), and open upside-down triangle (HP20). (b-c) Representative photographs of participants’ skin before and after device application (b), and after device removal (c); photographs are from HP11. (c) “Cleaned” means excess hydrogel was removed to determine if the skin had been discolored.

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Fig. 6
figure 6

Polarization for 6 of 24 h. (a) Results of participant-described grading for device related reactions to e-bandages. For each self-reported, graded reaction, study participants are represented by the following symbols: open circle (HP3), open square (HP5), and open right-side-up triangle (HP8). Data for the fourth participant, HP10, was excluded due to micropotentiostat failure (Supplementary Figure S5g). (b-c) Representative photographs of participants’ skin before and after device application (b) and device removal (c); photographs are from HP5. (c) “Cleaned” means excess hydrogel was removed to determine if the skin had been discolored.

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Fig. 7
figure 7

Polarization for 12 of 24 h. (a) Results of participant-described grading for device related reactions to e-bandages. For each self-reported, graded reaction, study participants are represented by the following symbols: open circle (HP18), open square (HP7), open right-side-up triangle (HP21), and open upside-down triangle (HP22).(b-c) Representative photographs of participants’ skin before and after device application (b) and device removal (c); photographs are from HP18. (c) “Cleaned” means excess hydrogel was removed to determine if the skin had been discolored.

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Fig. 8
figure 8

Polarization for 24 h. (a) Results of participant-described grading for device related reactions to e-bandages. For each self-reported, graded reaction, study participants are represented by the following symbols: open circle (HP23), open square (HP24), open right-side-up triangle (HP25), and open upside-down triangle (HP26). (b-c) Representative photographs of participants’ skin before and after device application (b) and device removal (c); photographs are from HP24. (c) “Cleaned” means excess hydrogel was removed to determine if the skin had been discolored.

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Three study participants wearing an e-bandage polarized for 6 h did not experience severe side effects (Fig. 6a); the fourth participant in this group, HP10, had a micropotentiostat that experienced technical failure immediately after being turned on (Supplementary Figure S5g); as such, this participant’s data was excluded from the group. With the data from the remaining three participants having no reported device-related issues, a replacement for HP10 was not enrolled. It was noted that hydrogel, but not skin, discoloration occurred in this group, as depicted when comparing Fig. 6b and c. Similar hydrogel discoloration findings were observed upon increasing polarization to 12 and 24 h and participants in these final two study groups reported no reactions to the devices (Figs. 7a-c and 8a-c, respectively). HP18 experienced a Grade 2 “other” mild itch across entire Tegaderm covered area, describing the sensation as “there’s something on my arm and I want to pick at it” (Fig. 7a).

Micropotentiostats involved in this study typically experienced no technical issues, successfully driving the programmed potential of −0.6 VAg/AgCl as indicated in Supplementary Figure S5. In this Figure, the current level—even if fluctuating—is indicative of electrochemical reactions occurring at the e-bandage working electrode surface, where the primary reaction should be H2O2 generation, as demonstrated previously20. Therefore, as long as the current remained in a low but variable range for the duration of the driven − 0.6 VAg/AgCl potential, it is reasonable to conclude that H2O2 is being produced, despite the possible presence of current noise (e.g., Supplementary Figure S5k). Overall, micropotentiostats successfully powered the e-bandages for the programmed duration in all study participants, except subject HP10. This indicates that normal human activities do not disturb the connection between micropotentiostat and its partner e-bandage when placed on the forearm. Notably, no premature device detachment occurred because of normal human activity, such as working out at the gym or showering, indicating a suitable protocol for device attachment to the skin.

Analysis of patient demographics across study groups

Participant demographics per participant are found in Supplementary Table S1. Ages ranged from 23 to 55 years across all groups (Fig. 9a). Within groups, the mean age (with standard deviation) in the non-polarized group was 35.0 ± 11.7 years, while the means and standard deviations for the 3-, 6-, 12-, and 24-hour polarization groups were 24.5 ± 1.5, 40 ± 8.6, 40 ± 9.4, and 33 ± 7.2 years, respectively. Evaluating the effect of age across groups resulted in a p-value of 0.0746; ε2 was 0.301. While the p-value was not significant, the effect size (i.e., ε2) suggests a moderate group effect. This indicates that age distributions may differ meaningfully across groups which could, in turn, impact the outcomes reported. However, the ε2 value should be cautiously interpreted as rank-based effect measures can be unstable in small-sample settings.

Fig. 9
figure 9

Participant demographics by study group. De-identified details regarding study participants (see Supplementary Table S1 for further details) by respective study group by age (a), race (b), ethnicity (c), and sex (d). Group 1 indicates those individuals assigned to 24 h of non-polarized e-bandages, while Groups 2, 3, 4, and 5 are those assigned to the e-bandages polarized for 3, 6, 12, or 24 h, respectively.

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Race had little variance across groups (Fig. 9b). For example, Caucasian/White participants represented 75% in the first two study groups (i.e., non-polarized and 3 h of polarization) and 100% of subjects in the remaining groups (Supplementary Table S2). The remaining 25% in the first two groups were Asian participants. With limited statistical power due to small sample sizes, it is unsurprising there is no statistically significant difference observed for race across groups (p-value = 1).

Similarly, there was little variation in ethnic distribution across groups (Fig. 9c, Supplementary Table S2) with 75% of the participants in the non-polarized and 12 and 24 h polarization groups reporting being non-Hispanic or Latino. The remaining 25% reported being either Hispanic or Latino (non-polarized group) or unknown/not reported (12 and 24 h polarization groups). In the remaining study groups (i.e., 3 and 6 h of polarization) all participants reported being non-Hispanic or Latino. There was no statistically significant difference observed for ethnicity across groups (p-value = 1).

In contrast, there was uneven distribution of sex across study groups. Females represented all participants in the non-polarized as well as the 6 and 12 h polarized groups (Fig. 9d, Supplementary Table S2). In the 24 h polarized group, females constituted 75% and males 25%. The only instance where sex was found to have equal representation occurred in the 3 h polarization group. Overall, there was a statistically significant difference in sex (p-value = 0.0361).

Discussion

With a growing number of individuals afflicted by chronic wounds, there is a need to develop therapeutics that promote the return of wounded skin to its original integrity. The described e-bandage is a potential strategy, being designed to administer H2O2—a naturally occurring agent in the wound bed—for extended durations to promote wound healing and/or prevent and/or treat wound infection. in vitro, ex vivo, and/or preclinical (in mice) efficacy for infection prevention and/or treatment with the e-bandage against clinical bacterial and yeast isolates has been demonstrated19,20,21,22,23,24. The current study sought to initiate advancement of this technology from the bench to clinical studies.

It was first necessary to evaluate what device materials would be in contact with human skin in a clinical setting and define biocompatibility. The e-bandage Elat® electrode material and cotton fabric separators (Supplemental Figure S1a) need to contact the skin for device functionality but had not been assessed for skin tolerance; the micropotentiostat, however, required protection from the skin as its function might be impacted by the presence of moisture such as sweat (Supplementary Figure S1b). With these considerations, micropotentiostats weres placed in biocompatible cases and the study focused primarily on e-bandage biocompatibility. Adults with healthy skin were chosen for this Phase 0/1 clinical trial due to the natural resilience of this organ when undamaged—if the e-bandage irritated healthy skin, it would undoubtedly harm sensitive, wounded skin. Importantly, inactive e-bandages did not evoke any study-limiting adverse events, demonstrating that the material used to build the e-bandages is not harmful to healthy skin under the conditions studied. Future studies should investigate tolerability of the e-bandage material on different skin types as well as on wounds.

The next goal was to confirm that extended exposure to low, micromolar concentrations of electrochemically generated H2O2 would not adversely affect skin. This is a first step given that (i) future wound healing, infection prevention and/or treatment studies with this device will require multiple application days; and (ii) while the H2O2 generated by the e-bandage is within non-toxic ranges, continuous, extended exposure of human skin to this electrochemically generated biocide has not been investigated. Across all study participants, there were no reports or findings suggesting that exposure to electrochemically generated H2O2 negatively impacted the skin itself. Of the participant data included, there was a single report of device-localized discomfort exclusively at the working electrode site; this occurred with polarization for 3h (Fig. 5, HP11). All remaining Grade 2 reports occurred from itchiness and/or redness across the Tegaderm-covered area for e-bandages without (Fig. 4, HP14) and with polarization for 3, 6, and 12 h (Fig. 5 [HP20, HP13], 6 [HP3], and 7 [HP18], respectively); these were considered primarily Tegaderm- rather than device-related outcomes. The e-bandage material itself or, in the polarized groups, the H₂O₂27,28 and/or discolored hydrogel may have contributed to the itchiness and/or redness. Reassuringly, no discomfort, itchiness or redness was reported with 24 h of polarization (Fig. 8), the longest duration studied.

Another observation was e-bandage-associated hydrogel discoloration, observed in all participants in the polarized groups and none in the non-polarized group. Previous murine experiments found e-bandage-related discoloration on the skin of some mice, which was easily removed by ethanol wipes. Affected mice did not display adverse side effects associated with this discoloration23. Results of the murine work, combined with those of the current study, suggest that the discoloration is not associated with harm to mammalian skin thus far. Nevertheless, discoloration is undersireable.

A limitation of this study is the small sample size for each study group (n = 3 for 6 h polarization and n = 4 for the remaining groups). Thus, there was limited power to detect statistically significant variances in participant demographics across groups unless pronounced (e.g., sex). Examination of percent distribution for categorical data indicated an overrepresentation of non-Hispanic white female participants. In a broader context, this likely reflects a participant pool with more homogeneous skin type. Therefore, while the e-bandage and its generated biocide might be safe in the tested context, it would be beneficial to study the safety of this device on individuals with different skin types, who may react differently to the device materials and/or electrochemically produced H2O2. Furthermore, though not statistically different, there were differences in the mean age between groups. For example, the 3 h polarization group had younger participants (24.5 ± 1.5 years) than the 6 and 12 h polarization groups (40.1 ± 8.6 and 40.4 ± 9.4, respectively). Therefore, it is important to consider that age as well as the presence (or lack thereof) of age-associated comorbidities that could impact e-bandage safety. Given the impact age-related comorbidities have on chronic wound healing and sensitivity to materials, future studies should consider age a stratification factor. Another limitation is that, while device function was consistent for this study, usability at other areas of the body was not evaluated. Considering that chronic wounds can occur in anatomical locations that experience significant movement and/or pressure (e.g., lower back, bottom of the foot) and take longer than 24 h to heal, assessing devices in more relevant areas and for longer periods is warranted. Finally, the study used a bespoke questionnaire and skin imaging analysis tools, and the data was not collected and reviewed in a blinded fashion by multiple operators. While these approaches are used to enhance objectivity and reliability of clinical research, the current pilot study sought to minimize variability through review by a single, trained operator. Even though this design choice supports internal consistency, it may limit generalizability and could introduce bias.

Collectively, this study suggests that the e-bandage and its electrochemically generated H2O2 does not incite limiting adverse effects on healthy human skin within the limited assessment performed, and for up to 24 h, motivating the advancement of this topical therapeutic towards future clinical trials.