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. 2025 Jun 10;5(4):520–528. doi: 10.1021/acsmeasuresciau.5c00033

Reproducibly Modified Elastin-like Polymer Gold Electrode Surfaces

Stanley Feeney †, Marissa Morales †, Galen Arnold ‡, Wynter Paiva ‡,§, Eva Rose M Balog ‡, Jeffrey Mark Halpern †,*
PMCID: PMC12371586  PMID: 40861911

Abstract

Elastin-like polymers (ELPs) have been used for a variety of biomedical applications, including drug delivery and tissue scaffolding. ELPs are useful due to their adjustable lower critical solution temperature and tunable structure for different applications. However, despite ample characterization of ELPs in aqueous solutions, the characterization of ELPs on surfaces is less well explored. For example, sources of inconsistency in ELP modification to surfaces have yet to be explored in detail. Surface modifications of large macromolecules often suffer from poor reproducibility and inconsistent measurements. We developed and optimized a method for modifying a gold electrode surface with ELPs using a thiol-gold interaction through a single cysteine residue near the N-terminus. The modification parameters were tuned for reproducible charge-transfer resistance of the surface, as measured by electrochemical impedance spectroscopy. The final optimized surface modification parameters, without dimethyl sulfoxide or other cosurfactant treatment, are 0.0125 mg/mL ELP for 30 min at 4 °C in 3.5 mM TCEP in ultrahigh-purity water at pH 7.4. The relative amount of cysteine modified to gold versus ELP solution concentration was determined via thiol reduction. Using these data, the source of poor reproducibility was confirmed to be nonspecific polymer interactions.

Keywords: elastin-like polymers, surface reproducibility, thiol modifications, electrochemistry, electrochemical impedance spectroscopy, surface optimization

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Introduction

Elastin-like polymers (ELPs) are genetically encoded, intrinsically disordered polypeptides that exhibit behavior closely resembling that of the naturally occurring protein, elastin. ELPs are classed as thermoresponsive polymers with a lower critical solution temperature (LCST), above which the polymer condenses and becomes insoluble and below which it dissolves in solution. Because of their biological nature and ability to be expressed and produced recombinantly in various organisms, ELPs are biocompatible, tunable, and benefit from monodisperse formulations, making them suitable to a wide variety of applications. − ELPs are extensively studied for their applications in aqueous solution due to the stimulus-response behavior, − but there is interest in developing surface attachment strategies for ELPs. − For example, ELPs in a surface-attached state have been explored for their potential in biosensing applications. − Initial reports suggest strategies such as applied electric field to impact surface mass loading and polymer orientation. , Thermoresponsive films have applications in many fields, such as wound dressings, temperature-sensitive coatings, and tissue scaffolding ,, and we expect surface-attached ELPs could be used in any field that takes advantage of thermoresponsive polymer films.

Thiol-gold modifications were primarily used in this work to bind ELP to electrochemical surfaces because of its commonly used and readily available chemistry. Two other common surface modification methods include electrochemical grafting of diazonium salts and N-heterocyclic carbene (NHC) modification. , Electrochemical grafting of diazonium salts relies on the transfer of electrons from the gold substrate to the diazonium cation, resulting in a very stable covalent bond. , Reproducibility of this modification is generally very good, with some variation of functional groups and applied voltage. , The downside to electrochemical diazonium modifications is safety hazards, including the instability of the diazonium salt and the common use of hazardous organic solvents. Another modification is the self-assembly of N-heterocyclic carbene (NHS) monolayers by strong σ-bonds to transition metals. NHC modification provides enhanced stability, especially in the presence of biofluids, where competitive inhibition by exogenous thiols can displace the thiols used in modification. The reproducibility of NHC modification can vary greatly depending on temperature, atmosphere, and deposition solution.

Consistent surfaces are particularly important because of how the structure dictates the properties of thermoresponsive polymer surfaces. For example, the thickness of layered poly­(N-isopropylacrylamide) (pNIPAM), a common, thermoresponsive synthetic ELP analog, has been shown to affect the adhesion of cells. , Surface attachment methods for pNIPAM by physical adsorption have resulted in poor stability and irreversibility due to weak connections to the substrate. ,

Ensuring a consistent surface has an impact on product performance for surfaces used in the wound dressing, tissue scaffolding, and biosensing industries. − A crucial step in developing a reproducible polymer surface is identifying the potential sources of poor reproducibility based on the properties of the polymer. Often, one difficulty hindering the translation of polymer-based electrodes from the laboratory (i.e., benchtop) environments into the field is due to the consistent fabrication of the surface. In this paper, we explored the generation of reproducible ELP surfaces by investigating the physisorption of ELP, the use of organic solvents, and modification protocols. Often, issues with reproducibility of thermoresponsive polymer surfaces can be attributed to nonspecific adsorption or environment-induced structural changes. , We attach ELP to gold surfaces via cysteine-containing ELP molecules, using isoleucine as a guest residue and a 40-repeat sequence polymer, denoted as I40. In the context of this paper, reproducibility is defined as the generation of the surface with a consistent charge-transfer resistance obtained by electrochemical impedance spectroscopy (EIS); a Randles circuit model can be applied to redox-mediated impedance spectroscopy data to obtain the charge-transfer resistance. − Additionally, we used cyclic voltammetry (CV) to induce thiol-gold bond cleavage to investigate the role of physisorbed ELPs as a source of poor reproducibility issues for generating surface-attached ELP gold electrodes. We believe that improvement in the reproducibility of these types of surfaces will increase their relevancy in their intended applications.

Experimental Section

Materials and Equipment

I40 was generated by the Balog Lab at the University of New England. Potassium ferricyanide and potassium ferrocyanide for the redox couple were obtained from Acros Organics. Potassium hydroxide (KOH) for CV stripping experiments was obtained from Alfa Aesar. Tris­(2-carboxyethyl)­phosphine HCl for surface modification was obtained from Thermo Scientific. Sodium hydroxide (NaOH) used for pH balancing was obtained from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) used for the treatment of modified electrodes was obtained from Fisher Chemical. Ultrahigh-purity deionized (UHP DI) water was generated with a Direct-Q 3 UV-R Ultra Pure (Type 1) water purification system obtained from Milli-Q.

Reference 600+ Potentiostat/Galvanostat/ZRA and the VistaShield Faraday Cage used for electrochemical measurements were obtained from Gamry Instruments. Electrochemical cells, 1.6 mm gold rod electrodes, Ag/AgCl reference electrodes, and platinum wire counter electrodes used in the electrochemical measurements were obtained from BASi Research Products. XP6 Microbalance was obtained from Mettler Toledo.

ELP Synthesis and Design

The I40 was designed and synthesized with a core structure consisting of 40 repeats of the pentapeptide VPGIG and a cysteine residue near the N-terminus to provide the thiol group for surface immobilization. The I40 was designed and synthesized as previously described. Briefly, the plasmid POE-W I40 was transformed into BL21­(DE3) E. coli and plated on 2XYT solid medium + carbenicillin. Starter cultures of nutrient-rich liquid medium + carbenicillin were inoculated with multiple colonies and shaken at 200 rpm at 37 °C for 2–4 h until visible growth was detected. Cultures were transferred to 1 L volumes of the same media in 2 L flasks, which were then shaken at 200 rpm at 37 °C for 24 h. Cells were harvested by centrifugation, and I40 was purified from the periplasmic fraction using inverse transition cycling. Purified I40 was lyophilized for long-term storage at −20 °C. Our full protocol used in the expression and purification of I40 is available on protocols.io (https://dx.doi.org/10.17504/protocols.io.5jyl8npw6l2w/v2).

ELP Surface-Immobilization

Prior to modification with I40, the 1.6 mm gold rod electrode was polished with a 3 μm diamond slurry, followed by 1 μm diamond slurry rinsing with methanol and UHP DI water. A final polish was done with a 0.55 μm alumina slurry. The electrode was rinsed thoroughly with UHP DI water for approximately 30 s before immersing in a prechilled polymer solution of I40 ELP in 3.5 mM TCEP balanced with NaOH to pH 7.4 at T = 4 °C and dissolved in UHP DI water. TCEP was used as a reducing agent to prevent the formation of disulfide bonds. A temperature of 4 °C was chosen, as this is below the LCST of I40 ELPs under these conditions and prevents the formation of coacervates that would impede surface modification. I40 ELPs were dissolved in TCEP in a refrigerator at 4 °C to reach final concentrations of 0.00625, 0.0125, 0.025, 0.035, and 0.05 mg/mL for 10 min, with occasional light vortexing. The gold electrode was submerged in 1 mL of the ELP solution at 4 °C. The electrode was then removed from the solution and rinsed with UHP DI water for 30 s and immediately transferred to the redox couple solution to measure the impedance response using EIS. This procedure is tabulated in Table .

1. Summarizing the Steps for Modification of Cysteine-Terminated ELPs to a Gold Electrode.

step # process time additional parameters
1 3 μm diamond polishing 1 min  
2 1 μm diamond polishing 1 min  
3 0.55 μm alumina polishing 1 min  
4 DI water rinse 0.5 min  
5 electrode incubation variable ELP in 0.5 mM TCEP, pH 7.4, 4 °C, aqueous
6 DI water rinse 0.5 min 30 s
7 electrochemical measurement: EIS ∼6 min 5 mM ferri/ferrocyanide, 0.5 M KCl, aqueous
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Electrochemical Measurements

All EIS measurements were performed at the open circuit potential over a frequency range of 100,000–0.05 Hz. A three-electrode array was used: (1) gold working electrode, (2) platinum wire counter electrode, and (3) Ag/AgCl reference electrode; the cell was purged with nitrogen for 10 min prior to adding the working electrode. Prior to all EIS measurements, the working electrode was rinsed with UHP DI water. A redox couple solution of 5 mM ferri/ferrocyanide [Fe­(CN)6]3–/4– with a 0.5 M potassium chloride (KCl) supporting electrolyte in UHP DI water was used at room temperature.

For CV experiments, the electrodes were rinsed with UHP DI water and moved to a solution of aqueous 0.5 M KOH solution that was purged for 10 min. CV was run with an initial voltage of 0 V vs Ag/AgCl and a scan rate of 100 mV/s from 0 to −1.4 V vs Ag/AgCl.

A Randle’s equivalent circuit model was used to fit the impedance data using Gamry EChem Analyst software (Figure ) to describe the combination of kinetic and diffusion processes contributing to the total impedance response. The Randle’s circuit is composed of resistive and capacitive components, including the solution resistance (R s) which corresponds to the uncompensated resistance of the bulk electrolyte, charge-transfer resistance (R ct) which corresponds to the resistance to the flow of electrons due to kinetic reduction and oxidation reactions, Warburg impedance (W) which corresponds to the semi-infinite diffusion, and a constant phase element (CPE) which corresponds to the dielectric layer and at the surface of the electrode. The Randle’s circuit was chosen so that the impedance caused by the addition of ELPs to the electrode could be interpreted by redox components.

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Randle’s circuit is used to quantify solution resistance (R s), charge-transfer resistance (R ct), Warburg impedance (W), and double-layer capacitance (C dl) as components of the impedance response. Image generated with a Gamry Echem Analyst.

All statistical comparisons were made using the Student’s t test. Student’s t test was used due to the assumption of a normal distribution of test variables.

Results and Discussion

Modification Strategies

Charge-transfer resistance is known to increase when proteins are added to the electrode surface. We expect an increase in charge-transfer resistance after I40 modification compared to unmodified electrodes because of the formation of an insulating I40 layer on the electrode surface. , We polished two electrodes and generated an impedimetric charge-transfer resistance value of ∼150 Ω. The low charge-transfer resistance indicates unrestricted access to the electrode surface by the redox couple. Two electrodes were modified with I40 for 24 h in our ELP modification solution. Charge-transfer resistance values of ∼42,000 and ∼26,000 Ω were recorded from two unique electrodes, indicating variability in the modification events (Figure ). We propose that the poor reproducibility of the impedance response at modification times longer than 30 min is influenced by this nonspecific adsorption. The increase in charge-transfer resistance includes ELPs attached via nonspecific adsorption due to electrostatic and van der Waals forces as well as ELPs attached via thiol-gold interactions (Figure ).

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Impedance response of an unmodified electrode (∼150 Ω) compared to two electrodes modified with 0.05 mg/mL I40 for t = 24 h (∼42,000 and ∼26,000 Ω) indicating poor reproducibility in the surface immobilization of I40. Bode plots are found in Figure S1.

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We hypothesized nonspecific interactions between surface-immobilized and solvated ELPs hindered the thiol-gold interaction during surface immobilization, contributing to the variability in the impedance response observed.

Incubation times and I40 concentrations were varied, with an expected change to intermolecular interactions and nonspecific adsorption, influencing the impedance response (Figure ). The concentration was varied between 0.0125, 0.025, and 0.05 mg/mL. We expected that a lower concentration of I40 would help to prevent intermolecular interactions hindering reproducibility caused by ELP aggregation or clustering during modification. Impedance is proportional to the concentration of I40, indicating less electrode surface area coverage with a lower concentration of I40. The incubation time was varied between 30 and 60 min. Likewise, we expected lower incubation times to decrease the nonspecific adsorption of ELP since the thiol-gold interaction between the cysteine and gold electrode would be faster than the nonspecific adsorption interactions. Essentially, lower incubation time provides enough time for the thiol-gold interaction to occur but less time for nonspecific adsorption to occur. As expected, lowering both the incubation time and the modification concentration resulted in modifications with lower impedances. We further investigated if the lower impedance resulted in less surface modification or less physisorbed ELP binding.

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Impedance response of electrodes modified with varying concentrations of I40 with a charge-transfer resistance value of ∼42,000 Ω for 0.05 mg/mL, ∼31,000 Ω for 0.025 mg/mL, and ∼22,750 Ω for 0.0125 mg/mL for 60 min modifications. A decreasing trend of charge-transfer resistance with I40 concentration was observed, indicating lower concentrations of I40 achieve lower surface area coverage. Bode plots are found in Figure S2.

Surface Treatment with DMSO

Following surface immobilization of I40, the modified electrodes were treated with a solvent (DMSO) to remove nonspecifically bound I40 from the electrode surface. DMSO should solvate and release nonspecifically bound I40 from the surface, while I40 immobilized via the thiol-gold bond should remain. The impedance response from two electrodes modified with 0.05 mg/mL I40 in 3.5 mM TCEP pH = 7.4 T = 4 °C for t = 24 h was recorded before solvent treatment. The impedance responses from the I40 modified electrodes were recorded after solvent treatment for 24 h (Figure A). For electrode one, an initial, untreated impedance response of 26,000 Ω was observed, followed by a decrease in charge-transfer resistance to 19,000 Ω after 24 h in DMSO. Electrode 2, modified with I40 under the same conditions, had an initial untreated impedance response of 43,000 Ω, followed by a decrease in charge-transfer resistance to 18,500 Ω after 24 h in DMSO. Before treatment, electrodes 1 and 2 had a difference of 17,000 Ω, while after treatment, the difference in charge-transfer resistance is reduced to 500 Ω. The decrease in charge-transfer resistance of the I40 modified electrode after soaking in DMSO indicates an increase in the available electrode surface area, implying the removal of ELPs from the surface. The ELPs removed from the surface would be those with the lowest binding affinity; namely, nonspecifically adsorbed ELPs. After the 24 h soak in DMSO, both electrodes have similar charge-transfer resistance values, indicating a probable shift in nonspecific physisorption that occurs with a charge-transfer resistance around 20,000 Ω.

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(A) Impedance response from I40 modified electrodes before and after treatment with DMSO with an average charge-transfer resistance response after treatment of ∼18,000 Ω. The reduced charge-transfer resistance indicates removal of some of the ELPs from the surface. (B) Impedance response after tuning reaction parameters from three unique modification events with an average charge-transfer resistance value of ∼14,700 Ω and the impedance response from two DMSO-treated electrodes. The improved reproducibility of the modified electrodes confirms that tuning of the modification procedure was successful. Bode plots are found in Figure S3A,B.

The impedance responses from (N = 3) unique I40 modification events at 0.0125 mg/mL in 3.5 mm TCEP for 30 min were compared to each other and to the results of the DMSO-treated surfaces in Figure . Decreasing the modification time to 0.5 h and tuning the modification concentration to 0.0125 mg/mL, it was observed that the charge-transfer resistance is much more reproducible. Because the DMSO-treated surfaces have been stripped of nonspecifically adsorbed I40 and because the tuned modification protocol produces charge-transfer resistance values close to the DMSO-treated ones, we hypothesize that the tuned modification surface has a similar surface coverage to the DMSO-treated surface. Hence, the reproducible modification of a gold electrode with I40 is successful with a modification concentration of 0.0125 mg/mL and a modification time of 30 min without the need for DMSO treatment. However, a further investigation into the nonspecific adsorption of ELP was necessary to explore the hypothesis that nonspecifically adsorbed ELPs were the cause of poor reproducibility.

Analysis of Chemisorption with CV

A higher charge-transfer resistance suggests the presence of ELPs on the surface of the gold, but it does not differentiate between chemisorbed and nonspecifically adsorbed ELPs. The charge-transfer resistance obtained by EIS directly after modification is related to the ELP concentration used in the modification (Figure ). The increasing trend shows that the amount of total ELPs adsorbed to the surface (both chemisorbed and nonspecifically adsorbed) is proportional to the modification concentration. The 0.05 mg/mL concentration has a much higher error bar than the others; this is likely due to a significant contribution of nonspecifically bound ELP to the total charge-transfer resistance.

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Charge-transfer resistance values of electrodes modified with varying concentrations of I40 ELP. Larger standard deviations at higher modification concentrations indicate worse reproducibility at those concentrations.

CV experiments were performed in the presence of 0.5 M KOH to observe the amount of thiol attachment for each modification concentration. CV peak analysis can be used to determine the amount of thiol-terminated molecules chemisorbed to gold electrodes. − The CV causes the reduction of thiol bonds attached to gold, decoupling the thiol-bonded ELP from the gold surface. Two peaks appeared in the cyclic voltammograms of ELP-modified electrodes, labeled A and B (Figure ). Peak A disappears in repeated voltage sweeps, indicating that the electron transfer corresponding to peak A is irreversible. Additionally, unlike Peak B, Peak A does not appear in bare electrodes; note that Peak B is shifted from the bare electrode to the modified electrodes. Peaks at voltages around −0.8 V vs Ag/AgCl, like the ones seen here, have been linked to thiol decoupling in the past. ,

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Cyclic voltammetry response of I40 ELP-modified gold electrodes at different concentrations. The inset shows Peak A, the peak associated with the decoupling of the thiol.

Peak area values are normalized with respect to a baseline (Figure S4). The value of Peak B also does not change with the modification concentration (Figure S5). This supports the claim that Peak A corresponds to the thiol decoupling from the gold surface. Therefore, we expect the area under the decoupling peak (Peak A), or the charge, is directly related to the electrons transferred and can be used to determine the relative amount of chemisorbed polymer.

The CV results were used to examine the trend in the amount of chemisorbed ELP at the different modification concentrations. The peak area values of Peak A were compared for different modification concentrations (Figure ). The initial downward trend of charge versus modification concentration suggests that there is some direct relationship between chemisorbed ELP and modification concentration. However, there is not a statistically significant difference between the peak area values of different modification concentrations. The total charge-transfer resistance, and therefore, the total ELP on the surface, increases according to Figure , but the amount of chemisorbed ELPs does not significantly change according to Figure . Hence, nonspecifically adsorbed ELPs must be increasing instead. Therefore, the hypothesis that nonspecific adsorption contributes to the charge-transfer resistance more than chemisorption is supported.

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Charge values of Peak A from the CV of electrodes modified with different concentrations. The values plotted here are in comparison to a baseline drawn from the anodic to the cathodic side of Peak A to normalize across potential electrode differences. Nonaveraged raw data are available in Figure S6.

EIS was run before and after the CV sweep to ensure that the surface was successfully modified and that the CV procedure results in expected thiol decoupling. The Nyquist plots change after CV is performed (Figure ). The sharp decrease in the resistance, indicated by the change in diameter of the semicircle of the Nyquist plot, indicates that the thiol decoupling was successful. A control experiment was performed by measuring the impedance of a modified electrode after a KOH soak without applying a voltage. This did not result in a decrease of charge-transfer resistance, indicating that ELP is being decoupled by CV and not by KOH alone.

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Electrode modified with 0.035 mg/mL I40 ELP (chosen randomly from modification concentrations above the tuned 0.0125 mg/mL concentration) for 30 min. Effects of the electrode being soaked in KOH without cyclic voltammetry as well as ELP being stripped from the surface with cyclic voltammetry in KOH are shown. Low charge-transfer resistance after KOH stripping indicates that the ELPs were removed from the surface, while no decrease after soaking in KOH indicates that CV is required for the ELP stripping. Bode plots are found in Figure S7.

CV was also performed on electrodes modified with 0.05 mg/mL of I40 ELP that were then treated with DMSO for 24 h. It was found that treatment with DMSO improved reproducibility from a relative standard deviation (σ/μ) of 42.3%RSD to 28.3%RSD for charge-transfer resistance and also increased the area under peak A by 835% to 1395 nC, indicating further chemisorption (Figure ). The improved reproducibility is possibly due to nonspecifically bound ELP molecules desorbing from the surface, making them available for further chemisorption in the organic solvent. The decrease in charge-transfer resistance from DMSO treatment as seen in both Figure and Figure would be consistent with the hypothesis that nonspecifically adsorbed ELPs contribute more to charge-transfer resistance than gold–thiol chemisorbed ELPs. Charge-transfer resistance is not proportional to the amount of surface modified by the cysteine of these ELPs; we hypothesize that greater charge-transfer resistance can be due to greater physisorption of ELP islands compared with a more chemisorbed surface (Figure ).

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Charge-transfer resistance and Peak A charge values for electrodes modified with 0.05 mg/mL ELP concentration for 30 min without and with DMSO for 24 h. The decrease in charge-transfer resistance after DMSO soaking (p < 0.1) suggests the overall surface coverage decreases after treatment, while an increase in Peak A area after DMSO soaking (p < 0.001) suggests that nonspecifically adsorbed ELPs may be able to reattach to the surface via gold–thiol interaction during treatment.

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In addition to Figure , where we hypothesize that charge-transfer resistance is directly proportional to I40 modified to a surface, we now also hypothesize that nonspecifically adsorbed I40 contributes more to overall charge-transfer resistance than thiol-bonded chemisorbed I40.

Conclusions

Reproducibility of ELP-modified surfaces was greatly improved through adjustment of the modification parameters. This was done by reducing the concentration of the I40 ELP used in the modification and reducing the modification time. In order to better understand the nature of poor reproducibility at higher modification concentrations, electrochemical stripping of the modified electrodes was performed by CV. The poor reproducibility was likely due to nonspecific adsorption of ELP molecules. In addition to removing nonspecifically adsorbed ELP, we hypothesize that the application of a DMSO treatment increased levels of chemisorption compared to nontreated surfaces, leading to a larger overall charge-transfer resistance. The charge-transfer resistance of the DMSO-treated surface was used as a benchmark to find the ideal modification concentration and time of 0.0125 mg/mL and 30 min for surfaces that can only be exposed to aqueous media. However, the non-DMSO-treated surface is likely to still have nonspecifically adsorbed ELP. This indicates that DMSO treatment may be used as an additional step in the modification process for surfaces that can be exposed to nonaqueous solvents. Broadly, this work suggests that reducing both the modification concentration and modification time and implementation of solvent treatments can improve reproducibility in thiol-gold surfaces that are prone to physisorption. As the reproducibility of these surfaces improves, their reliable application to industry becomes more feasible.

Supplementary Material

tg5c00033_si_001.pdf (250.5KB, pdf)

Acknowledgments

Support was provided by a National Science Foundation (NSF) EPSCoR award BIO-SENS (#2119237), NSF Eager CBET Award (#1638896 and #1638893), the University of New Hampshire’s Center for Integrated Biomedical and Bioengineering Research (CIBBR) through a grant from the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM113131, and Summer Undergraduate Research Experience fellowships provided by the University of New England’s College of Arts and Sciences. We also thank Laura Marvin for the production of a portion of the I40.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmeasuresciau.5c00033.

  • Additional experimental details, including nonaveraged data, further explanation of data analysis, and Bode plots (PDF)

∥.

S.F. and M.M. contributed equally to this work. CRediT: Stanley Feeney data curation, formal analysis, investigation, methodology, validation, visualization, writing - original draft, writing - review & editing; Marissa Morales conceptualization, formal analysis, investigation, methodology, writing - original draft; Galen Arnold resources; Wynter Paiva resources, validation; Eva Rose Murdock Balog resources, supervision, validation, writing - review & editing; Jeffrey Mark Halpern conceptualization, data curation, formal analysis, funding acquisition, project administration, resources, supervision, writing - review & editing.

The authors declare no competing financial interest.

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