Electrochemical Fabrication of Functional Gelatin-Based Bioelectronic

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Electrochemical Fabrication of Functional Gelatin-based Bio-electronic Interface Xianghong Peng, Yi Liu, William E Bentley, and Gregory F. Payne Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01491 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016

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Electrochemical Fabrication of Functional Gelatinbased Bio-electronic Interface Xianghong Peng,*,†,‡ Yi Liu, † William E. Bentley, †,§ and Gregory F. Payne*,†,§ †

Institute for Bioscience and Biotechnology Research, University of Maryland, College Park,

MD 20742, United States; §Fischell Department of Bioengineering, University of Maryland, College Park, MD 20742, United States; ‡Key Laboratory of Optoelectronic Chemical Materials and Devices, Ministry of Education, Jianghan University, Wuhan 430056, People’s Republic of China *Corresponding Authors: Prof. Gregory F. Payne

[email protected]

Prof. Xianghong Peng

[email protected]

ACS Paragon Plus Environment

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ABSTRACT Gelatin remains one of the most important biopolymeric material platforms because of its availability, safety, biocompatibility, biodegradability and stimuli-responsive properties. Here, we report a simple, rapid and reagentless anodic deposition method to assemble gelatin hydrogels from aqueous salt solutions onto an electrode surface. Results indicate that anodic reactions partially oxidize gelatin to yield a covalently crosslinked network that can perform multiple functions. First, anodically deposited gelatin remains activated allowing covalent protein grafting and thus enabling biofunctionalization for electrochemical biosensing. Second, the anodically deposited gelatin retains its thermally-responsive physical crosslinking properties that enable switching functions. Finally, the physical and chemical crosslinking mechanisms are reversible which enables self-healing functions. Thus, anodic deposition provides a facile method to assemble gelatin-based multifunctional matrices for diverse applications in bioelectronics.

INTRODUCTION There are increasing efforts to enlist controllable electrical signals to fabricate materials: to induce the formation of hierarchical structure and assemble components that confer function. Examples of the use of electrical inputs for soft matter fabrication include: (i) the electropolymerization of monomers to create conducting films (e.g., polythiophenes); (ii) the electrodeposition of pH-responsive polymers for protective coatings,1 composite films,2-5 and (bio)functionalized hydrogels;6-11 (iii) the creation of redox-responsive and redox-active films for bio-electronics;12-16 (iv) the coupling of electrical inputs with layer-by-layer assembly to create complex multilayers;17-21 and (v) the integration of redox chemistries for covalent assembly.17,22-


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While these examples demonstrate the broad potential for enlisting electrical inputs for

generating functional films and hydrogels, the materials and mechanisms available for such electrochemical fabrication are limited. Gelatin remains an important biopolymeric platform material. Because of its abundance and safety, gelatin has been exploited for various commercial products, while its compatibility enables its use in foods, pharmaceuticals, and medicine. Gelatin also possesses reversible thermally-responsive self-assembling properties that can be enlisted to confer additional functionalities. Recently, gelatin has been incorporated as one component in electrodeposited films for: electroplated metals/alloys;26-28 lead-free solders for electronics;29 composite antimicrobial films;30 multifunctional biofilms;31 and biocompatible surface coating.32 We contend that broader access to gelatin’s unique properties could be accessed if mechanisms were available to electrodeposit gelatin without the need for other components. In this paper, we report an anodic mechanism for the electrodeposition of a gelatin film. Our results indicate that anodic deposition is due to a partial oxidation of gelatin and the generation of a covalently crosslinked hydrogel network (presumably resulting from the formation of Schiff bases). Importantly, these partially oxidized gelatin films are activated allowing for the facile conjugation of proteins (also presumably due to Schiff base formation). We demonstrate protein conjugation using the readily visualized model red fluorescent protein mCherry and an enzyme glucose oxidase. Furthermore, we demonstrate that gelatin’s thermal-responsiveness and the reversibility of the physical and chemical crosslinking mechanisms allows for the creation of self-healing switches. We anticipate this simple, rapid, and reagentless electrochemical fabrication method will enable diverse applications in biosensing and smart bioelectronics.


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EXPERIMENTAL SECTION Materials. The following materials were purchased from Sigma-Aldrich: gelatin from porcine skin (type A), sodium chloride (99.5%), chitosan from crab shells (85% deacetylation), D-(+)-glucose (≥99.5%), glucose oxidase (GOx) from Aspergillus niger, gold wire (0.25 mm dia., ≥ 99.9%). Sodium acetate anhydrous (≥ 99.0%) was obtained from Fisher Scientific. Platinum wire (99.95%) was purchased from Surepure Chemetals Inc. (Florham Park, NJ). Water was de-ionized with Millipore SUPER-Q water system until final resistivity >18 MΩ cm was reached. The red fluorescent protein mCherry was engineered using a previously published method.33 Fluorescein-labeled chitosan was prepared by reacting NHS-fluorescein with chitosan using previously published method.34 The gold-coated silicon wafer was fabricated in University of Maryland NanoCenter. Sample preparation. Gelatin solution (10 % w/v) was prepared by dissolving gelatin powder in 37 ºC acetate buffer (10 mM, pH 5.6) containing 4 M NaCl. For electrochemical fabrication of gelatin, the gold electrode (gold wire or gold-coated silicon wafer) was first cleaned with piranha solution (H2SO4 : H2O2 =7 : 3 v/v) for 15 min and washed thoroughly with DI water, followed by drying in air. Details about the electrodeposition process are provided in Supporting Information (Scheme S1). Briefly, the clean electrode was immersed in the gelatin solution and connected to the power source (2400 Sourcemeter, Keithley) using alligator clips, and the gold electrode was biased to serve as the anode (4 - 20 A/m2) while a platinum wire served as the counter electrode.

After electrodeposition, the gelatin-coated electrode was

removed from the deposition solution and rinsed with DI water. mCherry (20 µg/mL) was dissolved in phosphate buffer (0.1 M, pH 7.0), and conjugation of mCherry on anodicallydeposited gelatin film was carried out at room temperature (22 ºC) for 3 h, followed by rinsing


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with 37 ºC buffer for 2 h. GOx (500 U/mL) was dissolved in phosphate buffer (0.1 M, pH 7.0), and conjugation of GOx on anodically-deposited gelatin film was carried out at 4 ºC for 12 h, followed by rinsing at with buffer at room temperature for 30 min. Instrumentation. X-ray photoelectron spectroscopy (XPS) measurements were performed on a Kratos AXIS 165 spectrometer with a monochromatic A1 Kα (1486.7 eV) x-ray source. Peak fittings were performed using CasaXPS software and a Shirley-type background was applied to all the spectra. A Jasco 4100 series Fourier Transform Infrared Spectroscopy (FTIR) with an attenuated total reflection (ATR) cell (Jasco Inc., Easton, MO) was used for chemical analysis of the dried gelatin films. Fluorescence images of gelatin films were obtained using an Olympus MVX10 MacroView microscope, and images were obtained using an Olympus DP72 digital camera connected to the microscope. Confocal images were taken with Zeiss LSM-310 laser-scanning microscope. Fluorescence of biodegraded gelatin in the well-plate was measured with a Molecular Devices SpectraMax M2. Electrochemical measurements such as i – t curves were carried out with a CHI6273C Electrochemical Analyzer (CH Instruments, Austin, TX). Measurements were performed using three-electrode configurations with Ag/AgCl (3 M NaCl) as a reference electrode and Pt wire as a counter electrode.

RESULTS AND DISCUSSION Figure 1 illustrates a putative mechanism for gelatin’s anodic deposition.35,36 A working electrode (i.e., gold wire) is immersed into a buffered gelatin solution (10 % gelatin, 10 mM acetate buffer, pH 5.6) containing a high concentration of NaCl (4 M). An anodic potential is applied (20 A/m2 for 5 minutes) to oxidize Cl− to Cl2 which is then hydrolyzed to form the


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diffusible oxidant hypochlorous acid (HOCl). HOCl is also a weak acid (pKa 7.5) and since HOCl is considered to be the more reactive oxidant (vs OCl−), acetic acid is incorporated into the deposition solution to control pH conditions to favor the HOCl species. We hypothesize that HOCl partially oxidizes gelatin to generate carbonyls (e.g., aldehydes) that can undergo Schiff base crosslinking reactions.37,38 The photograph in Figure 1 shows that a transparent hydrogel film is formed on the electrode surface when anodic oxidation is performed in the presence of gelatin and NaCl.

Figure 1. Experiment and putative mechanism of anodic electrodeposition of gelatin.

Chemical evidence for anodic gelatin oxidation was obtained from XPS and FTIR measurements of gelatin films (films were peeled from the gold wire for analysis). The left XPS spectra in Figure 2a compare the O 1s regions for oxidized gelatin and control gelatin. The anodically deposited gelatin has an increased C=O peak relative to C–OH peak consistent with a partial oxidation of the film.36 [See Supporting Information for detailed XPS measurements (Figure S1).] The FTIR spectra of Figure 2a compare the control and oxidized gelatin films and


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shows slightly broad peaks in the amide region and small changes in the fingerprint region, consistent with a covalent modification of gelatin.

Figure 2. (a) Chemical evidence for gelatin oxidation. (b) Physical evidence for covalent crosslinking (repeated swelling and de-swelling above gelatin’s melting temperature indicates that the gel is chemically crosslinked). Physical evidence that the electrodeposited gelatin is covalently crosslinked is shown in Figure 2b. In this experiment, anodic deposition was performed on a gold-coated silicon wafer (4 A/m2 for 5 minutes). The schematic and photographs illustrate that the deposited hydrogel swells upon immersion in phosphate buffer (0.1 M, pH 7.0) and de-swells upon drying. The two plots in Figure 2b show that separate films were subjected to repeated swelling and drying cycles with swelling being performed either below or above gelatin’s melting temperature (~28 °C. We did not specifically study the film's adhesion mechanism and strength, however we should note


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that the ability to repeatedly swell/de-swell these films suggests adhesion is reasonably stable.). Below gelatin’s melting temperature (22 °C), swelling and de-swelling is reversible for multiple cycles until the film ultimately delaminates from the flat electrode surface after 11 cycles. Above gelatin’s melting temperature (37 °C), swelling is considerably greater suggesting that gelatin’s physical crosslinks (i.e., triple helices) have been melted and the structure is retained solely by chemical (i.e., covalent) crosslinks formed during anodic oxidation. While repeated swelling and de-swelling cycles were observed at 37 °C, these covalent crosslinks were not entirely stable consistent with the reversibility of Schiff bases.35 Anodic deposition generates gelatin films with three useful characteristics. First, the deposited gelatin possesses thermally responsive properties as illustrated by the swelling curves in Figure 3a. Experimentally, gelatin films were anodically deposited onto a gold wire (same conditions as Figure 1) and then dried. After drying, the thickness of the gelatin-coated wire was measured (from photomicrographs) and then the wire was incubated in phosphate buffer at progressively higher temperatures. The results in Figure 3a indicate that swelling increased with increasing temperature and was repeatable over three tests.

The right-most plot in Figure 3a

indicates that the slope of the swelling vs temperature curve changes near the gelatin’s melting temperature. This thermally-responsive swelling is consistent with results in Figure 2b. The second characteristic of anodically-deposited gelatin, is that it remains partially activated for subsequent grafting for (bio)functionalization. This activation is illustrated in Figure 3b which shows the anodically deposited gelatin was rinsed and then immersed in a solution containing the model fluorescent protein mCherry (20 µg/mL). The first fluorescence image in Figure 3b shows the deposited gelatin has bright red fluorescence consistent with mCherry grafting. [Note: Figure S2 of Supporting Information shows that treatment of the


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electrodeposited gelatin with the NaBH4 reductant prevents mCherry binding consistent with the loss of activated aldehydes required for Shiff base formation.] Next, a wire with mCherrymodified-gelatin film was immersed in a solution containing fluorescein-labeled chitosan (0.9 % chitosan dissolved in acetic acid pH 5). The final fluorescence images in Figure 3b indicate that this hydrogel possess both red and green fluorescence consistent with mCherry and chitosan grafting.

Figure 3. Properties of anodically-deposited gelatin. (a) Thermally-responsive swelling behavior suggests both physical and covalent crosslinks. (b) Deposited


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gelatin is activated for covalent conjugation to proteins or chitosan. (c) Deposited gelatin is biodegraded by protease.

The third characteristic feature of the anodically deposited gelatin is its biodegradability.39,40 Figure 3c shows that a wire coated with mCherry-modified gelatin was immersed in a solution containing the protease trypsin.41 The optical and fluorescence images in Figure 3c show that trypsin digestion removes the gelatin film from the wire electrode and releases mCherry’s red fluorescence into the solution. Finally, we demonstrate 3 functionalities of anodically deposited gelatin. First, Figure 4 shows that anodically deposited gelatin is activated for protein grafting to enable biofunctionlization. For this proof-of-concept, we first anodically-deposited gelatin onto a gold wire (7.5 A/m2 for 90 sec) and then contacted the coated wire with a solution of the standard biosensing enzyme glucose oxidase (GOx). Figure 4 shows the expected proportional increase in output current with glucose concentration for this GOx-functionalized wire (Figure S3 of Supporting Information shows GOx activity is reasonably stable over time). These results demonstrate that the gelatin’s anodic deposition facilitates biofunctionalization of a wire electrode which is a particularly convenient platform for electrochemical biosensing in remote locations and unconventional formats (e.g., embedded in fabrics).42

Figure 4. Activation of film allows protein biofunctionalization for electrochemical biosensing.


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The second function demonstrated in Figure 5 is thermally-responsive switching. For this experiment, we prepared a switch from two electrodes each with anodically-deposited gelatin (4 A/m2 for 2 min). The “active element” was subsequently functionalized with GOx. These two elements were individually pre-incubated in buffer (30 min) to melt any physical crosslinks (37 ⁰C for the active element and 55 ⁰C for the inactive element).

After pre-incubation, the two

elements were clamped together and refrigerated (10 min) to bond the elements by re-forming the physical crosslinks. In this experiment, the GOx-active element serves as the working electrode and the inactive element physically blocks access of substrates (glucose and O2). This switch was then immersed in a buffered glucose solution (6 mM, pH 7) at 22 ⁰C.

After the

initial transient, the low-temperature i-t plot shows a small, constant current (“off” state) which is consistent with visual observations that the elements show no tendency to separate at the low temperature. The switch was then transferred to a warm glucose solution and Figure 5 shows a more complex i-t response. The higher initial current is presumably due to the temperature effect on GOx kinetics, while the increasing current is due to a swelling of the hydrogel interface between elements thus allowing access to the substrates (a “partially-on” state).

The abrupt

increase in current (≈ 3 min) occurred when the two elements were visually observed to separate (the inactive element fell off the switch to yield a “fully-on” state). Qualitatively, the results in Figure 5 suggest that thermally-responsive switching is due to gelatin’s physical crosslinks, although the slow switching through a partially-on state suggests reversible Schiff base reactions may participate in crosslinking.43-45


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Figure 5. Thermally-responsive properties enable switching.

The third function demonstrated in Figure 6 is self-healing. The schematic (Figure 6a) suggests healing could involve both physical (e.g., triple helix formation) and chemical (e.g., Schiff base formation) mechanisms. To demonstrate healing, we prepared a switch (similar to the one shown in Figure 5) from two anodically-deposited gelatin (4 A/m2 for 2 min) electrodes, one of which was functionalized with GOx (active element).

These two elements were

individually pre-incubated in buffer (30 min) to melt any physical crosslinks (37 ⁰C for the active element and 55 ⁰C for the inactive element). After pre-incubation, the two elements were clamped together and refrigerated (10 min) to bond the elements by re-forming the physical crosslinks. Then, the switch was repeatedly incubated in glucose (6 mM) solutions at low (22


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⁰C) and then high temperatures (37 ⁰C). At the end of each high-temperature incubation, we turned on the switch by physically separating the elements (as indicated by the sharp rise in current). Next, the two elements were clamped together and refrigerated (10 min) to re-heal the interface and regenerate the switch. The self-healing process was repeated 3 times. The plots in Figure 6b show that healing of the gelatin interface enables regeneration of a switch that was stably off at low temperature and could be turned on at higher temperatures.

Figure 6. The reversibility of crosslinking enables self-healing.

CONCLUSIONS In conclusion, we demonstrate a novel anodic electrodeposition mechanism that allows the simple, rapid and reagentless assembly of a gelatin interface at an electrode surface. The anodically deposited gelatin remains activated for subsequent protein-based biofunctionalization (e.g., for electrochemical biosensing). Further, gelatin’s thermal-responsiveness and the reversibility of the crosslinking mechanisms allows for the creation of self-healing switches. We envision that anodic deposition allows smart bioelectronic interfaces to be generated that can


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access both the molecular recognition capabilities of biology and the speed and sensitivity of electronic signal processing.

FIGURES Figure 1. Experiment and putative mechanism of anodic electrodeposition of gelatin. Figure 2. (a) Chemical evidence for gelatin oxidation. (b) Physical evidence for covalent crosslinking (repeated swelling and de-swelling above gelatin’s melting temperature indicates that the gel is chemically crosslinked). Figure 3. Properties of anodically-deposited gelatin. (a) Thermally-responsive swelling behavior suggests both physical and covalent crosslinks. (b) Deposited gelatin is activated for covalent conjugation to proteins or chitosan. (c) Deposited gelatin is biodegraded by protease. Figure 4. Activation of film allows protein biofunctionalization for electrochemical biosensing. Figure 5. Thermally-responsive properties enable switching. Figure 6. The reversibility of crosslinking enables self-healing.

ASSOCIATED CONTENT Supporting Information. Electrodeposition process; XPS analysis of anodically-deposited gelatin; evidence that proteins (e.g., mCherry) are covalently conjugated on anodic deposited gelatin films; and stability of conjugated GOx on anodic gelatin film. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Phone: 301-405-8389 FAX: 301-314-9075 *E-mail: [email protected] Phone: (86)2784226806 FAX: (86)2784225198


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors gratefully acknowledge financial support from the National Science Foundation (CBET-1435957), the Department of Defense (Defense Threat Reduction Agency; HDTRA1-131-0037) and the Foundation of Science and Technology Bureau of Wuhan [No. 2014010101010016]. We thank Drs. Hsuan-Chen Wu and Chen-Yu Tsao for providing the protein mCherry. We also thank Dr. Karen Gaskell for assistance with XPS analysis, Ms. Melissa Rhoads for assistance with confocal measurements, and Ms. Rouyang Xu for assistance with FTIR measurements.

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TOC Graphic Electrochemical Fabrication of Functional Gelatin-based Bio-electronic Interface

Xianghong Peng,*,†,‡ Yi Liu, † William E. Bentley, †,§ and Gregory F. Payne*,†,§


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Electrochemical Fabrication of Functional Gelatin-Based Bioelectronic

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