Electrodeposition of a Biopolymeric Hydrogel: Potential for One-Step

Article pubs.acs.org/Biomac

Electrodeposition of a Biopolymeric Hydrogel: Potential for One-Step Protein Electroaddressing Kelsey M. Gray,†,‡ Benjamin D. Liba,†,‡ Yifeng Wang,§ Yi Cheng,∥ Gary W. Rubloff,∥ William E. Bentley,‡,⊥ Alexandra Montembault,#,○,□ Isabelle Royaud,△ Laurent David,#,○,□ and Gregory F. Payne*,‡,⊥ ‡

Institute for Bioscience and Biotechnology Research, University of Maryland, 5115 Plant Sciences Building, College Park, Maryland 20742, United States § School of Material Science and Engineering, Wuhan University of Technology, Wuhan, 430070, People's Republic of China ∥ Department of Materials Science and Engineering and Institute for Systems Research, University of Maryland, College Park, Maryland 20742, United States ⊥ Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States # Université de Lyon, F-69361, Lyon, France ○ CNRS, UMR 5223, Ingénierie des Matériaux Polymères, F-69622, Villeurbanne, France □ Université Claude Bernard Lyon 1, 15 bd Latarjet, F-69622, Villeurbanne, France △ Département SI2M, Institut Jean Lamour, Nancy Université, CNRS UMR 7198 Ecole des Mines, Parc de Saurupt, CS 14234 54 042 Nancy, Cedex, France ABSTRACT: The electrodeposition of hydrogels provides a programmable means to assemble soft matter for various technological applications. We report an anodic method to deposit hydrogel films of the aminopolysaccharide chitosan. Evidence suggests the deposition mechanism involves the electrolysis of chloride to generate reactive chlorine species (e.g., HOCl) that partially oxidize chitosan to generate aldehydes that can couple covalently with amines (presumably through Schiff base linkages). Chitosan’s anodic deposition is controllable spatially and temporally. Consistent with a covalent cross-linking mechanism, the deposited chitosan undergoes repeated swelling/deswelling in response to pH changes. Consistent with a covalent conjugation mechanism, proteins could be codeposited and retained within the chitosan film even after detergent washing. As a proof-of-concept, we electroaddressed glucose oxidase to a side-wall electrode of a microfabricated fluidic channel and demonstrated this enzyme could perform electrochemical biosensing functions. Thus, anodic chitosan deposition provides a reagentless, single-step method to electroaddress a stimuli-responsive and biofunctionalized hydrogel film.

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INTRODUCTION Electrodeposition is a process in which imposed electrical “signals” are employed to direct the assembly of thin films. Electrodeposition has been widely used with metals and alloys to create protective coating and fabricate integrated circuits. The extension of electrodeposition to soft matter (i.e., polymers) is attracting increasing attention because it offers broad opportunities for a diverse range of applications as illustrated by the following examples. Electrodeposition provides a major route for the synthesis of conducting polymers such as polypyrrole, polyaniline and polythiophene.1−3 Electrodeposition paints (EDPs) enjoy widespread use because they allow arbitrarily complex surfaces to be coated while offering economic, safety, health, and environmental benefits.4 Components that can be blended into the deposition solution can be codeposited and this capability enables the fabrication of composite films for fields that range © 2012 American Chemical Society

from medicine (e.g., hydroxyapatite-containing coatings for bone implants)5−9 to electronics (nanoparticle-containing films with enhanced electronic properties).10−12 Polymer electrodeposition is often achieved from aqueous solution under ambient conditions and thus codeposition allows films to be fabricated to contain labile biological components (e.g., proteins and cells) for biosensor applications and biological experimentation.13−19 The electrical signals that induce deposition can be imposed with exquisite spatiotemporal selectivity, thus, allowing the sequential assembly of different components at separate electrode “addresses” (e.g., for chipbased arrays).20,21 Finally, electrodeposition can be achieved on patterned electrodes without the need for external access to the Received: January 25, 2012 Revised: March 7, 2012 Published: March 13, 2012 1181

dx.doi.org/10.1021/bm3001155 | Biomacromolecules 2012, 13, 1181−1189

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surface (vs printing) or line-of-sight (vs photolithography), and thus, electrodeposition is ideally suited for electroaddressing components within fully packaged (i.e., covered) microfluidic channels for lab-on-a-chip applications.22,23 Currently, there are only a few known mechanisms for polymer electrodeposition. Electro-polymerization employs electrochemical reactions (e.g., anodic oxidation) to simultaneously polymerize a monomer and generate an insoluble polymer film.1−3 Electropolymerization is extensively used to create conducting polymer films (e.g., polypyrrole). Alternatively, previously synthesized stimuli-responsive polymers can sometimes be electrodeposited.24 To our knowledge, electrodeposition paints (EDPs) were the first example of the electrodeposition of pre-existing polymers.4 Typically, EDPs are pH-responsive polymers that are soluble in the deposition solution but become insoluble at the electrode surface due to pH gradients generated by electrolytic reactions. For instance, EDPs with carboxylate groups (COO−) “recognize” the local low pH at the anode and “respond” by undergoing protonation (to form COOH) and precipitation (or gelation) at the anode surface. EDPs with protonated ammonium groups (e.g., NH3+) recognize the high pH at the cathode to become neutral and insoluble. Nearly 10 years ago, it was discovered that pH-responsive film-forming biological polymers could also be electrodeposited by neutralization mechanisms.5,19,25−27 To our knowledge, the aminopolysaccharide chitosan was the first biopolymer to be electrodeposited by a cathodic neutralization mechanism,5,19,25−29 and this was followed by the discovery of anodic deposition of the acidic polysaccharide alginic acid.30 The broad interest in biopolymer electrodeposition spurred the investigation of alternative deposition mechanisms.31 For instance, a mechanism was discovered for the reversible electrodeposition of Ca2+-alginate hydrogels and this mechanism permits the codeposition of viable bacterial populations.32−35 Also, the Ca2+-alginate deposition mechanism allows for the codeposition of the thermally responsive and neutral polysaccharide agarose.36 In addition to macromolecules, low molecular weight peptide-based hydro-gelators37−39 were reported to electrodeposit through a neutralization mechanism. The above mechanisms for biopolymer electrodeposition all rely on the use of an electrical signal to trigger a reversible sol− gel transition, with the resulting deposits being physical gels. For instance, chitosan undergoes cathodic electrodeposition because the high cathodic pH neutralizes chitosan’s primary amines and induces gelation of this pH-responsive film-forming polysaccharide. The cathodic deposit is stable under neutral and basic conditions but redissolves if the film is exposed to acidic conditions that reprotonate the amines. Here we investigated a new method; the electrodeposition of chitosan at an anode. In contrast to chitosan’s cathodic electrodeposition, we provide evidence that anodic deposition involves a chemical modification of chitosan that leads to the formation of a covalently cross-linked chemical gel. Scheme 1 outlines the putative mechanism. Specifically, Scheme 1 proposes that the anodic oxidation of Cl− generates Cl2, which can undergo reactions in the bulk aqueous phase to form various reactive chlorine species.40−45 These reactive chlorine species diffuse into the bulk where they react to partially oxidize chitosan.46 It is well-known that polysaccharides such as chitosan can undergo partial oxidation reactions47−53 that in some cases generate aldehyde moieties.54−56 While the oxidation chemistry is not fully elucidated, Scheme 1 suggests

Scheme 1. Putative Mechanism for Anodic Electrodeposition of Chitosan: Anodic Oxidation Generates Reactive Chlorine Species That Partially Oxidize Chitosan, Enabling CrossLinking and Conjugation Reactions

two putative sites for chitosan’s oxidation. If aldehydes are generated by chitosan’s partial oxidation, then these moieties should form Schiff bases with primary amine moieties53,57 to generate a covalently cross-linked chitosan network. Interestingly, Schiff bases are sometimes reduced to form more stable linkages, but this does not appear necessary for chitosan, as glutaraldehyde (without reducing agents) is commonly used to generate cross-linked chitosan gels.58−60 Importantly, if the reactions proposed in Scheme 1 can impart aldehyde functionality to chitosan, then it should also be possible to covalently conjugate proteins through nucleophilic amino acid residues (e.g., through the ε-amine of lysine residues).61,62 In this case, the anodic reactions would serve to induce chitosan’s electrodeposition while simultaneously activating the film for protein conjugation. We provide evidence for such single-step protein conjugation using the standard biosensing enzyme glucose oxidase (GOx).

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EXPERIMENTAL SECTION

Materials. The following materials were purchased from SigmaAldrich; chitosan from crab shells (340000 molecular weight and 15% degree of acetylation, as reported by the manufacturer), GOx from Aspergillus niger (181300 U/g), pH indicator dye (pH 0−5 Universal Indicator), NHS-rhodamine, and Schiff’s reagent for aldehydes. Preparation of Chitosan. Chitosan was dissolved in stoichiometric amounts of either acetic acid (HAc) or HCl. Fluorescently labeled chitosan was prepared by reacting NHS-rhodamine (2.5 mg/ mL) with chitosan dissolved in HAc (1% chitosan, pH 5.5). After reaction, the labeled chitosan was precipitated by raising the pH, and the precipitates were extensively washed to remove unreacted dye. Electrodeposition of Chitosan. Chitosan was electrodeposited by both a cathodic deposition method (this served as a control) and an anodic deposition method (the focus of this investigation). Cathodic chitosan deposition occurs through a reversible sol−gel transition involving a neutralization mechanism. Cathodic deposits were generated from 1.0 or 2.1% chitosan solutions dissolved in HCl (pH 5.5) using a constant current density (4 A/m2). Anodic deposits are formed by the putative mechanism of Scheme 1. For anodic deposition, chitosan was dissolved in HAc with 0.15 M added NaCl (pH 5.5) and a constant current density (4 A/m2) was applied. Two electrodes were used for both cathodic and anodic deposition and these electrodes were biased using a DC power supply (2400 Sourcemeter, Keithley). 1182


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Electrodeposition was performed using gold-coated silicon wafers (termed “chips”) and a fluidic device. Chips (1 × 2 cm) were prepared by thermal evaporation to deposit films of 16 nm chromium and 120 nm of gold onto silicon wafers which were then diced and cut (in some cases the gold was patterned onto the wafers). For anodic deposition onto chips, the gold surface of one chip served as the working electrode while the gold surface of a second chip served as the counter electrode. An overview of the fluidic device is described later while further details of device fabrication have been reported elsewhere.29 For deposition in a fluidic channel, two patterned gold surfaces on opposite sidewalls served as the working and counter electrodes. The imaging software used to characterize the thickness and volume of the deposited chitosan was CellSense (Olympus). Protein Conjugation to Chitosan. Proof-of-concept biosensing experiments were performed using GOx as a model biosensing enzyme. GOx was codeposited with anodic chitosan gels by preparing a deposition solution containing GOx (680 U/mL), chitosan (1% dissolved in HAc), and NaCl (0.15 M). This solution was added to the fluidic channel and deposition was initiated by applying a constant anodic current (4 A/m2) with the opposing electrode on the opposite wall of the channel serving as the counter electrode. After deposition, the channel was rinsed with water and then phosphate buffer (0.1 M, pH 7.0). For sensing, sugar-containing test solutions were then intermittently added to the channel while the underlying gold electrode was biased to +0.6 V for H2O2 detection (the opposing electrode served as the counter electrode and a Ag/AgCl wire was partially inserted into the channel to serve as a quasi-reference electrode). The biosensing measurements were made using a CHI627C electrochemical analyzer (CH Instruments, Inc., Austin, TX). Raman Spectroscopy. Raman spectra were obtained from a high resolution confocal Raman microscope (Horiba Jobin-Yvon LabRamHR 800) using a 633 nm laser source.

Figure 1. Electrodeposition of chitosan at cathode and anode. Scheme of experiment and photograph of the anodic gel deposited onto a goldcoated silicon anode (4 A/m2 for 4 min) from a 1% chitosan solution (dissolved in HAc with 0.15 M NaCl). Table indicates conditions required for hydrogel electrodeposition; (+) indicates a stable hydrogel and (−) indicates an unstable deposit easily removed by washing.

illustrated in Scheme 1 begins with the electrochemical oxidation of Cl− . 2Cl− → 2e− + Cl2

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(1)

The sodium chloride electrolysis reaction is well-known and forms the basis for industrial chlor-alkali processes. Evidence that this reaction occurs in our system is provided by cyclic voltammetry (CV) measurements with solutions containing combinations of acetic acid, chitosan (1%), and NaCl (0.15 M). Figure 2a shows that, as the potential is swept above +1 V (vs Ag/AgCl), oxidation currents are only observed if NaCl is included in the solution. [Note: the observed differences cannot simply be attributed to the conductivity of the added NaCl as CVs for potassium ferricyanide from 0 to +0.5 V vs Ag/AgCl were the same for the HAc buffer with and without NaCl; not shown.] Anodically generated Cl2 is known to undergo hydrolysis reactions to yield hypochlorous acid (HOCl) which at higher pH can be deprotonated to form hypochlorite (OCl−).

RESULTS AND DISCUSSION Initial Demonstration of Anodic Deposition. We observed that chitosan could be reproducibly deposited on the anode only under a limited set of experimental conditions. Specifically, we prepared chitosan solutions (1% chitosan, pH 5.5.) by dissolving with either HCl or acetic acid (HAc) and, in some cases, we added NaCl (0.15 M). Electrodeposition was investigated by immersing gold-coated silicon wafers into the individual solutions (as illustrated by the schematic in Figure 1) and applying a potential to achieve a constant current density (4 A/m2) for 4 min. After disconnecting the power, the gold surfaces were visually examined. As illustrated by the table in Figure 1, we observed stable hydrogel films when chitosan was electrodeposited onto the cathode from solutions prepared with HCl. In contrast, the cathodic deposits from HAc solutions were irreproducible, easily removed by rinsing and thus termed unstable. [We should note that other groups routinely electrodeposit chitosan from HAc solutions onto cathodes although using somewhat different deposition conditions (e.g., ethanol-containing solutions27,63 or higher voltages64).] Also illustrated by the table in Figure 1 is that anodic deposition of chitosan was only observed when chitosan was dissolved in HAc and when NaCl was included in the deposition solution (these requirements are discussed in the following section). The photograph in Figure 1 shows an anodically deposited chitosan hydrogel film. In summary, the results in Figure 1 indicate that chitosan can be induced to undergo anodic electrodeposition under a limited set of experimental conditions. Chemical Evidence for Anodic Deposition Mechanism. The proposed mechanism for anodic deposition

Cl2 + H2O ⇌ HOCl + Cl− + H+

(2)

HOCl ⇌ OCl− + H+

(3)

Figure 2b shows the equilibrium distribution of these reactive chlorine species as a function of pH.65 Importantly, the HOCl form is considered to be the most reactive oxidant.66−69 An important difference between HCl and HAc is that the latter is a weak acid that provides buffering capacity. To demonstrate this difference in buffering, we filled a fluidic channel with a 1% chitosan solution dissolved in either HCl or HAc (the fluidic device is described later). Each solution contained 0.15 M NaCl and the pH indicator dye. Figure 3 shows that before applying a potential the pH-indicator dye shows that both fluids were at a similar pH (5.5) and no pH gradients were obvious. A potential was then applied to a side wall gold electrode to achieve a constant current density (4 A/ m2) for 90 s after which the fluids were photographed. Figure 3 1183


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Figure 2. Anodic generation and distribution of reactive chlorine species. (a) Cyclic voltammogram (CV) shows oxidation occurs only in the presence of NaCl, presumably through the Cl2-generating reaction. (b) Equilibrium distribution of reactive chlorine species as a function of pH.

is capable of buffering the solution near the anode, and this is important because it provides environmental conditions favoring the HOCl species. [Interestingly, HAc also provides buffering capacity at the cathode which limits cathodic deposition via a pH gradient induced neutralization mechanism.] The above results support the hypotheses in Scheme 1 that an electrochemically generated reactive chlorine intermediate (e.g., HOCl) oxidizes chitosan to generate carbonyl groups (presumably aldehydes). To test this aspect of the mechanism, we performed a colorimetric Schiff test for aldehyde detection.70 Specifically, we anodically deposited chitosan (1% in HAc and 0.15 M NaCl; 4 A/m2 for 5 min), immersed the deposited film in cold ethanol, added the Schiff reagent, and observed that the film underwent an immediate change in color. The photograph in Figure 4a shows this color approximately 2 min after initiating the Schiff test. The control in Figure 4a is a thick chitosan gel deposited at the cathode from an HCl solution (2.1% chitosan; 0.15 M NaCl; 4 A/m2 for 5 min). As expected, no color change was observed for this control. The positive Schiff test for the anodic, but not cathodic, chitosan deposit supports the conclusion that the deposition mechanisms and the nature of the hydrogels are

Figure 3. Buffering effect of acetic acid. Chitosan solutions containing HAc maintain a near constant pH (≈5) adjacent to the anode during electrodeposition and this pH favors the HOCl species. Chitosan solutions containing HCl show a steep pH gradient adjacent to the anode.

shows that a substantial pH gradient is observed for the HClcontaining solution with the pH near the electrode reaching values of 1.0. In contrast, no pH gradient is generated near the anode for the chitosan solution prepared in HAc. Thus, acetate

Figure 4. Evidence for partial oxidation of chitosan. (a) Schiff test provides colorimetric evidence for the partial oxidation of chitosan and formation of aldehyde substituents. (b) Raman bands for primary amines (1080−1140 cm−1 and 3300−3500 cm−1) are diminished for the anodic gel vs the cathodic gel and also diminished when anodic deposition is performed for a longer time. 1184


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Figure 5. Controllable anodic deposition within a fluidic channel. (a) Schematic of microfabricated fluidic device with 1 × 1 mm fluidic channel and 1 mm wide sidewall electrodes (for clarity a cutaway of the PDMS cover is shown). (b) Spatial control of anodic deposition is demonstrated by depositing fluorescently labeled chitosan at two different electrode addresses at separate times. (c) Quantitative control of anodic deposition is demonstrated by depositing chitosan for varying times and at two different current densities (4 and 8 A/m2).

opposing electrode directly across the channel serves as the counterelectrode. The device’s base and cover are generated from polydimethylsiloxane (PDMS), which allows optical imaging of the deposited film as illustrated in Figure 5a. Initial studies to anodically deposit chitosan on the sidewall address were performed by introducing the deposition solution into the channel. To facilitate visualization we prepared our deposition solution with rhodamine-labeled chitosan (1% dissolved in HAc with 0.15 M NaCl). After filling the channel, the individual electrodes were sequentially biased at a constant current density (4 A/m2) for 90 s. After deposition, the channel was rinsed with deionized water. The fluorescence photomicrographs in Figure 5b show that deposition is localized to the sidewall electrode addresses (no deposits were observed on the opposing counterelectrodes). These images indicate that chitosan can be anodically deposited at an electrode address within a covered fluidic device. In separate studies, we used the fluidic device to examine the ability to control anodic chitosan deposition. In these studies, the channel was filled with the chitosan deposition solution (1% chitosan dissolved in HAc with 0.15 M NaCl and 5 μM rhodamine-labeled dextran to enhance visualization) and an anodic potential was applied to an individual electrode (4 A/ m2) for a controlled time. After deposition, the channel was rinsed with deionized water and emptied, and the deposited film was imaged. This procedure was repeated for several deposition times and also for a second current density of (8 A/ m2). The imaging software and approach used to assess the dimensions of the deposited hydrogel films were described previously for studies of the cathodically deposited chitosan films.29 However, there is one difference between the analysis of the cathodic and anodic chitosan deposits. The cathodic deposits from our previous study (using HCl and no added salt) were somewhat opaque allowing the edge of the film and the surrounding liquid to be discerned and this enabled in situ observation of the growing film.29 In contrast, the anodic deposits reported here are transparent and the edge between

different. Further, the positive Schiff test supports the proposed anodic deposition mechanism of Scheme 1, which indicates that chitosan is partially oxidized to generate aldehyde moieties. In addition to the Schiff test, we used Raman spectroscopy in an effort to obtain direct evidence that the anodically deposited chitosan has undergone chemical modifications. Anodic gels (1%, HAc, 0.15 M NaCl) were deposited on gold-coated wafers for varying times, washed with water and phosphate buffer (pH 7.0) then dried overnight (37 °C). These anodic gels were compared to cathodic gels (1%, HCl, 0.15 M NaCl) deposited for 5 min, washed with water and phosphate buffer (pH 7.0) then dried overnight (37 °C). Figure 4b shows double vibrational bands at 1080−1140 cm−1 and 3300−3500 cm−1 that are consistent with the primary amines found in chitosan.71 Consistent with chemical modification of chitosan, these amine bands were smaller for the anodic (vs the cathodic) gel. Further, as the deposition time for anodic deposition was increased, these bands were observed to progressively decrease. These Raman results provide supportive evidence that the anodic deposits are chitosan and that the chitosan is chemically modified upon anodic deposition. In summary, the results in Figures 2−4 provide several pieces of independent evidence supporting the mechanism for chitosan’s anodic deposition illustrated in Scheme 1. Further supporting this mechanism was a visual observation that a gel is formed when an acetate-buffered sodium hypochlorite (NaOCl) solution (pH 5) was added to a solution of chitosan (not shown). Electrodeposition in a Fluidic Channel. A major motivation for our electrodeposition studies is to create methods that allow electroaddressing within microfabricated fluidic devices. For these studies, we employ the test device illustrated in Figure 5a,29,33,35 which is fabricated from two glass slides, each patterned with 1 mm wide parallel gold lines. These slides are positioned to be 1 mm apart to form a channel with the gold lines forming sidewall electrode addresses and their leads. During deposition, one of the sidewall electrodes is connected to the power supply to serve as the anode, while the 1185


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the film and surrounding solution could not be discerned: thus the channel needed to be emptied to visualize the edge of each anodically deposited film. Figure 5c demonstrates that the volume and thickness of the anodically deposited chitosan systematically increases with deposition time. Further the volume and thickness increased with current density. Important to note is that the observed thickness spans a considerable fraction of the 1 mm wide channel. Figure 5c also shows how far the anodically deposited film extends beyond the edge of the electrode. The extension of the gel past the edge of the electrode presumably reflects the diffusion of the anodically generated HOCl and the edge thickness is well-correlated to the film’s middle thickness. In summary, the results in Figure 5 indicate that anodic deposition is rapid (occurring in minutes) and spatially controllable. As an aside, it is interesting to note that the literature often uses the terms “electrodeposition” and “electrophoretic deposition” interchangeably when describing the cathodic deposition of chitosan. The latter term suggests that the electric field contributes to the transport of positively charged chitosan chains from the bulk solution toward the negative electrode (cathode). Recent measurements indicate that the electrophoretic migration of chitosan to the cathode makes a relatively small contribution to deposition suggesting that the majority of the deposited gel is formed from chitosan chains that were present near the electrode when the pH gradient was suddenly imposed.29 The term “electrophoretic deposition” would appear inappropriate for describing chitosan’s anodic deposition since the positively charged anode should act to repel chitosan chains and suppress anodic deposition. Thus, we believe our results indicate that the generation and diffusion of reactive chlorine species, and chitosan’s oxidation and crosslinking all occur faster than field-induced chain migration, which would deplete chitosan from the vicinity of the anode. Repeated Swelling of the Anodically-Deposited Chitosan. The hypothesis in Scheme 1 suggests that anodically deposited chitosan is covalently cross-linked through Schiff base linkages. If so, then it should be possible for this chemically cross-linked network to be swollen, but not dissolved, by acidic conditions that protonate chitosan’s amines.72,73 To test this possibility, we anodically deposited chitosan onto a sidewall electrode (1% chitosan in HAc with 0.15 M NaCl; 4 A/m2 for 90 s) and then sequentially contacted this hydrogel film with acid (1 M HAc, 20 min) and base (1 M NaOH, 20 min). Between each acid/base treatment, the contents of the channel were removed leaving only the deposited gel which was then imaged. Figure 6 shows the volume of the anodically deposited gel after each swelling (acid treatment) and deswelling (base treatment) step. The results indicate that the gel undergoes a 3-fold change in dimensions upon swelling and deswelling and these changes can be repeated for multiple cycles. However, over long times, the gel’s swelling becomes less which is consistent with visual observations that the hydrogel film has begun to lose its structural integrity. This loss in structural integrity is consistent with the reversible nature of the putative Schiff base cross-links. In summary, these results support the hypothesis that the anodically deposited chitosan hydrogel films are covalently cross-linked and can undergo a limited number of swelling/ deswelling cycles. Biofunctionalization of Anodically-Deposited Chitosan. In a final proof-of-concept experiment we demonstrated biofunctionalization of the gel using the model electrochemical

Figure 6. Stimuli-responsive properties of the anodic deposit. (a) Scheme illustrating that the anodic chitosan gel deswells in base and swells in acid. (b) Result shows repeatable, although not indefinite, swelling/deswelling (≈3-fold volume change) for anodically deposited chitosan gel.

biosensing enzyme, GOx. GOx (680 U mL−1) was added to the deposition solution (1% chitosan in HAc with 0.15 M NaCl) and anodically codeposited (4 A/m2 for 90s) onto a sidewall electrode address. After washing the channel with phosphate buffer (0.1 M, pH 7.0) we examined the electrochemical biosensing capabilities of the codeposited enzyme via the standard enzyme-catalyzed glucose oxidation and subsequent electrochemical detection of the H2O2 product. glucose + O2 → gluconic acid + H2O2

(4)

H2O2 → O2 + 2H+ + 2e−

(5)

For detection, we used a three-electrode system as illustrated in Figure 7a: the electrode functionalized with chitosan and GOx served as the working electrode; the electrode directly across the channel served as the counterelectrode; and an Ag/ AgCl wire was partially inserted into the channel to serve as a quasi-reference electrode. For detection, we applied a constant anodic potential (+0.6 V) to the working electrode and measured the output current. The channel was initially filled with phosphate buffer (0.1 M, pH 7.0) to allow the output current to stabilize and then this solution was replaced with test solutions containing sugars. Fluids were introduced into the channel using a syringe. In our initial test, we sequentially introduced fluids with increasing concentrations of glucose (0.5, 1.0, 1.5, and 2.0 mM). As shown in Figure 7b, each fluid replacement led to a spike in output current followed by a period in which the output current stabilized to a constant steady state value. Figure 7b shows that the steady state output current increased with the solution’s glucose concentrations. After addition of the last glucose solution, we injected buffer into the channel and observed that the output current returned to its original, nearzero value. These results indicate that GOx can be anodically codeposited at an electrode address within a covered microfluidic channel and that this codeposited enzyme retains its biological activity. Next, we injected solutions containing high concentration of alternative sugars (8 mM of either sucrose or fructose). Figure 1186


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Figure 7. Codeposition of catalytically active protein. (a) Schematic diagram illustrating electrochemical biosensing with codeposited GOx. (b) Plot shows quantitative response in output current for fluids containing varying concentrations of glucose, and little response for controls containing 8 mM of either sucrose or fructose. An intermediate washing step was performed with phosphate buffer containing 0.01% Tween to remove noncovalently bound enzyme. (c) Standard curve for data in (b).

oxidative cross-linking mechanism of Scheme 1. Anodic chitosan deposition shares many of the benefits of biopolymeric films deposited through neutralization mechanisms: (i) deposition is rapid occurring in seconds to minutes; (ii) deposition is reagentless (except for NaCl) and, thus, provides a simple and safe method for thin film generation; (iii) deposition is spatially and temporally selective allowing for programmable assembly (e.g., with chips and microfluidic devices); and (iv) deposition should be “bio-friendly” as it proceeds from aqueous solution under ambient conditions and yields a hydrogel matrix. In contrast to biopolymeric physical gels that are deposited through a neutralization mechanism, the anodically deposited chitosan is stable over a range of pHs presumably because it is a covalently cross-linked chemical gel. As a result, these anodically deposited chemical gels possess pH-responsive swelling/deswelling properties that can be contrasted with chitosan’s cathodically deposited physical gels that dissolve at low pHs. The anodic-deposition of chitosan offers two characteristics that could be useful for technical applications. First, the ability to address active proteins at electrode surfaces (presumably through covalent linkages) provides a simple means to assemble components for multiplexed electrochemical biosensing. Second, the stimuli-responsive swelling/deswelling properties may provide a means to employ chemical stimuli to exert a mechanical force (e.g., for microfluidic valving) or to trigger the release of entrapped components (e.g., vesicles). Thus, we believe this novel electrodeposition method may provide a complementary approach to generate hydrogels for integrating biology74−77 and performing actuation functions78,79 in microfluidic devices.

7b shows no appreciable change in output current after these sugars were injected. To ensure the enzyme retained activity, we injected a solution of glucose (0.5 mM) and observed a spike and stabilization of the output current similar to that observed for the initial introduction of glucose (0.5 mM). This result indicates that the codeposited GOx retains its chemoselectivity for detecting glucose. Scheme 1 hypothesizes that anodic reactions partially oxidize chitosan to generate aldehyde groups that are able to react with primary amines to form Schiff base linkages. Potentially, these aldehydes would be able to react with the amines of proteins (i.e., GOx) to covalently conjugate them to the chitosan network. To provide evidence that GOx may be covalently conjugated to chitosan, we performed three sequential washes (each for 5 min) by injecting phosphate buffer (0.1 M, pH 7.0) containing 0.01% of the detergent Tween. These washes are expected to remove (or at least partially remove) any physically bound GOx. After these washes, we sequentially injected the same test solutions containing sugars. The output currents observed in Figure 7b were nearly identical to those observed prior to performing the washing steps. This observation provides evidence that anodic codeposition may also result in the covalent conjugation of the protein to the network. Finally, Figure 7c summarizes the results in Figure 7b by showing the standard curve between the steady state output current and sugar concentration. As expected, this curve is nearly linear for glucose while minimal output currents are observed for sucrose and fructose. Also, the glucose standard curves are similar before and after washing with Tweencontaining buffer.

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CONCLUSIONS We report the anodic electrodeposition of chitosan hydrogel films, and provide physical and chemical support for the 1187


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from Robert W. Deutsch Foundation, National Science Foundation (EFRI-0735987), Defense Threat Reduction Agency (BO085PO008), and the Office of Naval Research (N000141010446).

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