Characterization of Electroconductive Blends of Poly (HEMA-co

Biomacromolecules 2009, 10, 2539–2549

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Characterization of Electroconductive Blends of Poly(HEMA-co-PEGMA-co-HMMA-co-SPMA) and Poly(Py-co-PyBA) Gusphyl Justin† and Anthony Guiseppi-Elie*,†,‡ Center for Bioelectronics, Biosensors and Biochips (C3B), Clemson University Advanced Materials Center, 100 Technology Drive, Anderson, South Carolina 29625, and Departments of Chemical and Biomolecular Engineering and of Bioengineering, Clemson University, Clemson, South Carolina 29634 Received April 27, 2009; Revised Manuscript Received June 19, 2009

Electroconductive hydrogels (ECH) prepared as blends of UV-cross-linked poly(hydroxyethylmethacrylate) [p(HEMA)]-based hydrogels and electropolymerized polypyrrole (PPy) were synthesized as coatings on microlithographically fabricated interdigitated microsensor electrodes (IMEs) and microdisc electrode arrays (MDEAs). Hydrogels were synthesized from tetraethyleneglycol diacrylate (TEGDA), hydroxyethylmethacrylate (HEMA), polyethyleneglycol monomethacrylate (PEGMA), N-[tris(hydroxymethyl)methyl]-acrylamide (HMMA), and 3-sulfopropyl methacrylate potassium salt (SPMA) to produce p(HEMA-co-PEGMA-co-HMMA-co-SPMA) hydrogels. The conductive polymer was synthesized from pyrrole and 4-(3′-pyrrolyl)butyric acid by electropolymerization within the electrode-supported hydrogel. ECH films produced with different electropolymerization charge densities were investigated using cyclic voltammetry, electrical impedance spectroscopy, differential scanning calorimetry (DSC), and thermal gravimetric analysis (TGA). Polymer morphology was studied by SEM. The ECH demonstrated the desired characteristics of high electrical conductivity (low impedance), as well as high thermal stability compared to pure hydrogel. Signal enhancement was achieved by modifying the surface of an MDEA biotransducer with the ECH, with a 10-fold increase in the voltammetric current response associated with the ferrocene monocarboxylic acid (FcCO2H) redox reaction.

1. Introduction Electroconductive hydrogels are blends or conetworks of inherently conducting electroactive polymers (CEPs) and highly hydrated hydrogels. First described by Wallace et al.1 and later by Guiseppi-Elie et al.,2 these polymeric networks offer the promise of engineered biocompatibility associated with the hydrogel component and the low electrical interfacial impedance, both ionic and electronic, associated with the inherently conductive polymer component. Since the early work, ECH have been the subject of growing attention.3 Electroconductive hydrogels have been fashioned as the biorecognition membrane layer in various biosensors.4,5 An electrically conducting hydrogel comprising a poly(hydroxyethyl methacrylate) [poly(HEMA)] and polypyrrole (PPy) conetwork was investigated for potential applications to implantable electronic devices. Such devices may include neuronal prosthetic and recording devices (NPDs and NRDs),6-9 electrostimulated drug release devices (ESRDs),10-12 and implantable electrochemical biosensors.13-18 For such implantable devices, there is a need for soft polymeric materials that are also electrically conducting to serve as low impedance and noncytotoxic interfaces between the device and native living tissue. The material should be capable of inhibiting the detrimental effects associated with the inflammatory response that eventually lead to (fibrous) device encapsulation. This may be achieved either through the electrostimulated release of bioactive agents at the site of implantation or through resistance to protein * To whom correspondence should be addressed. Phone: +1-864-6561712. Fax: +1-864-656-1713. E-mail: [email protected]. † Clemson University Advanced Materials Center. ‡ Clemson University.

adsorption. For implantable electrochemical biosensors, such as the subcutaneous or intramuscular glucose and lactate sensors described by Guiseppi et al.,13 Schuvailo et al.,19 Petrou et al.,20 and Moussy et al.,21-24 it is important that the electrode surface resists protein adsorption and denaturation (fouling) and eventual fibrous capsule formation, as this can lead to an appreciable reduction in the current response and consequently a low signalto-noise ratio.25 It has also been proposed that initial protein adsorption is a principal precursor to the inflammatory cascade.18,26 Increasingly, however, the evolving view is that it is protein denaturation pursuant to adsorption that may be the carrier of molecular cues (appropriate amino acid sequences) that provoke the inflammatory cascade.27,28 Blends and conetworks of hydrogels and conductive electroactive polymers (CEP) are promising, stimuli-responsive, multifunctional materials with its CEP component molecularly engineered to reduce interfacial electrical impedances. Blends and conetworks of poly(HEMAco-PEGMA) and a cross-linker, tetraethylene glycol diacrylate (TEGDA), have previously been formulated for electrochemical biosensor applications.13,29 By varying the ratios of the individual monomers within the hydrogel composition, one can influence the hydration characteristics, as well as the mechanical strength or rigidity of the final polymerized material. It is expected that appropriate ECH hydration and modulus that simulate or mimic the characteristics of living tissue are both amenable to reduced fibrous encapsulation once implanted. Polypyrrole was selected as the CEP of interest to form the conetwork with the acrylate-based hydrogel. Polypyrrole has been previously studied and was selected on the basis of its known biocompatibility9,30,31 and its relative ease of polymerization (chemical oxidation or electropolymerization).

10.1021/bm900486d CCC: $40.75  2009 American Chemical Society Published on Web 08/25/2009

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Biomacromolecules, Vol. 10, No. 9, 2009

Table 1. Monomer and Prepolymer Constituents and Composition of the Hydrogel Cocktaila composition (mol %) components

poly(HEMA)

poly(HEMA)-PPy

HEMA TEGDA PEGMA 3SPMA IIMMA poly(HEMA) DMPA PyBA Py ethylene glycol water

84.0 3.0 5.0* 0 5.0 2.0* 1.0 0 0 10 wt %/wt 10 wt %/wt

62.5 3.0 5.0* 5.0 5.0 2.0* 1.0 1.5 15 10 wt %/wt 10 wt %/wt

a

*Represents mol % of repeat units.

This paper reports on the electrical, electrochemical, and impedimetric characterization of electroconductive hydrogels of tetraethyleneglycol diacrylate cross-linked poly(HEMA-coPEGMA-co-HMMA-co-SPMA) that have been fashioned into conetworks with poly(pyrrole-co-4-(3′-pyrrolyl)butyric acid). The former hydrogel network was prepared as spun-applied membranes on chemically modified interdigitated microsensor electrodes and microdisc electrode arrays and UV polymerized. The polypyrrole copolymer was synthesized by electropolymerization of pyrrole and 4-(3′-pyrrolyl) butyric acid monomer that were physically entrapped within the p(HEMA)-based hydrogel while supplemented by pyrrole monomer that was in the bathing solution. In this system, the 3-sulfopropyl methacrylate potassium salt (SPMA) of the hydrogel component serves as the counteranion to the positively charged, oxidized form of the conductive electroactive polypyrrole. The 4-(3′pyrrolyl) butyric acid (PyBA) of the conductive, electroactive component serves as a hydrophilic monomer that can potentially establish electrostatic interactions with the HMMA monomer of the hydrogel component. In this way, molecular intimacy is designed into the two polymer system that favors chemical compatibility and blend formation. The influence of electropolymerization charge density on the membrane morphology, electrical/electrochemical characteristics, thermal and optical absorption characteristics were also studied.

2. Materials and Methods 2.1. Hydrogel Composition and Preparation. The following components were used in the preparation of a base nonconducting hydrogel and an electrically conducting hydrogel conetwork (Table 1). Hydroxyethyl methacrylate (HEMA), tetraethyleneglycol diacrylate (TEGDA, technical grade), N-[tris(hydroxymethyl)methyl]-acrylamide (HMMA, 93%), 2,2-dimethoxy-2-phenylacetophenone (DMPA, 99%), pyrrole monomer (reagent grade, 98+%), 4-(3′-pyrrolyl) butyric acid (PyBA), and 3-sulfopropyl methacrylate potassium salt (SPMA) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Poly(ethyleneglycol)(200)monomethacrylate (PEGMA) was purchased from PolySciences, (Warrington, PA). The diacrylate and methacrylate reagents were passed through an inhibitor removal column (Sigma-Aldrich, St. Louis, MO) before use for removal of the polymerization inhibitors hydroquinone and monomethyl ether hydroquinone polymerization. Pyrrole monomer was passed through an alumina silicate column for inhibitor removal. The hydrogel cocktails were prepared by mixing HEMA, TEGDA, PEG(200)MA, HMMA, and DMPA in a typical ratio 86:3:5:5:1 mol % for the native hydrogel. For the ECH formulation, 3-sulfopropyl methacrylate potassium salt (SPMA), pyrrole monomer, and 4-(3-pyrrolyl) butyric acid (PyBA) were also included. The mixtures were added to a 1:1 (v/v) solution of ethylene glycol/water (20 wt % of initial mixture).

Justin and Guiseppi-Elie 2.2. IME and MDEA Surface Cleaning, Modification, and Hydrogel Application. The cleaning, modification, and derivatization of electrode surfaces for the application of hydrogel films has previously been described.32,33 The interdigitated microelectrode (IME 1550.5 M Au U; 10 µm line and space, 5 mm long digits, 50 digits per bus; 0.05 cm2) and microdisc electrode array (MDEA 050 Au; 50 µm diameter disks, 5,184 disks) devices were purchased from ABTECH Scientific (Richmond, VA). Trichloroethylene (spectrophotometric grade, g99.5%, Sigma-Aldrich), acetone (g99.9%, Sigma-Aldrich), 2-propanol (g99.8%, Sigma-Aldrich),ammoniumhydroxidesolution(ACSreagent,28.0-30.0% as NH3, Sigma-Aldrich), hydrogen peroxide solution (ACS reagent, 30 wt. % in H2O, Sigma-Aldrich), ethanol (CHROMASOLV, SigmaAldrich), and anhydrous toluene (99.8%) are essential to the cleaning procedure for the electrode surfaces and were used as received. All IMEs and MDEAs were cleaned and functionalized for subsequent attachment of the hydrogel layer. Both the IME and MDEA devices were cleaned by sequential ultrasonic washing in boiling trichloroethylene (3 min, 86.7 °C), acetone (1 min; 56.2 °C), and 2-propanol (1 min; 82.4 °C) and then washed profusely in room temperature (RT) deionized water. To remove adventitious chemisorbed organic residues, the MDEA devices were treated for 10 min in the UV-ozone cleaner (UV_Clean, Boelkel Industries, PA), washed by ultrasonication in 2-propanol, and then washed profusely in room temperature deionized water. To remove residual organic/ionic contamination and to produce a uniform, reproducible layer of -OH groups on the surface of the Si3N4 (activation), the electrodes were immersed in a (5:1:1, v/v/v) 60 °C solution of DI H2O/NH4OH/H2O2 (RCA clean), held for about 10 s, quenched in DI water for 1 min, and then washed profusely with running DI water, followed by exposure to radiofrequency (RF) glow discharge plasma for 10 min using a Harrick Plasma Cleaner/Sterilizer PDC 32G (Ithaca, NY), periodically allowing humidified air to bleed into the chamber. 3-Mercapto-1-propanol (95%) and (3-aminopropyl)trimethoxysilane (γ-APS, 97%) used for modification of the gold and nitride surfaces for covalent hydrogel attachment were purchased from Sigma-Aldrich Co (St. Louis, MO). The gold surface of the cleaned and activated devices was chemically modified with 0.01 M 3-mercapto-1-propanol (in anhydrous ethanol) and stored at room temperature overnight to introduce pendant alkylhydroxy surface functionalities. The pendant alkylhydroxy and surface -OH groups of the silicon nitride surface were subsequently functionalized by treatment with 3-(aminopropyl)trimethoxysilane (γ-APS; 0.01 M, in anhydrous toluene, RT, 2 h) to introduce pendant alkylamino surface functionalities. The devices were cured at 40 °C for 20 min, then 110 °C for 20 min, and then 40 °C for 20 min for a total of 1 h. For the final functionalization and to establish a continuous path of covalent bonding between the device surfaces and the hydrogel layer, the devices were incubated for 2 h in acryloyl(polyethyleneglycol)-N-hydroxysuccinamide (Acrl-PEG-NHS, MW ) 3500) solution (0.001 M) made up in 0.1 M HEPES (pH ) 8.5), prepared under UV filtered conditions. Acrl-PEG(3500)-NHS was purchased from Jenkem Technology U.S.A. (Allen, TX). Under UV-free conditions, the final hydrogel cocktail was sonicated, sparged with nitrogen gas, and applied evenly to the surface of the Acryl-PEG functionalized devices using a spin coater (1000 rpm for 10 s, followed by 3500 rpm for 30 s). The mixture was immediately irradiated with UV light (366 nm, 2.3 W/cm2, 5 min) in a UV crosslinker (CX-2000 CROSSLINKER, UVP, Upland, CA) under an inert nitrogen atmosphere to effect polymerization of the hydrogel component. Finally, the electrodes with the base hydrogel were conditioned and unreacted monomer extracted by sequential immersion in ethanol/ deionized water mixtures (100% ethanol, 75:25; 50:50; 25:75; 100% DI water; v/v) for a minimum of 1 h each. The electrodes with the pyrrole monomer containing hydrogel were immersed in a saturated pyrrole solution for subsequent electropolymerization of PPy. 2.3. Electropolymerization of PPy, Electrochemical Impedance Spectroscopy, and Cyclic Voltammetry. Electropolymerization of polypyrrole was achieved using either of two techniques: (1) chrono-

Electroconductive Hydrogel Blends


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Scheme 1. Surface Modification Procedure and the Final ECH Membrane Covalently Attached to a Microdisc Electrode Array (MDEA) Surface

amperometry, which involves applying a 0.70 V (vs Ag/AgCl) potential for a defined period of time; (2) chronopotentiometry, which entails fixing the current at 1 mA and defining a specific charge limitation for electropolymerization. The hydrogel coated electrode surfaces were bathed in a saturated pyrrole monomer solution (approximately 0.4 M pyrrole monomer in 0.1 M Tris/0.1 M KCl solution, pH ) 6.1) for approximately 1 h prior to electropolymerization to ensure adequate hydration of the gel and equilibration of the pyrrole monomer and electrolytes between the solution and hydrogel phases. Tris buffer solutions (0.1 M adjusted to pH ) 7.2 and pH ) 6.1 as needed) were made from Tris(hydroxymethyl)aminomethane or Trizma (99.8+%, A.C.S., Sigma-Aldrich). A PAR 283 Potentiostat/Galvanostat (AMETEK Princeton Applied Research, Oakridge TN) coupled with a Solartron 1260 Frequency Response Analyzer (FRA, AMETEK Solartron Analytical, Oakridge, TN) linked to a Dell PC controlled by EG&G PowerSine and ZView and ZPlot softwares was used for electropolymerization of PPy within the hydrogel membrane and for electrochemical impedance spectroscopy (EIS) for the evaluation of impedance changes associated with electropolymerization of PPy, respectively. Multiple scan rate cyclic voltammetry (MSRCV) was performed using a three-electrode (working, counter, and reference) electrochemical setup using the PAR 283 under the control of the PowerSuite software or the BAS 100 Potentiostat/Galvanostat Electrochemical Analyzer under the control of the BAS 100 software (Bioanalytical Systems, West Lafayette, IN). EIS and MSRCV were both performed in a 0.1 M Tris/0.1 M KCl buffer solution (adjusted to pH ) 7.2) made from Tris(hydroxymethyl)aminomethane (99.8+%, A.C.S., Sigma-Aldrich) and potassium chloride (Sigma-Aldrich). In both instances, a coiled platinum wire or platinum mesh (Alfa Aesar, Ward Hill, MA) was used as the auxiliary electrode, while a silver/silver chloride (Ag/AgCl) electrode (ABTECH Scientific, Richmond, VA) was used as the reference. To investigate the voltammetric current response for an ECH-modified MDEA biotransducer, MSRCV was also performed in a solution of 1 mM ferrocene monocarboxylic acid (FcCO2H, 97%, Sigma-Aldrich) made up in 0.1 M Tris/0.1 M KCl buffer solution. Scheme 1 illustrates the surface modification procedure and the final ECH membrane covalently attached to a microdisc electrode array (MDEA) surface. Throughout this paper, Acryl-PEG functionalized electrodes, MDEA or IME, will be denoted with an asterisk (*), for example, MDEA* or IME*.

2.4. UV-Vis Spectroscopy. The various hydrogel formulations were coated onto indium tin oxide (ITO) surfaces. Because ITO has a conductive surface, electropolymerization of PPy can occur as described previously, following cleaning and functionalization for attachment of the hydrogel films. The fact that ITO is transparent permits its use in UV-Vis spectroscopy. The absorbance spectra of the following surfaces were obtained using a Perkin-Elmer Lambda 950 UV Vis spectrophotometer (Perkin-Elmer, Waltham, MA): (i) ITO, (ii) ITO*|Gel, (iii) ITO|PPy, and (iv) ITO*|Gel-P(Py-co-PyBA) (three charge densities), where the asterisk (*) indicates an Acryl-PEG functionalized surface. 2.5. Thermal Analysis and Microscopy Characterization. Thermogravimetric analysis (TGA) was performed using a Mettler Toledo TGA/SDTA851e, while differential scanning calorimetry (DSC) was performed using a TA Instruments DSC 2920 Modulated DSC. Hydrogel samples were initially fully hydrated and then lyophilized overnight. Approximately 10 mg of each sample was weighed for use with the TGA and DSC instrumentation. Scanning electron microscopy (SEM) of the hydrogel and hydrogel-PPy membranes were performed on a Hitachi S-4800 UHR FE-SEM and conducted at the central Electron Microscopy Facility at Clemson University’s Advanced Materials Center. The samples were first freeze fractured in liquid nitrogen and then sputter coated with platinum using a Hummer Sputter Coater.

3. Results and Discussion 3.1. Synthesis of Electroconductive Hydrogels. The hydrogel cocktail of UV polymerizable monomers uniquely contains 4-(3′-pyrrolyl)butyric acid (PyBA). Its role is to serve as a comonomer and to become coelectropolymerized with the free pyrrole (Py) monomer, thereby forming poly(pyrrole-co4-(3-pyrrolyl)butyric acid) [poly(Py-co-PyBA)], which serves as the inherently conductive and electroactive component of the polymer blend. The PyBA monomer being polar and hydrophilic imparts hydrophilic compatibility between the electroactive and hydrogel components of the polymer blend and so favors better distribution of the [poly(Py-co-PyBA)] within the p(HEMAco-PEGMA-co-HMMA-co-SPMA). There is also the potential, under appropriate pH conditions, for the carboxylate form of the PyBA to have an electrostatic interaction with the quaternary

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Justin and Guiseppi-Elie

Figure 1. Top: Schematic illustration of (A) the p(HEMA-co-PEGMA-co-HMMA-co-SPMA) hydrogel following UV-polymerization and of (B) the p(HEMA-co-PEGMA-co-HMMA-co-SPMA)-P(Py-co-PyBA) hydrogel conetwork following electropolymerization. Bottom: Illustration of functional groups that make up ECH conetwork.

form of HMMA. The hydrogel cocktail also uniquely contains the monomer 3-sulfopropyl methacrylate potassium salt (SPMA) that is incorporated into the hydrogel component. The monomer serves as the counteranion to the positively charged, oxidized form of the conductive electroactive poly(Py-co-PyBA)]. The hydrogel cocktail of UV polymerizable monomer was spun applied to the surfaces of the chemically modified and derivatized IME* and MDEA* chips at a final spin speed of 3500 rpm for 35 s. For the viscosity of this cocktail, the spin speed of 3500 rpm produced a ∼5 µm thick film as measured by freeze facture SEM. Pyrrole (Py) and Py with PyBA were electropolymerized onto the surfaces of uncoated as well as within the membrane layer of hydrogel-coated IME* and MDEA* chips. This allowed the direct comparison of the electrical/electrochemical performance and properties of PPy films with those of the ECH conetwork of Gel-PPy films. Six different levels of PPy electropolymerization corresponding to 0, 25, 50, 75, 100, and 200 mC of charge passed were studied. Chips with the hydrogel layer and no electropolymerization charge (0 mC) served as controls and chips with no hydrogel membranes (IME* and MDEA*) served as blanks. Figure 1 is a schematic illustration of the hydrogel and ECH conetwork formed by this process of UV cross-linking followed by electropolymerization. Figure 2 shows the chronopotentiograms produced by galvanostatic (1 mA, fixed current) electropolymerization of Py to form IME*|PPy and IME*|Gel-P(Py-co-PyBA) chips (in the context of the electrically conducting gel, PPy will throughout this article be used generically to refer to the Py-co-PyBA copolymer). The electropolymerization of pyrrole and 4-(3pyrrolyl)butyric acid is an acid favored reaction. The kinetics of such reactions are favored under slightly acidic conditions; accordingly, the pH was adjusted to 6.1. Because the oxidoreductase family of enzymes of interest tend to have slightly acidic pH optima (e.g., 5.4 for GOx), there is not expected to

Figure 2. Differing chronopotentiometric profiles associated with the electropolymerization to form PPy on (A) IME* and (B) IME*|Gel. The PPy polymerizes more readily within the hydrogel layer compared to on an unmodified chip.

be a performance issue. Galvanostatic electropolymerization has the advantage of fixing the kinetics of electrochemical discharge of the Py monomer and allowing the ready control of charge density for electropolymerization. However, the electrode potential is free to wander according to the value needed to support that impressed current. Figure 2 shows that each of the two IME* groups (uncoated and hydrogel-coated) cluster with clearly characteristic patterns. For the uncoated IME*, PPy electropolymerization resulted in a decrease in potential between 0.8 V and 0.7 V, with a 50% drop in the potential occurring within the first 14 s (Figure 2A) for the 200 mC chip. For the



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Figure 3. Optical micrographs of electropolymerized PPy films grown on IME* (left) and on IME*|Gel devices (right). Electropolymerization charge of 25, 50, 100, and 200 mC cm-2.

IME*|Gel-P(Py-co-PyBA) (200 mC) chip; however, the initial potential decrease proved to be a lot sharper and over a slightly larger potential drop (0.8 and 0.65 V), with a 50% decrease in the potential occurring within the first 2 s of electropolymerization for the 200 mC (Figure 2B). It is clear that PPy electropolymerization within the hydrogel membrane leads to more favorable electrode polarization than it does when directly electropolymerized onto IME* interdigitated gold surfaces. This more rapid fall to lower potentials (ca. 0.65 V vs ca. 0.72 V) indicates more favorable electropolymerization kinetics for the formation of P(Py-co-PyBA) within the hydrogel compared to the formation of pure PPy. The presence of PyBA and SPMA within the original spun applied hydrogel membrane likely supports the more rapid initial electropolymerization. Also, the hydrogel milieu, while about 35% hydrated in DI water,34 likely supports a more favorable partitioning of Py into the near surface membrane layer that favors electropolymerization. 3.2. ECH Morphology by Optical Microscopy and SEM. Optical micrographs of the IME*|PPy and IME*|Gel-P(Py-coPyBA) chips show clear differences in the amount and distribution of electropolymerized PPy (Figure 3). At 25 mC, both chips show electropolymerized PPy originating at the digits. With increasing charge, the PPy grows laterally becoming more diffuse. At 50 mC, the growth of PPy can be seen, in the case of IME*|Gel-P(Py-co-PyBA), to bridge the digits but to remain diffuse at the IME*|PPy chip. Merging of PPy between the digits does not occur until 100mC for the IME*|PPy. Ultimately, growth within the hydrogel membrane layer produces a more uniform, homogeneous PPy film. These images appear to support the view that PPy grows within the hydrogel matrix to form a polymer blend or conetwork. SEM images of a base hydrogel, a uniformly polymerized ECH and a multilayered PPy-Gel film are shown in Figure 4. Differences in the gross hydrogel morphology can be seen between the base hydrogel and the ECH. The multilayered PPy-

Gel film was produced potentiostatically by the application of higher electrode potentials (0.8 V vs Ag/AgCl) and leads to the formation of a distinct PPy layer beneath the previously spun applied hydrogel layer. For the formation of a contiguous layer of PPy within the hydrogel membrane, lower potentials (between 0.7 and 0.75 V vs Ag/AgCl) were used. The inclusion of the more soluble PyBA within the hydrogel formulation also facilitates the formation of the PPy within rather than beneath the hydrogel layer. 3.3. Electrical Properties of Electroconductive Hydrogels. The electrical properties of electroconductive hydrogels were investigated by a form of linear polarization resistance (LPR) in solution. This technique can be used to determine the I-V characteristics of electrified interfaces that for purely capacitative systems can reveal the double layer capacitance and for purely resistive systems reveal the resistance. An electrode immersed in an electrolyte solution and subjected to minor potential perturbations ((5 mV) away from the quiescent potential using extremely low scan rates, ν (typically 10 mV/s), leads to a hysteresis-shaped plot with some degree of separation between the positive and negative currents over the defined potential range.33,35 The current difference, ∆I, between the positive and negative currents may be used in the following equation to determine the double layer capacitance, CDL

CDL )

∆I 2ν

(1)

For electrodes modified with a conductive electroactive polymer, both the positive and negative currents increase almost linearly indicating Ohmic behavior and a narrow hysteresis profile is typically observed. The observed hysteresis in the LPR response is an indication that the system is not entirely resistive, but that a capacitive component still exists associated with the

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Figure 4. Top-left: SEM of an unmodified hydrogel membrane layer that was UV polymerized onto a functionalized MDEA device surface. Bottom-left: SEM of an electrically conducting hydrogel membrane layer following electropolymerization to form PPy on an MDEA and grown within the hydrogel. Right: SEM of PPy formation at interface between electrode and applied hydrogel. This structure occurs normally in the absence of the bifunctional monomer or at higher potentials (0.85 V vs Ag/AgCl).

Figure 6. Conductivity of polymer-modified IME chips as determined using a linear polarization resistance (LPR) technique. Increasing conductivity (associated with increasing gradient) with increasing charge for IME*|PPy. Little change observed for the IME*|Gel-P(Pyco-PyBA); however, the conductivity is initially higher than the IME*|PPy chips for charge densities of 25, 50, and 75 mC and lower for 100 and 200 mC (IME 1050 cell constant is 0.04 cm-1).

Figure 5. Linear polarization resistance (LPR) of (A) Au*|Gel (before electropolymerization), and (B) Au*|Gel-P(Py-co-PyBA) (after electropolymerization) showing the 3 orders of magnitude change in the current and the change from predominantly capacitative to predominantly resistive behavior.

charging of an electrified interface. Figure 5 shows typical LPR plots for IME*|Gel (Figure 5A) and IME*|Gel-P(Py-co-PyBA) (Figure 5B). The comparison of the I-V profiles obtained for the hydrogel and the ECH demonstrates the shift in the nature of the system from one that initially comprised of both capacitive and resistive components, to one that is predominantly resistive. Based on a linear response approximation, the resistance at the IME*|Gel-PPy|solution can be determined.

Similar I-V curves were obtained for both systems as a function of the electropolymerization charge for the IME*|PPy and IME*|Gel-P(Py-co-PyBA) at the five levels of PPy electropolymerization studied. Resistances obtained in this way were converted to conductance values and these used to obtain conductivity using the IME device cell constant (0.04 cm-1). Figure 6 is a plot of the measured conductivity of the IME*|PPy and the IME*|Gel-P(Py-co-PyBA) systems as a function of electropolymerization charge. An increase in conductivity with increasing PPy electropolymerization charge or content is observed for the IME*|PPy from 8 µS cm-1 (at 25 mC) to 76 µS cm-1 (at 200 mC). Here the initial conductivity reflects that of the buffer solution and the sharp increase reflects the coalescence of the PPy film. For the IME*|Gel-P(Py-co-PyBA), the conducitivity remains relatively constant, with an average value of 54 ((5) µS cm-1. The current system described here uses a polymeric hydrogel network of pendant sulfonates as a multivalent macro anion. The results of the electrical conductivity of the electroconductive



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Figure 7. IME*|PPy and IME*|Gel-P(Py-co-PyBA): Planar EIS (coplanar counter and working electrodes). Bode impedance magnitude and phase plots for the IME*|PPy (left) and IME*|Gel-P(Py-co-PyBA) (right) measured using the coplanar arrangement of counter and working electrodes.

Figure 8. IME*|PPy and IME*|Gel-P(Py-co-PyBA): Trans EIS (external counter electrode). Bode impedance magnitude and phase plots for the IME*|PPy (left) and IME*|Gel-P(Py-co-PyBA) (right) measured using an external counter electrode.

hydrogel, based on the LPR technique, are quite interesting, as they suggest that the quantity of PPy (charge density) necessary to achieve an equivalent conductivity is lower when incorporated within a hydrogel matrix compared to the alternative, that is, direct electropolymerization onto an electrode. The exact reason for this phenomenon is unclear; however, it may be postulated that the lower resistances are due to the greater ease in electronic and ionic one-dimensional hopping along the PPy network within the gel, compared to directly electrodeposited PPy. Greater ease of electronic hopping and ionic charge transfer would be associated with faster redox switching speeds for the PPy within the gel. 3.4. Electrochemical Impedance Spectroscopy (EIS). The design of the IME 1550 devices, which comprises two independently addressable electrodes with multiple interpenetrating fingers, permits the ability to conduct two-electrode impedance spectroscopy measurements within a single plane (planar impedance measurements) and, when short-circuited, permits the ability to conduct two-electrode impedance spectroscopy measurements across the electrode solution interface (trans impedance measurements). For trans impedance spectroscopy measurements, the two independently addressable electrodes of the IME chip are shorted and used as a single working electrode. An external counter electrode (platinum wire) would be used as the second electrode. Planar EIS measurements were performed between the two independently addressable electrodes of the IME chip. This IME design is useful, as it allows the ability to measure impedances within a single plane of the PPy

film or ECH film in a planar EIS configuration. In the trans EIS configuration, insight into the impedance characteristics across the PPy and ECH films and the nature of the PPy|solution and ECH|solution interfaces can be obtained. Bode impedance and phase plots obtained in the planar EIS configuration for the IME*|PPy (Figure 7) show that PPy does not completely merge across the digits of the IME devices until 100 mC of charge was passed, leading to the formation of a perfect resistor (θ ) 0 across frequencies, |Z| ∼ 10 Ohms). For the IME*|Gel-P(Py-co-PyBA) in planar EIS configuration, the Bode plots almost immediately demonstrate completely resistive behavior. After 25 mC of charge is passed, the impedance has already dropped to 100 Ohms, and at 50 mC, complete resistive behavior is attained, with a resistance of 10 Ohms. This supports the hypothesis that PPy is polymerizing through the gel to form a true conetwork of gel and PPy, and that this polymerization occurs more readily within the gel, resulting in an almost immediate bridging of the gaps between the IME fingers compared to PPy electropolymerization directly onto the electrodes. In Figure 8, the Bode impedance plot for the trans EIS configuration shows a decrease in |Z| with PPy polymerization (by about 1 order of magnitude) for both the IME*|PPy and IME*|Gel-P(Py-co-PyBA) chips. The impedance plots are very similar across all charge densities, particularly for the IME*|Gel-P(Py-co-PyBA) chips. The phase plot demonstrates a deviation from the unmodified chip behavior at between 102 and 103 Hz, indicating a shift toward more resistive behavior within that frequency domain with the increasing PPy

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Figure 9. Nyquist plot and Bode impedance magnitude and phase plots for the IME*|PPy and IME*|Gel-P(Py-co-PyBA) (100 mC) measured using an external counter electrode and at offset potentials of -500, 150, and +800 mV vs Ag/AgCl. Table 2. Equivalent Circuit Parameters for IME*|Gel-PPy and IME*|PPy (100 mC) electrode Gel-PPy PPy

offset potential (mV) -500 150 800 -500 150 800

Rsol (Ω) 71.2 73.1 78.9 65.8 65.2 60.9

error (%) 1.05 1.20 1.52 2.08 2.16 2.01

Rct (kΩ) 137 15 5 96 16 9

content. This frequency range is physiologically relevant as it corresponds to the frequency range of neural potential signals.7 Although similar trends were observed between the IME*|PPy and IME*|Gel-P(Py-co-PyBA) chips, the impedance and phase plots for the Gel-P(Py-co-PyBA) membranes were noticeably much tighter than for PPy alone. In considering the phase plots, a phase angle of -23° was obtained for the PPy membrane at 1 kHz frequency, but only -8° for the Gel-P(Py-co-PyBA) membrane at 25 mC. At increasingly higher charge, the phase angle of the PPy film approaches that of the ECH film, with the two eventually becoming almost equal in value (-9° for the ECH membrane and -10° for the PPy membrane) at 200 mC and 1 kHz frequency. In the context of implantable neural signal recording electrodes, this is an important result to consider. Previous groups have applied PPy directly to the surface of implantable neural electrodes with the goal of decreasing the interfacial impedance.7-9 A problem with this approach is the fact that PPy tends not to be very mechanically strong and can break or peel off the device surface with insertion into the tissue bed. By providing a hydrogel matrix for the copolymerization of PPy, a mechanically stronger material can be applied to the electrode surfaces. Hydrogels, such as our poly(HEMA-co-PEGMA-co-HMMA), are also biocompatible and decrease the incidence of protein adsorption.13 Therefore, an ECH film such as the one described in this paper, may serve well for such implantable device applications, as it would possess desirable properties such as good biocompatibility, low protein adsorption, high mechanical strength (determined by qualitative inspection and comparison), and high hydration (typical of such hydrogels) without compromising the impedance that would otherwise be obtained with just PPy, a fact that is clearly shown in the Bode impedance and phase plots for the trans EIS configuration. Also, with less PPy content required for achieving similar impedances, when incorporated into the hydrogel compared to directly onto the electrode surfaces, it is possible that one may be able to optimize the system to reduce the PPy content further, thus facilitating as little impact as possible on the mechanical properties of the hydrogel film.

error (%) 33.70 4.41 3.54 41.46 8.25 5.19

CPE-T -5

8.30 × 10 5.61 × 10-5 5.66 × 10-5 7.60 × 10-5 5.74 × 10-5 4.48 × 10-5

error (%)

CPE-P

error (%)

2.00 2.74 4.60 2.08 4.55 4.90

0.84 0.85 0.81 0.83 0.83 0.86

0.66 0.78 1.22 1.20 1.30 1.26

In Figure 9, the Bode phase plot indicates a shift toward resistive behavior with increasing offset potential. Similar impedances and phase shifts were observed for the IME*|GelP(Py-co-PyBA) and IME*|PPy. These results support the earlier conclusion that the conductivity of PPy is not compromised by its incorporation into the hydrogel. Table 2 lists the equivalent circuit parameters for the IME*|PPy and the IME*|Gel-P(Pyco-PyBA) systems, based on a Randles-like network of solution resistor, Rsol, in series with a constant phase element, CPE, which is in parallel to a charge transfer resistor, Rct.32 A CPE-P value of 1 is indicative of a pure capacitor. For the IME*|PPy, the value of Rsol does not vary greatly across the various offset potentials, and the same is true for the IME*|Gel-P(Py-coPyBA). This is expected, as the concentration of electrolyte in the solution is assumed to be constant, because the same stock solution of 0.1 M Tris/0.1 M KCl was used in each case. However, the average Rsol for the IME*|Gel-P(Py-co-PyBA) (74.4 ( 4.0 Ω) is slightly higher than that of the IME*|PPy chip (64.0 ( 2.6 Ω). This difference may be accounted for by the presence of the hydrogel, which contributes to a slightly lower diffusion coefficient of ions and as a consequence a higher solution resistance within the diffusion boundary layer. In the case of Rct, there is clearly a decreasing trend, with Rct values of 5 kΩ for the Gel-P(Py-co-PyBA) and 9 kΩ for the PPy. There is an appreciably large decrease in Rct for both electrode systems, which is consistent with the fact that PPy is insulating in its reduced state (at -500 mV vs Ag/AgCl) and conducting in its oxidized state (at +800 mV vs Ag/AgCl). 3.5. Cyclic Voltammetry (CV). Cyclic voltammetry measurements of the IME*|PPy and IME*|Gel-P(Py-co-PyBA) indicate increasing oxidation and reduction currents with increasing electropolymerization charge density (Figure 10A,B). The magnitude of the currents for the IME*|PPy were about twice as large as those of the IME*|Gel-P(Py-co-PyBA) chip. The smaller currents for the latter are reflective of the reduced ionic mobility within the hydrogel. The apparent diffusion coefficient, Dappt, of ferrocene monocarboxylic acid (FcCO2H) through a base hydrogel has previously been



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Figure 10. Top: Increasing voltammetric current is associated with increasing PPy electropolymerization charge for both the IME*|PPy (A) and the IME*|Gel-P(Py-co-PyBA) (B). Peak currents are slightly smaller for the IME*|Gel-P(Py-co-PyBA). Bottom: Increasing voltammetric current is associated with increasing scan rate (10-500 mV/s) for both the IME*|PPy (C) and the IME*|Gel-P(Py-co-PyBA) (D).

determined.36 It was shown in this study that with the application of a hydrogel to the surface of an MDEA chip, a decrease in the peak current response, as well as a decrease in the gradient of the Randles-Sevcik-like plot (Ip vs υ1/2) occurs. From the gradients of the unmodified and hydrogelmodified chips, the diffusion coefficients can be calculated. A 2 orders of magnitude decrease in the Dappt was observed for FcCO2H within the gel compared to in solution. In the case of the IME chips, the decrease in the current response was not quite as great with application of the ECH film, compared to the PPy modified IMEs, indicating the important contribution of the PPy conetwork within the gel toward increasing the overall charge passed. Multiple scan rate cyclic voltammetry (MSRCV) also reveals the expected oxidation and reduction current dependence on scan rate. Increasing currents are associated with increasing scan rates for both the IME*|PPy and the IME*|Gel-PPy (Figure 10C,D). 3.6. UV-Visible Spectroscopy. UV-visible electronic absorption spectroscopy of hydrogel-modified planar indium tin oxide coated glass coverslips indicate, as expected, greater absorbance associated with the hydrogel-polypyrrole films compared to the films without electropolymerized polypyrrole (Figure 11A). ITO* and ITO*|Gel demonstrated very similar absorbances within the 375-800 nm region (Figure 11A, traces a and b). Electropolymerization of PPy within the gel resulted in an increase in absorbance (Figure 11A, traces c-f), with the greatest absorbance occurring for the hydrogelP(Py-co-PyBA) film associated with the greatest charge density (100 mC). ITO*|PPy (100 mC) demonstrated similar absorbance values to the ITO*|Gel-P(Py-co-PyBA) (100 mC) between 375 and 425 nm, with some deviation at higher wavelengths. Increasing the charge density during polymerization increases the amount of the electroconductive component within the film and consequently results in increased absorbance within the wavelength region of interest. The lack of a well-defined peak in the case of these absorbance spectra may be an indication of the oxidation state of polypyrrole.35 However, the clear increase and broadening in the near-

infrared (NIR) region is generally associated with increased conductivity. 3.7. Thermal Analysis: TGA and DSC. TGA revealed that both the native hydrogel and hydrogel-P(Py-co-PyBA) compositions were relatively stable between room temperature (25 °C) and 300 °C (Figure 11B). Beyond 200 °C, the native hydrogel began to demonstrate some decomposition, with dramatic decomposition occurring at about 400 °C. The hydrogel-P(Py-co-PyBA) composite maintained overall greater thermal stability than the hydrogel, similarly demonstrating dramatic decomposition of the sample at about 410 °C. The greater thermal stability of the composite relative to the hydrogel and the maintenance of a single decomposition temperature suggest a molecular level association between the two polymer constituents. The final higher residual mass of the composite following decomposition at about 430 °C also suggest a molecular level association between the two polymer constituents. Figure 11C shows thermograms obtained using DSC associated with the Gel and Gel-P(Py-coPyBA) formulations. Similar double peaks were obtained between 150 and 250 °C for the two samples. However, both transitions in the Gel-P(Py-co-PyBA) blend appear shifted toward higher temperatures and the higher temperature peak appears sharper. 3.8. Integration of ECH Membrane with Microdisc Electrode Arrays. Cyclic voltammetry at hydrogel-modified microdisc electrode arrays (MDEAs) has been recently described and explored for potential application in implantable biosensors and deep brain stimulation electrodes.33,35,36 Modification with electroconductive hydrogels presents the opportunity for reduced interfacial impedance. Gold MDEA 050s (50 µm diameter, 5184 discs, d/r ) 4; A ) 0.1 cm2) were coated with Gel, PPy, and Gel-P(Py-co-PyBA)) and studied by cyclic voltammetry (CV) in 1.0 mM ferrocene monocarboxylic acid (FcCO2H) made up in 0.1 M Tris/0.1 M KCl buffer solution. The hydrogel layer being fully hydrated and supportive of ready diffusive transport of

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Figure 11. (A) UV-Vis spectroscopy of (a) ITO*|Gel; (b) ITO; (c) ITO*|Gel-P(Py-co-PyBA) (25 mC); (d) ITO*|Gel-P(Py-co-PyBA) (50 mC); (e) ITO*|Gel-P(Py-co-PyBA) (100 mC); (f) ITO|PPy (100 mC). (B) TGA thermograms of native hydrogel and electrically conducting hydrogel GelP(Py-co-PyBA) (25-500 °C). (C) DSC thermograms of native hydrogel and electrically conducting hydrogel Gel-P(Py-co-PyBA) (0-230 °C).

Figure 12. MSRCV of MDEA 050 (in 1.0 mM FcCO2H in 0.1 M Tris/0.1 M KCl solution) (A) at the unmodified microdisc array electrode; (B) at the MDEA coated with the hydrogel membrane layer; (C) with an electropolymerized PPy membrane layer (50 mC); and (D) with an electroconductive hydrogel of Gel-P(Py-co-PyBA) (50 mC).

(FcCO2H) was shown (Figure 12B) to not adversely affect the cyclic voltammograms at the MDEA surface shown in Figure 12A. With the hydrogel coating, currents are only modestly attenuated, but the change in shape of the voltammograms reflects the shift toward increased radial diffusion. When coated with PPy, there is an apparent order of magnitude increase in the voltammetric current response (Figure 12C). However, there are no clearly defined redox peaks that can be associated with FcCO2H. When coated with Gel-P(Py-co-PyBA), the order of magnitude increase in the voltammetric response remains evident, but there now emerges clearly defined redox peaks for FcCO2H. An increase in the redox peak current for FcCO2H is directly attributable to the presence of the polypyrrole within the hydrogel film

that serves as an extension of the total electroactive surface area. Some portion of the response may be attributable to the electroactive properties of the conductive polymer component itself. However, the redox properties of PPy-SO3results in peaks that are centered about 200 mV versus Ag/ AgCl.37 Consequently, the responses shown here are likely due principally from the redox reaction of FcCO2H and the increased electroactive surface area engendered by the PPy component of the film. High peak currents are desirable for microbiosensor applications where limits of detection, as well as sensitivity are crucial. By applying an ECH film to a biosensor surface, it may be possible to significantly enhance the redox signal, limits of detection, and sensitivity, although the signal-to-noise ratio may not be improved.


4. Conclusion The electropolymerization of pyrrole has been shown to be associated with faster polymerization kinetics when occurring within a hydrogel matrix that contains PyBA and pendant sulfonate groups. The presence of P(Py-co-PyBA) within the poly(HEMA-co-PEGMA-co-HMMA-co-SPMA) membrane results in a dramatic decrease in the absolute impedance of the system by about 3 orders of magnitude compared to the native gel applied onto functionalized gold electrode surfaces. By increasing the amount of total charge passed during electropolymerization, greater amounts of PPy on the electrode surfaces and increased PPy content within the hydrogel conetworks were achieved. The goal of increasing the PPy content would be to further lower the absolute impedances at the electrode-solution interface. However, an analysis of the trans EIS (external counter electrode) indicates that increasing the PPy content grown within the hydrogel film does not significantly decrease the absolute impedance. Hence, reduced interfacial impedances may be achieved with minimal growth of PPy within the hydrogel coating. Coplanar EIS data and SEM images confirmed that polypyrrole grew throughout the hydrogel film and not simply at the interface between electrode and gel. Equivalent circuit modeling of the two systems (Gel vs Gel-P(Py-co-PyBA)) indicates that the hydrogel membrane contributes a greater apparent solution resistance value, Rsol. The charge transfer resistances, Rct, however, are comparable when the PPy component is in its oxidized state. The presence of poly(Pyco-PyBA) within the poly(HEMA)-based hydrogel network served to stabilize the blend or conetwork leading to increased mechanical strength/rigidity compared to a pure polypyrrole or pure hydrogel film. The thermal degradation properties of the ECH were enhanced compared to the hydrogel only and reflect the molecular compatibility between the poly(Py-co-PyBA) and the poly(HEMA)-based hydrogel. Use of high potentials (g0.8 V vs Ag/AgCl) in the chronoamperometric polymerization of PPy within the hydrogel matrix lead to formation of a multilayer structure with PPy growth occurring at the interface between the hydrogel and the electrode surface. The use of lower oxidation potentials or application of a chronopotentiometric/ galvanostatic technique that fixes current (kinetics) and total charge leads to a true blend or conetwork of PPy and hydrogel as evaluated by SEM. This structure was conserved regardless of the electropolymerization technique used: galvanostatic or potentiometric. Application of ECH films to MDEA transducer devices leads to enhanced voltammetric currents in the presence of a reversible redox active species such as FcCO2H. Electroconductive hydrogels appear to be highly suitable candidates to serve as coatings for implantable biosensors and neuronal prostheses. Acknowledgment. The authors would like to thank Dr. Stephen Foulger (MSE and COMSET) and Xiuxian He (Chemistry), both at Clemson University, for access to equipment. This work was supported by the U.S. Department of Defense (DoDPRMRP) Grant PR023081/DAMD17-03-1-0172 and by the Consortium of the Clemson University Center for Bioelectronics, Biosensors and Biochips (C3B).

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BM900486D

Characterization of Electroconductive Blends of Poly (HEMA-co

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