Synthesis of Water-Soluble Electroactive Ferrocene-Grafted Poly (p

Conjugated polymers as sensing materials have received considerable attention because of the high sensitivity of their optical and conducting properti...
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Notes Synthesis of Water-Soluble Electroactive Ferrocene-Grafted Poly(p-phenylene-ethynylene) via Phase Transfer and Its Biosensing Application Cuihua Xue,† Zhen Chen,† Fen-Tair Luo,‡ Kumaranand Palaniappan,† David J. Chesney,† Jian Liu,† Jian Chen,§ and Haiying Liu*,† Department of Chemistry, Michigan Technological University, Houghton, Michigan 49931, Institute of Chemistry, Academia Sinica, Taipei, Taiwan 115, Republic of China, and Zyvex Corporation, 1321 North Plano Road, Richardson, Texas 75081 Received December 4, 2004 Revised Manuscript Received January 24, 2005

Introduction Conjugated polymers as sensing materials have received considerable attention because of the high sensitivity of their optical and conducting properties to analytes.1 In particular, water-soluble conjugated polymers have been proven to be very useful in sensor applications for ions, peptides, proteins, bacteria, and nucleic acids.2,3 Conventional strategies for making conjugated polymers water soluble consist of introducing hydrophilic ionic side chains to the polymer in order to overcome π-π stacking interactions between the hydrophobic polymer backbones via electrostatic repulsion and enhance enthalpic interactions with water. Ionic groups such as carboxylic,3c sulfonic,4a ammonium,4b or phosphonate4c have been employed on side chains to conjugated polymers. Recently, neutral groups such as sugar5a and highly branched hydroxyls5b have been successfully grafted into the polymer for highly water-soluble poly(p-phenyleneethynylene)s. Here we report, for the first time, another effective approach to prepare water-soluble, electroactive ferrocene-grafted PPE (polymer B) via phase transfer. It is based on complexation of the ferrocene groups of the polymer A with β-CD or β-CD-modified gold nanoparticles (Scheme 1). Water-soluble redox conjugated polymers are very useful in establishing good contact between redox enzymes and the polymers, and to efficiently transfer electrons between redox enzymes and an electrode for biosensor applications. The water-soluble, polycationic polymer B was conjugated with polyanionic glucose oxidase (GOx) through electrostatic interaction. Immobilization of this polymer/enzyme complex on amineterminated monolayers at a gold electrode was achieved via a cross-linker, glutaraldehyde, which involves formation of imine bonds with amine groups from the enzyme residues and the monolayers. Electron transfer in redox polymer/ * To whom correspondence should be addressed. E-mail: [email protected]. † Michigan Technological University. ‡ Academia Sinica. § Zyvex Corporation.

enzyme composite film is achieved by hopping between immobile redox centers through collisions of mobile reduced and oxidized redox centers. The composite film displays a large charge-transfer diffusion coefficient of 7.3 × 10-7 cm2 s-1. The long, flexible, and hydrophilic tethers facilitate electron transfer between the enzyme and the electrode because the mobility of the tethered redox centers increases the rate of electron transferring collisions.6 Experimental Section Instrumentation. 1H NMR and 13C NMR spectra were recorded on a 400 MHz Varian Unity Inova spectrometer in the indicated solvents at the indicated fields. Chemical shifts are expressed in parts per million (δ) using residual solvent peaks as internal standards. Chloroform (δ 7.24 ppm for 1H and 77.00 ppm for 13C) was employed as an internal standard for chloroform-d. Splitting patterns are expressed as s (singlet), d (doublet), t (triplet), q (quartet), bs (broad singlet), or m (multiplet). UV spectra were taken on a HewlettPackard 8452A Diode Array Spectrophotometer. Fluorescence spectra were obtained on a 1681 steady-state Spex Fluorolog fluorometer. Fluorescence quantum yields of the polymers were measured in dilute chloroform solution and calculated by using quinine sulfate in 0.1 N sulfuric acid as the reference absolute quantum efficiency (φn ) 55%).7 The molecular weights of the polymers were determined by gel permeation chromatography (GPC) using a Waters Associates model 6000A liquid chromatograph. The mobile phase was HPLC grade THF which was filtered and degassed by vacuum filtration through a 0.5 µm Fluoropore filter prior to use. The polymers were detected by a Waters model 440 ultraviolet absorbance detector at a wavelength of 254 nm and a Waters model 2410 refractive index detector. The polymer solutions were prepared at approximately 1 mg/mL concentration. Molecular weight was measured relative to polystyrene standards. The electrochemical experiments were conducted by using a CH Instruments model 660 Electrochemical Workstation and a conventional three-electrode (Pt auxiliary, Ag/AgCl reference) cell with a modified gold electrode as a working electrode. All experimental solutions were thoroughly deoxygenated by bubbling argon through the solution for at least 10 min, and in stationary potential experiments, a gentle flow of argon was also used to facilitate stirring. The glucose stock solutions were prepared in 0.1 M phosphate buffer (pH 7.0) and allowed to mutarotate overnight before use. Synthetic Procedures. All reagents, solvents, and GOx were obtained from Aldrich, Acros, Sigma, Lancaster, or Fluka. All solvents were dried by standard procedures. All other chemicals were analytically pure and were used without further purification. All reactions and manipulations were

10.1021/bm049231e CCC: $30.25 © 2005 American Chemical Society Published on Web 03/04/2005

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Scheme 1. Synthetic Route to a Water-Soluble Electroactive Ferrocene-Grafted Poly(p-phenyleneethynylene)

conducted under an argon atmosphere using Schlenk techniques or in an argon-atmosphere glovebox. 2,5-Diiodo-1,4-bis{2-2-[(2-hydroxyethoxy)-ethoxy]ethoxy}-ethoxy}benzene (2). 2,5-diiodo-1,4-hydroquinone (1) (20.6 g, 57.0 mmol) in dry DMF (100 mL) was added to a stirred suspension of K2CO3 (23.6 g, 172 mmol) and KI (2.50 g, 15 mmol) in dry DMF (150 mL) under argon. A solution of 2-[2-(2-chloroethoxy)ethoxy]ethanol (24.0 g, 143 mmol) was added to the mixture, and the temperature was raised to 70 °C and stirred for 10 days. After cooling to room temperature, the mixture was filtered and the residue was washed with THF (100 mL). The solvent was removed in vacuo and a large amount of water (1000 mL) was poured into the residue to precipitate the product. The precipitate was collected by filtering, washing with distilled water and freeze-drying. Recrystallization of the crude product from EtOAc-hexane yielded compound 2 as pale yellow solid. 1 H NMR (400 MHz, CDCl3): δ 7.24 (s, 2H), 4.10 (t, J ) 4.3 Hz, 4H), 3.87 (t, J ) 4.8 Hz, 4H), 3.77 (t, J ) 4.6 Hz, 4H), 3.76∼3.62 (m, 10H), 3.62 (t, J ) 5.2 Hz, 4H) ppm. HRMS: Calcd, 625.9873 (C18H28I2O8). Found, 625.9846. Compound 3. 2,5-Diiodo-1,4-bis{2-2-[(2-hydroxyethoxy)ethoxy]-ethoxy}-ethoxy}benzene (2) (2.00 g, 3.19 mmol) was dissolved in dry THF (20 mL), and 3-bromopropionyl chloride (1.5 mL, 14.9 mmol) and pyridine (1.0 mL) then were added. The reaction mixture was stirred at room temperature for 15 h. The solvent was removed, and the residue was diluted with ethyl acetate (50 mL) and washed with 10% HCl (30 mL × 2) and brine (30 mL × 3) and dried over anhydrous MgSO4. The solvent was evaporated and the crude compound was purified by column chromatography on silica gel with methylene chloride-EtOAc (10: 1, V/V) to give the target compound 3 as a faint yellow oil.1H NMR (400 MHz, CDCl3): δ 7.22 (s, 2H), 4.27 (t, J ) 5.0 Hz, 4H), 4.09 (t, J ) 5.0 Hz, 4H), 3.87 (t, J ) 5.0 Hz, 4H), 3.67∼3.76 (m, 16H), 2.80 (t, J ) 6.4 Hz, 4H) ppm. 13C NMR

(400 MHz, CDCl3): δ 37.46, 38.93, 64.04, 69.06, 69.62, 70.28, 70.70, 71.13, 86.37, 123.43, 153.08, 170.23 ppm. HRMS: Calcd, 893.8622 (C24H34Br2I2O10). Found, 893.8635. 1,4-Bis{2-[2-(2-methoxyethoxy)-ethoxy]-ethoxy}-2,5-diethynylbenzene (4). Compound 4 was prepared according to a published procedure using ethanol as a reaction solvent instead of methanol.8 1H NMR (400 MHz, CDCl3): δ 6.96 (s, 2H), 4.11 (t, J ) 5.0 Hz, 4H), 3.83 (t, J ) 4.8 Hz, 4H), 3.74-3.72 (m, 4H), 3.64-3.61 (m, 8H), 3.52-3.50 (m, 4H), 3.34 (s, 6H), and 3.30 (s, 2H). 13C NMR (400 MHz, CDCl3): δ 153.70, 117.94, 113.22, 82.42, 79.19, 71.58, 70.69, 70.34, 70.19, 69.21, 69.14, and 58.63. HRMS: Calcd for C24H34O8, 450.2254 (M+), Found, 450.2249. Thioacetic Acid s-(4-Iodo-phenyl)ester (5). Compound 5 was prepared according to the literature.9 1H NMR (300 MHz, CDCl3): δ 7.75 (d, J ) 8.30 Hz, 2H), 7.14 (d, J ) 8.26 Hz, 2H) and 2.46 (s, 3H). 13C NMR (300 MHz, CDCl3): δ 192.94, 138.25, 135.83, 127.65, 95.88 and 30.17. Polymer 1. Monomer 3 (1.0 equiv), monomer 4 (1.1 equiv), and compound 5 (0.1 equiv) were polymerized for polymer 1 by using the Sonogashira cross-coupling reaction in the presence of 5% (PPh3)4Pd and 5% CuI in DMF and diisopropylamine at room temperature for 4 days. An additional amount of compound 5 (0.3 equ.) was added to the mixture, which reacted for another 24 h at room temperature. Polymer 1 was precipitated in 500 mL of distilled water, filtered, washed with ethanol, and dried under vacuum at room temperature. 1H NMR (400 MHz, CDCl3): δ 7.03 (s), 7.00 (s), 4.32-4.08 (m), 3.89-3.85 (m), 3.793.61 (m), 3.52-3.48 (m), 3.33-3.32 (m) ppm. 13C NMR (400 MHz, CDCl3): δ 166.36, 153.65, 131.25, 128.53, 92.54, 91.47, 72.19, 71.31, 71.00, 70.84, 69.93, 63.95, 59.27, 35.97. Gel permeation chromatography analysis (mobile phase: THF, polystyrene standards) indicated that Mn of polymer 1 was 15 000 g/mol and its polydispersity was 1.86.

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Scheme 2. Synthesis of Monomers and PPEs

Polymer A. Bromoalkyl polymer 1 (0.30 g) was reacted with excess (dimethylaminomethyl)ferrocene (0.30 g) in 30 mL of DMF solution at 60 °C under argon atmosphere for 3 days. When the resulting mixture was cooled to room temperature, the polymer A was precipitated with 600 mL of ethyl ether, filtered, washed with ethyl ether, and dried under vacuum at room temperature. 1H NMR (400 MHz, CDCl3): δ 7.03 (s), 7.00 (s), 4.26-4.16 (m), 3.89 (s), 3.783.63 (m), 3.51-3.50 (m), 3.33 (s), 2.25 (s) ppm. 13C NMR (400 MHz, CDCl3): δ 166.13, 153.58, 130.96, 128.23, 96.47, 94.38, 91.56, 71.87, 70.99, 70.66, 70.52, 69.65, 69.09, 63.63, 58.95, 46.34, 34.17. Polymer B. Method 1: β-cyclodextrin-saturated water solution (1 mL) was added to 1 mL of chloroform solution containing 0.1 mg of polymer A and stirred gently overnight. Method 2: β-cyclodextrin-saturated water solution (20 mL) was added to 10 mL of THF solution containing polymer A and stirred for an hour. Then, 20 mL of water was added to the residue after the solvents were removed under reduced pressure. The water solution of polymer B was obtained by filtration after 2-h sonication of the mixture. Polymer B was obtained by dialysis against water for 2 h to remove excess β-cyclodextrin, and lyophilization. Through complexation of its ferrocene groups with β-CDs, polymer A was made water soluble (about 0.5 mg/mL). Construction of Glucose Biosensors. Gold electrodes (2 mm diameter) were used for electrochemical measurements. Prior to the modification and measurements, they were cleaned according to the published procedure.10 Self-assembled monolayers of cystamine at gold electrodes were prepared by incubating in 0.1 M cystamine ethanol solution overnight, followed by rinsing with ethanol and water.11 Glucose biosensors were prepared by syringing a 10-µL aliquot of 0.5 mg/mL polymer B onto the electrode surface modified with cystamine. Then, 3 µL of a 10 mg/mL (50 mM phosphate buffer, pH ) 9.0) solution of GOx was added onto the electrode and stirred with a syringe needle. Next, 2 µL of a 3.5% solution of glutaraldehyde was added to the electrode and stirred. The electrode was allowed to cure for 5 h under air.12 For glucose biosensors coated with Nafion, a 4-µL aliquot of a 0.5% Nafion solution in 95% ethanol was syringed onto the electrode. When the film is dried at room temperature for 30 min, an additional 4-µL aliquot of 0.5% Nafion was

applied. After drying, the biosensors were put in a 0.1 M phosphate buffer (pH 7.0) overnight at 4 °C. Results and Discussion Synthesis of Ferrocene-Grafted Poly(p-phenyleneethynylene)s. β-Cyclodextrin (β-CD) and β-CD-modified gold nanoparticles are known to form strong inclusion complexes with ferrocene derivatives via noncovalent interactions in the β-CD hydrophobic cavity.13 Poly(p-phenyleneethynylene) (PPE) bearing ferrocene groups through hydrophilic ethylene glycol linker is made water soluble by complexation of its ferrocene groups with β-CDs or β-CD-modified gold nanoparticles. Our strategy is to prepare water-soluble, electroactive PPEs by synthesis of ferrocene-bearing PPEs in organic solvent and subsequent solubilization of them in water by β-CDs or β-CD-modified nanoparticles. Bromobearing PPE (polymer 1) was synthesized by a palladiumcatalyzed Sonogashira coupling polymerization of a diiodobenzene derivative (3) with a diethynyl benzene derivative (4) at room temperature for 4 days, according to Scheme 2. Thioacetic acid S-(4-iodo-phenyl) ester (5) was added to ensure that the polymer has well-defined, protected thiol polymer end groups for polymer immobilization to the gold surface. Swager et al. reported the synthesis of PPEs bearing alkyl bromide as the polymer end capping groups by heating a mixture of aryl acetylenes, aryl halides, and catalyst (5 mol % of CuI and (PPh3)4Pd) in 30:70 diisopropylamine: toluene at 70 °C for 14-16 h.14 They reported that the alkyl bromide group remained intact in the presence of the diisopropylamine at the elevated temperature.14 This synthetic route provides facile synthesis of bromo-bearing PPEs, which allows for post-polymerization functionalization of the polymers with a variety of groups such as amine, carboxylic acid, thiol, and ammonia through good leaving groups of the alkyl bromide.15 Based on this strategy, introduction of ferrocene to PPE was achieved by post-polymerization functionalization of polymer 1 via the quaternization reaction of bromoalkyl groups with (dimethylaminomethyl)ferrocene in DMF solution at 60 °C (Scheme 2). To allow for covalent attachment of ferrocene to the PPE backbone via the hydrophilic ethylene glycol linker, we used a synthesis strategy based on the use of intermediate 2, which was obtained by the alkylation of 2,5-diiodo-1,4-hydroquinone (1) with 2-[2-(2-chloroethoxy)ethoxy]ethanol in K2CO3/DMF solution in the presence of a small amount of KI. Monomer

Notes

Figure 1. Two phases of distilled water and the polymer A in chloroform (photo A), and phase transfer of the polymer A from chloroform to water by complexation of its ferrocene groups with β-CDs (photo B).

3 was obtained by coupling the intermediate 2 with 3-bromopropionyl chloride in THF solution in the presence of pyridine (Scheme 2). Monomers 3 and 4 were polymerized via the Sonogashira cross-coupling polycondensation reaction in the presence of the end-capping agent 5 to produce polymer 1. The polymerization was carried out using 5% (PPh3)4Pd and 5% CuI in DMF and diisopropylamine at room temperature for 4 days. An additional amount of compound 5 was added to the mixture, which reacted for another 24 h. Polymer 1 is readily soluble in common solvents such as THF, chloroform, methylene chloride, ethyl acetate, DMF, and DMSO. Gel permeation chromatography analysis (mobile phase: THF, polystyrene standards) indicated that Mn of polymer 1 was 15 000 g/mol and its polydispersity was 1.86. Polymer 1 displays maximum absorption and emission peaks at 420 and 490 nm in its chloroform solution, respectively, arising from the π-π* transition of the conjugated polymer backbone. Its fluorescence quantum yield was 0.62 by using quinine sulfate in 0.1 N sulfuric acid as the reference absolute quantum efficiency (φn ) 55%).7 Polymer A was prepared by a quaternization reaction of the bromoalkyl groups of polymer 1 with excess (dimethylaminomethyl)ferrocene in DMF solution at 60 °C under argon atmosphere for 4 days. It was purified by precipitation with a large amount of ethyl ether to remove excess (dimethylaminomethyl)ferrocene. Polymer A is readily soluble in common solvents such as ethanol, acetone, THF, chloroform, and methylene chloride, ethyl acetate, DMF and DMSO, but is insoluble in water. It displays the same maximum absorption and emission peaks as polymer 1. However, its quantum efficiency is 0.11, much smaller than that of polymer 1, caused by electron-transfer quenching with ferrocene as an electron donor. Figure 1 shows photos of polymers A and B in chloroform and water solutions. Addition of distilled water to its chloroform solution forms two phases since polymer A is insoluble in water (Figure 1A). However, addition of β-CDsaturated water solution to the dilute polymer in chloroform causes polymer A to transfer from chloroform to water under gentle stirring overnight because of strong complexation of its side chain ferrocene groups with β-CDs (Figure 1B). The

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Figure 2. Absorption and emission of polymer B in distilled water at an excitation wavelength of 440 nm.

positive charges adjacent to the ferrocene groups might facilitate the phase transfer reaction because the positive charges might enhance not only enthalpic interactions with water but also complexation chances of ferrocene groups with β-CDs. The cationic nature and the water solubility of polymer B are important features since they promote strong complexation with anionic enzymes via electrostatic interactions. The stability of such complexes is important since they serve as the active layers of specific sensors. Polymer B displays maximum absorption and emission peaks at 440 and 500 nm (Figure 2), respectively, a red shift compared with polymer A. This red shift might be due to enhanced coplanar confirmation of the polymer or polymer aggregation in aqueous solution.16 Its fluorescence quantum yield was 0.08 by using quinine sulfate in 0.1 N sulfuric acid as the reference absolute quantum efficiency (φn ) 55%).7 Polymer A is also readily transferred into water from its chloroform solution through complexation of its ferrocene groups with β-CD-modified gold nanoparticles.6 However, the polymer complex water solution with β-CD-modified gold nanoparticles exhibits no fluorescence, which is presumably due to effective quenching from the gold nanoparticles. Biosensor Application. As a biosensing application, we select glucose oxidase to demonstrate how water-soluble polymer B can facilitate electron transfer between an enzyme and the electrode. It can be extended to other redox enzymes. The cationic nature and the water solubility of polymer B promote strong complexation with anionic enzymes such as glucose oxidase via electrostatic interactions. The complex of glucose oxidase and polymer B is immobilized to gold electrodes modified with cystamine monolayers via a crosslinker, glutaraldehyde. The cross-linker forms imine bonds with amine groups from lysine and arginine residues of the enzyme present on the enzyme periphery and the monolayers.17 A Nafion coating is used to enhance the biosensor stability since it may prevent leaching of the enzyme or the polymer. In the absence of glucose, the enzyme gives no response and only the polymer electrochemistry is observed. Cyclic voltammetry displays the linear relationship of peak current versus square root of scanrate for glucose biosensors, which indicates propagation of charge in the volume of the polymer network by a diffusion-like process (Dct, charge-transfer

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Figure 3. Cyclic voltammetric responses of glucose biosensor in the absence and presence of glucose in 0.1 M phosphate buffer (pH 7.0) containing 0.1 M NaNO3 at a scan rate of 10 mV/s.

coefficient), such as electron hopping between neighboring redox sites or counterion motion. The charge-transfer diffusion coefficient is 7.3 × 10-7 cm2 s-1, calculated from linear ip vs V1/2 plot.18 The high mobility of the tethered redox ferrocene groups facilitates electron transfer since the rate of electron transferring collisions increases with the mobility of the tethered redox centers.6b The conjugated polymer backbone might be helpful for electron transfer.19 Upon gradual addition of glucose to the solution, the cyclic voltammetric responses of the glucose biosensor change dramatically, displaying a gradual increase in oxidation current and decrease in the reduction current (Figure 3). Comparison of the voltammograms in the absence and presence of glucose indicates that the polymer-bound ferrocene/ferricinium moieties allow close contact with the FAD/ FADH2 centers of the glucose oxidase and facilitate a flow of electrons from the enzyme to the electrode. Thus, glucose oxidase is reduced by the glucose diffusing into the film (eq 1). The reduced enzyme is oxidized and reactivated by Fc+ sites (eq 2), and the electrons are then transported through the ferrocene polymer film to the electrode surface. The disappearance of a reduction peak indicates that the film is homogeneously maintained in the reduced state by the transfer of electrons from GOx(FADH2) to Fc+ sites, resulting in a bioelectrocatalytic oxidation current (eq 3). The long, flexible, hydrophilic tethers may facilitate a more intimate interaction between the ferrocene moieties and the FAD/(FADH2), centers of glucose oxidase and provide better electrical wiring between the relays themselves. GOx(FAD) + glucose f GOx(FADH2) + gluconolactone (1) GOx(FADH2) + 2Fc+ f GOx(FAD) + 2Fc

(2)

2Fc -2e f 2Fc+ (at the electrode)

(3)

where GOx(FAD) and GOx(FADH2) represent oxidized and reduced forms of GOx and Fc+ and Fc stand for oxidized and reduced forms of ferrocene, respectively. Glucose biosensors coated with a Nafion film are more stable than those without Nafion overcoating. The Nafion

Figure 4. Calibration curves for biosensors as a function of glucose concentration at the applied potential of 0.4 V (vs Ag/AgCl).

coating may function to preserve film integrity and prevent the leaching of enzyme or polymer. It also extends the range of linear response from 10 to 22 mM by reducing substrate mass transport (Figure 4). Biosensors with the Nafion coating display less sensitivity to pH since the optimal pH range is from 6 to 9. Lowe et al. reported reagentless glucose biosensors by immobilization of glucose oxidase in ferrocene-modified pyrrole polymers.20 The enzyme was entrapped in polypyrroles, which were prepared by electropolymerization of N-substituted pyrrole monomers bearing ferrocene groups. Ferrocene-pyrrole conjugates were efficient oxidants of reduced glucose oxidase. However, the glucose biosensors failed to show any response to glucose in the absence of oxygen after 2 days of use. They attributed this failure to possible physical or chemical modification occurring on aging of polymer films.20 The biosensors, reported here, exhibit excellent storage characteristics. The maximal current of biosensors decreased by 5% after two-month dry storage at 4 °C. Biosensors with a Nafion coating lost about 10% of their initial glucose response when they were stored in 0.1 M phosphate buffer (pH 7.0) at 4 °C over the same period. We are investigating methods to increase the stability of the biosensors by preparing water-soluble PPEs bearing ferrocene and aldehyde groups so that we can covalently attach enzymes to the redox conjugated polymers. Conclusions It has been shown that ferrocene-grafted PPE can be synthesized in organic solution and subsequently solubilized in water through complexation of ferrocene groups with β-CDs or β-CD-modified nanoparticles. This β-CD-facilitated complex formation offers a new general approach to prepare water-soluble redox conjugated polymers. The watersoluble electroactive PPE provides promising biosensing applications to facilitate electron transfer between enzymes and electrodes. Acknowledgment. The authors greatly acknowledge startup funds, Research Excellence Fund of Michigan Technological University, NSF-NER, Zyvex Corporation, DAR-

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PA, and NASA (Grant No. NNJ04JA18C) for support of this work. We thank reviewers for their very valuable comments, and Professor Rudy Luck for his help in using the glovebox. We would like to thank Professor David H. Waldeck for his very helpful advice.

(7) (8)

(9)

Supporting Information Available. Figure showing the emission spectra of polymers 1 and A at an excitation wavelength of 420 nm. This material is available free of charge via the Internet at http://pubs.acs.org.

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