Strong Interaction between Imidazolium-Based Polycationic Polymer

Jan 22, 2012 - Quantitative monitoring of neurochemicals involved in physiological and pathological processes in the cerebral systems is of great impo...
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Strong Interaction between Imidazolium-Based Polycationic Polymer and Ferricyanide: Toward Redox Potential Regulation for Selective In Vivo Electrochemical Measurements Xuming Zhuang,†,‡ Dalei Wang,‡ Yuqing Lin,‡ Lifen Yang,‡ Ping Yu,*,‡ Wei Jiang,*,† and Lanqun Mao*,‡ †

School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, The Chinese Academy of Sciences (CAS), Beijing 100190, China

‡

S Supporting Information *

ABSTRACT: This study effectively demonstrates a strategy to enable the ferricyanide-based second-generation biosensors for selective in vivo measurements of neurochemicals, with glucose as an example. The strategy is based on regulation of redox potential of ferricyanide mediator by carefully controlling the different adsorption ability of ferricyanide (Fe(CN)63‑) and ferrocyanide (Fe(CN)64‑) onto electrode surface. To realize the negative shift of the redox potential of Fe(CN)63‑/4‑, imidazolium-based polymer (Pim) is synthesized and used as a matrix for surface adsorption of Fe(CN)63‑/4‑ due to its stronger interaction with Fe(CN)63‑ than with Fe(CN)64‑. The different adsorption ability of Fe(CN)63‑ and Fe(CN)64‑ onto electrodes modified with a composite of Pim and multiwalled carbon nanotubes (MWNTs) eventually enables the stable surface adsorption of both species to generate integrated biosensors and, more importantly, leads to a negative shift of the redox potential of the surface-confined redox mediator. Using glucose oxidase (GOD) as the model biorecognition units, we demonstrate the validity of the ferricyanide-based second-generation biosensors for selective in vivo neurochemical measurements. We find that the biosensors developed with the strategy demonstrated in this study can be used well as the selective detector for continuous online detection of striatum glucose of guinea pigs, by integration with in vivo microdialysis. This study essentially paves a new avenue to developing electrochemical biosensors effectively for in vivo neurochemical measurements, which is envisaged to be of great importance in understanding the molecular basis of physiological and pathological events.

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uses of oxidases as the biorecognition elements always necessitate an efficient electronic communication between oxidases and electrode.3 The difficulty in directly conducting oxidases onto electrode eventually makes the bioelectrocatalysis of substrate normally occur with O2 or artificial mediators as the electron acceptor for oxidases.4 Consequently, the oxidasebased electrochemical biosensors have mainly been developed through either the detection of hydrogen peroxide generation or oxygen consumption (i.e., so-called first-generation biosensors),5 both involved in the enzyme reactions, or through the uses of artificial mediators to shuttle the electronic communication between oxidases and electrode (i.e., so-called second-generation biosensors). Compared with the firstgeneration biosensors, the second-generation biosensors with mediated electron transfer of oxidases are more useful for in vivo neurochemical measurements. This is because the secondgeneration biosensors normally have lower oxygen dependency and less interference from hydrogen peroxide endogenously

uantitative monitoring of neurochemicals involved in physiological and pathological processes in the cerebral systems is of great importance in understanding the molecular basis of physiological and pathological events.1 Among the methods employed for accomplishing such a purpose, electrochemical methods with enzyme-based electrochemical biosensors remain particularly attractive. This is because the use of enzymes as the biorecognition elements in neurochemical biosensing, on one hand, enables the electroinactive neurochemicals such as glutamate, glucose, lactate, and hypoxanthine electrochemically detectable and, on the other hand, endows the as-prepared electrochemical biosensors with excellent analytical properties such as high selectivity and good sensitivity. These properties of the enzyme-based biosensors substantially make them especially useful for in vivo neurochemical measurements both as tissue-implanted probes for in vivo voltammetry and as a selective online detector for continuous neurochemical monitoring coupled with in vivo microdialysis.2 Generally, two kinds of enzymes (i.e., flavin adenine dinucleotide (FAD)-containing oxidases and dehydrogenases) are often employed to construct electrochemical biosensors with different electronic transducing pathways, of which the © 2012 American Chemical Society

Received: October 17, 2011 Accepted: January 21, 2012 Published: January 22, 2012 1900

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existing in the cerebral systems.6 Nevertheless, the use of mediators to shuttle the electron transfer between oxidases and electrode yet makes this kind of electrochemical biosensor far from in vivo neurochemical measurements. Among the mediators employed so far for shuttling the electron transfer between oxidases and electrode, ferricyanide (Fe(CN)63‑) remains the most popular because of its good electrochemical property and reactivity with FAD-containing oxidases.7 While the ferricyanide-mediated second-generation biosensors have been well studied and used in various electrochemical investigations, the good water solubility and high formal potential of Fe(CN)63‑/4‑, unfortunately, substantially make it difficult to apply this kind of electrochemical biosensor for in vivo neurochemical measurements. This is because, for in vivo measurements, the mediator should be stably confined onto electrode surface to generate integrated biosensors: dissolving the mediator into solution phase not only makes it difficult to conduct in vivo voltammetric measurements with a biosensor implanted into brain regions but also leads to the measurements being expensive with an environmental burden.8 More importantly, the redox potential of the solution-phased Fe(CN)63‑/4‑ generally overlaps with those for the oxidation of electrochemically active neurochemicals endogenously existing in the cerebral systems such as ascorbate, uric acid, catecholamines, and their metabolites.9 This property substantially invalidates the ferricyanide-mediated second-generation biosensors for in vivo selective neurochemical measurements in the cerebral systems. This study demonstrates a strategy effectively to enable the well-studied ferricyanide-based second-generation biosensors for selective in vivo measurements of neurochemicals, with glucose as an example. The strategy is based on the fundamental electrochemical phenomena on regulation of redox potential of electrochemical species by carefully controlling different adsorption ability of reactants and products onto electrode surface, as first reported by Wopschall and Shain.10 To realize the negative shift of the redox potential of Fe(CN)63‑/4‑, imidazolium-based polymer (Pim) is synthesized and used as a matrix for surface adsorption of Fe(CN)63‑/4‑ due to its stronger interaction with Fe(CN)63‑ than with Fe(CN)64‑. The stronger interaction of Pim with Fe(CN)63‑ than with Fe(CN)64‑ eventually enables the stable surface adsorption of both species to generate integrated biosensors and, more importantly, leads to a negative shift of the redox potential of the surface-confined redox mediator. The latter property substantially validates the ferricyanide-based second-generation biosensors for selective in vivo neurochemical measurements because of the good differentiation of the potential of the biosensors from those for the oxidation of electroactive neurochemicals. Although some methods have been used for surface confinement of Fe(CN)63‑,11 the chemical complexity of the cerebral systems yet makes it difficult to apply those kinds of biosensors for in vivo neurochemical measurements, in spite of their applications in other research fields. This study actually paves a new avenue to developing electrochemical methods effectively for in vivo neurochemical measurements, which is envisaged to be of great importance in understanding the molecular basis of physiological and pathological events.

Potassium ferricyanide (K3Fe(CN)6) was obtained from Beijing Chemical Co. (Beijing, China). D(+)-glucose and glucose oxidase (GOD, EC 1.1.3.4, from Aspergillus niger) were purchased from Sigma and used as supplied. Azo-bisisobutryonitrile (AIBN) was obtained from Aldrich. Bovine serum albumin (BSA) was obtained from Proliant. Multiwalled carbon nanotubes (MWNTs) were purchased from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China) and used as received. Artificial cerebrospinal fluid (aCSF) used as the perfusion solution for in vitro experiments and in vivo microdialysis was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), and CaCl2 (1.1 mM) into water. Other chemicals were of at least analytical grade reagents and were used as received. All the solutions were prepared with Milli-Q water. Synthesis of Poly(1-Vinyl-3-Butylimidazolium Chloride). Synthesis of poly(1-vinyl-3-butylimidazolium chloride) (Pim) was performed according to the previous report.12 Briefly, 1-vinylimidazole (9.6 g, 102 mM) and 1-chlorobutane (29.6 g, 320 mM) were added to a three-necked flask, and the mixture was then stirred vigorously for 75 h in an oil bath of 70 °C under nitrogen atmosphere. After cooling to room temperature, the top phase was decanted and the bottom viscous liquid was first washed by ethyl acetate for three times and then filtered and dried under vacuum at 50 °C overnight to obtain [Vbim][Cl] as a white solid. [Vbim][Cl]: 1H NMR (400 MHz, D2O) δ= 0.92 (t, 3H), 1.34 (m, 2H), 1.88 (m, 2H), 4.23 (t, 2H), 5.42 (dd, 1H), 5.80 (dd, 1H), 7.14 (dd, 1H), 7.57 (s, 1H), 7.76 (s, 1H) (NMR data, Figure S1, Supporting Information). Pim was synthesized by free radical polymerization, as reported previously.12 Typically, [Vbim][Cl] (3.0 g), AIBN (0.015 g), and chloroform (30 mL) were mixed into a threenecked flask in an oil bath of 60 °C under nitrogen atmosphere, and the mixture was then stirred for 18 h. After that, the mixture was cooled to room temperature and was allowed to precipitate with addition of ethyl ether. The resulting precipitate was redissolved into water, and the solution was dialyzed (cutoff, 10 000) in water for 2 days. The solution left in the dialysis bag was concentrated with a rotary evaporator at 45 °C, and the obtained product was dried under vacuum at 50 °C overnight to give poly(1-vinyl-3-butylimidazolium chloride) (NMR data, Figure S2, Supporting Information). Electrodes, Thin-Layer Flow Cells, and Apparatus. Glassy carbon (GC) electrodes used for in vitro voltammetric experiments (3 mm, diameter) and for online in vivo measurements (6 mm, diameter) in a thin-layer radial electrochemical flow cell were both purchased from Bioanalytical Systems Inc. (BAS, West Lafayette, IN). Both kinds of GC electrodes were polished first on emery paper and then with aqueous slurries of alumina powder (0.3 and 0.05 μm) on a polishing cloth and finally rinsed with Milli-Q water under an ultrasonic bath for 10 min. The thin-layer radial electrochemical flow cell consists of a thin-layer radial flow block with a 50 μm gasket, GC electrode (6 mm, diameter) as working electrode, stainless steel as auxiliary electrode, and Ag/AgCl electrode (3 M NaCl) as reference electrode. To stably confine the synthetic water-soluble Pim onto GC electrode, MWNTs were mixed with Pim to form uniform Pim/MWNT composite (Figures S3 and S4, Supporting Information) in terms of their hydrophobic interactions. The composite was found to be stably confined onto electrode

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EXPERIMENTAL SECTION Reagents and Solutions. Potassium ferrocyanide (K4Fe(CN)6) was purchased from Kanto Chem. Co. Inc. (Japan). 1901

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Figure 1. (A) UV−vis spectra of pure Pim (black curve), pure K3Fe(CN)6 (blue curve), and the mixture of Pim and K3Fe(CN)6 (red curve). Inset, digital pictures of aqueous solutions of K3Fe(CN)6 (left) and the mixture of Pim and K3Fe(CN)6 (right). Both solutions were centrifuged at 5000 rpm for 5 min. (B) UV−vis spectra of pure Pim (black curve), pure K4Fe(CN)6 (blue curve), and the mixture of Pim and K4Fe(CN)6 (red curve). Inset, digital pictures of aqueous solution of K4Fe(CN)6 (left) and the mixture of Pim and K4Fe(CN)6 (right). Both solutions were centrifuged at 5000 rpm for 5 min.

Throughout the surgery, the body temperature of the animals was maintained at 37 °C with a heating pad. Immediately after the surgery, the guinea pigs were placed into a warm incubator individually until they recovered from the anesthesia and were allowed to recover for at least 24 h prior to the surgeries for in vivo microdialysis. To investigate the validity of the ferricyanide-based second-generation biosensors prepared in this study for selective in vivo measurements of glucose in the cerebral systems, the biosensor was fixed into the thin-layer electrochemical flow cell and used as the selective detector for online monitoring of glucose in the microdialysates continuously sampled from the striatum of guinea pigs (Figure S5, , Supporting Information). Prior to online measurements, the microdialysis probe was implanted in the striatum and was allowed to equilibrate for at least 90 min by continuously perfusing aCSF. The microdialysates were continuously sampled with microinjection pump (CMA 100, CMA Microdialysis AB, Stockholm, Sweden) with aCSF as the perfusion solution at 2 μL/min and were monitored in the electrochemical flow cell.

surface. To prepare Pim/MWNT nanocomposite, 1 mg of MWNTs and 50 mg of synthetic Pim were mixed into 5 mL of water, and the resulting mixture was then sonicated for 1 h to form a homogeneous dispersion. Five and 10 μL of the asformed dispersion were dip-coated onto GC electrodes with diameters of 3 mm and 6 mm, respectively. The electrodes (denoted as Pim/MWNT-modified electrodes, hereafter) were then dried at ambient temperature and used for electrochemical measurements. To construct glucose biosensors, aqueous dispersion of Pim/MWNT composite (4 μL), K3Fe(CN)6 (4 μL, 1 mM), GOD (4 μL, 10 mg/mL), BSA (2 μL, 1%), and gluteraldehyde (2 μL, 1%) were mixed thoroughly. Five and 10 μL of the resulting mixture were dip-coated onto GC electrodes with diameters of 3 mm and 6 mm, respectively. The electrodes were finally dried at 4 °C before electrochemical measurements. In vitro electrochemical experiments were performed with a computer-controlled electrochemical analyzer (CHI 1030, Chenhua, Shanghai, China) with a conventional electrochemical cell with the as-prepared GC electrodes as working electrode, a platinum wire as counter electrode, and an Ag/ AgCl electrode (KCl-saturated) as reference electrode. All electrochemical experiments were performed at room temperature. Ultraviolet−visible (UV−vis) spectra were recorded on a TU-1900 spectrometer (Beijing, China), and FT-IR spectra were obtained on a Tensor-27 FT-IR spectrometer (Bruker) with KBr pellet. X-ray photoelectron spectroscopy (XPS) was performed on an ESCALab220i-XL electron spectrometer from VG Scientific using 300 W Al Kα radiation. In Vivo Microdialysis and Online Electrochemical Measurements. Surgery for in vivo microdialysis was performed as reported previously.13 Briefly, adult male guinea pigs (300−400 g) obtained from Health Science Center, Peking University, were housed on a 12:12 h light−dark schedule with food and water ad libitum. The animals were anaesthetized with chloral hydrate (345 mg/kg, i.p.) and positioned onto a stereotaxic frame. The microdialysis guide cannulas (CMA, dialysis length, 4 mm; diameter, 0.24 mm) were carefully implanted in the striatum (AP = 0 mm, L = 3 mm from bregma, V = 4.5 mm from dura) using standard stereotaxic procedures. The guide cannula was fixed with three skull screws and dental cement. A stainless steel dummy blocker was inserted into the guide cannula and fixed until the insertion of the microdialysis probe (CMA, dialysis length, 4 mm; diameter, 0.24 mm).

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RESULTS AND DISCUSSION

Interactions of Fe(CN)63‑ and Fe(CN)64‑ with Pim. The different interactions of Pim with Fe(CN)63‑ and Fe(CN)64‑ were investigated by UV−vis spectroscopy, as displayed in Figure 1. In the wavelength employed, the aqueous solution of Fe(CN)63‑ exhibits absorption at about 260, 302, 319, and 420 nm.14 The addition of Pim into the Fe(CN)63‑ solution results in the red-shift of the absorption peaks (Figure 1A). Whereas, the addition of Pim into the solution of Fe(CN)64‑ did not lead to obvious shift of absorption peak at 218 and 332 nm (Figure 1B). These results suggest the stronger interaction between Pim and Fe(CN)63‑, as compared with that between Pim and Fe(CN)64‑. The different interaction activity of Pim with Fe(CN)63‑ and with Fe(CN)64‑ was also simply verified through the possible formation of aggregation in the aqueous solutions of Fe(CN)63‑ and Fe(CN)64‑ upon the addition of Pim into both solutions. The addition of Pim into the aqueous solutions of Fe(CN)63‑ led to formation of aggregation (Inset, Figure 1A), while no aggregation was formed with the addition of Pim into the aqueous solutions of Fe(CN)64‑ (Inset, Figure 1B). To further confirm the strong interaction between Pim and Fe(CN)63‑, XPS was conducted, as shown in Figure 2. The N1s 1902

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(CN)6,15b,19 respectively (Figure S6, Supporting Information). Upon the formation of the composite with Pim, the stretching vibration of CN of K3Fe(CN)6 at 2117 cm−1 was red shifted to 2108 cm−1, while no obvious shift was observed for the stretching vibration of CN of K4Fe(CN)6 at 2030 cm−1. These results suggest that the interaction between Pim and K3Fe(CN) 6 was stronger than that between Pim and K4Fe(CN)6. Nevertheless, in the FT-IR spectra of the composite formed with Pim and K3Fe(CN)6/K4Fe(CN)6 mixture, a weak absorption at 2030 cm−1 was observed (Figure 3, red curve), indicating that K4Fe(CN)6 was still adsorbed into the Pim matrix, presumably through weak interaction, for example, electrostatic interaction, between Pim and K4Fe(CN)6. Moreover, a comparison of the FT-IR spectra recorded for Pim (black curve) and for the composite of Pim and K3Fe(CN)6/K4Fe(CN)6 (red curve) reveals that the absorption of Pim at 3092 and 3137 cm−1 was shifted to 3060 and 3128 cm−1, respectively, upon the formation of the composite. This result again demonstrates that Pim and K3Fe(CN)6 could strongly interact with each other presumably through formation of hydrogen bonds, according to the previous report.17c,18 In addition, the stretching vibration of the C−N bonds in imidazole ring of Pim at 1161, 1550, and 1637 cm−1 was redshifted upon the formation of the composite with K3Fe(CN)6, implying that π−π stacking interaction may also exist between the imidazole of Pim and the CN group in K3Fe(CN)6. All the results described above substantially validate our strategy to control different surface absorption ability of Fe(CN)63‑ and Fe(CN)64‑ through using the synthetic Pim to strongly interact with Fe(CN)63‑ (e.g., electrostatic, hydrogen bonding, and/or π−π stacking interactions) and weakly interact with Fe(CN)64‑ (e.g., electrostatic interaction). This property eventually enables the stable adsorption of both species onto electrode surface and, more importantly, leads to a negative shift of the redox potential of Fe(CN)63‑/4‑, as described below. Surface Absorption of Fe(CN)63‑/4‑ with a Negatively Shifted Redox Potential. Absorption of Fe(CN)63‑/4‑ onto the electrodes modified by Pim/MWNT composite was verified by cyclic voltammetry. Figure 4A depicts typical cyclic voltammogram (CV) obtained with Pim/MWNT-modified GC electrode in 0.10 M KCl solution containing 1 mM Fe(CN)63‑. Two redox waves were observed at the potentials of +0.16 V and +0.24 V. The wave at +0.24 V was ascribed to the redox process of solution-phased Fe(CN)63‑ at the electrode since, in an independent experiment, we observed a pair of well-defined redox waves at +0.24 V at GC electrode in the same solution (data not shown). Moreover, this wave disappeared after the electrode was taken out from the Fe(CN)63‑ solution, rinsed with water, and recycled in pure phosphate buffer containing no Fe(CN)63‑. The wave at +0.16 V was attributed to the electrochemical process of Fe(CN)63‑/4‑ adsorbed onto electrode surface. This assignment was essentially based on the facts that such a redox wave was still clearly recorded when the electrode was first immersed into K3Fe(CN)6 solution, taken out from the Fe(CN)63‑ solution, rinsed with water, and then recycled in the pure phosphate buffer (Figure 4B). Moreover, the currents of the redox wave increase with an increase in the time employed for immersing the Pim/MWNTmodified electrodes into 1 mM K3Fe(CN)6 in 0.10 M KCl solution, as typically depicted in Figure 4B, suggesting the adsorption of Fe(CN)63‑ onto electrode surface through the strong interaction between Pim and Fe(CN)63‑ described above.

Figure 2. XPS results of N1s for Pim (black curve), K3Fe(CN)6 (blue curve), and Pim/Fe(CN)63‑ composite (red curve).

spectra of Pim (black curve) and K3Fe(CN)6 (blue curve) were at 399.9 and 398.5 eV, respectively.15 Pim/K 3Fe(CN)6 composite was prepared by first mixing the K3Fe(CN)6 and Pim into water and then centrifuging the resulting mixture at 5000 rpm for about 5 min. The obtained composite was rinsed with water and dried under vacuum at 80 °C. Upon the formation of Pim/K3Fe(CN)6 composite, the binding energy of N1s for Pim and Fe(CN)63‑ was shifted to 401.5 and 397.8 eV (red curve), respectively. This result demonstrates that there is charge transfer interaction between N atoms in cyanide of Fe(CN)63‑ and in imidazolium of Pim, in which the cyanide might serve as electron-acceptor and the imidazolium ring as electron-donator.16 Figure 3 displays FT-IR spectra of the synthetic Pim, mixture of K3Fe(CN)6 and K4Fe(CN)6 (ca. 1:1), and composite of Pim

Figure 3. FT-IR spectra for Pim (black curve), mixture of K3Fe(CN)6 and K4Fe(CN)6 (blue curve), and composite of Pim and K3Fe(CN)6/ K4Fe(CN)6 (red curve).

and K3Fe(CN)6/K4Fe(CN)6. The composite of Pim and K3Fe(CN)6/K4Fe(CN)6 was prepared by first mixing the K3Fe(CN)6/K4Fe(CN)6 mixture with Pim into water and then centrifuging the resulting mixture at 5000 rpm for about 5 min. The obtained composite was rinsed with water and dried under vacuum at 80 °C. As displayed in Figure 3 (black curve), Pim exhibits absorption at 3092, 3137, 1161, 1550, and 1637 cm−1, of which the absorption at 3092 and 3137 cm−1 was assigned to the stretching vibration of C2−H in an imidazole ring,17 and the absorption at 1161, 1550, and 1637 cm−1 was attributed to the stretching vibration of the C−N bonds in imidazole ring.18 The mixture of K3Fe(CN)6 and K4Fe(CN)6 shows absorption at 2117 and 2030 cm−1 (blue curve), which was assigned to the stretching vibration of CN of K3Fe(CN)6 and K4Fe1903

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Figure 4. (A) CVs obtained at the Pim/MWNT-modified GC electrode in 0.10 M KCl solution in the absence (black curve) and presence (red curve) of 1 mM K3Fe(CN)6. Scan rate, 100 mV s−1. (B) CVs obtained at the Pim/MWNT-modified GC electrodes in 0.10 M phosphate buffer (pH 7.0). The electrodes were first immersed into 0.10 M KCl solution containing 1 mM K3Fe(CN)6 for different times of (from inner to outer) 0, 0.5, 1, 3, 5, 8, 10, and 15 min and then taken out of solution and rinsed with water. Scan rate, 100 mV s−1. (C) CVs obtained at MWNT-modified GC electrode (black curve) in 0.10 M phosphate buffer (pH 7.0) containing 1 mM K3Fe(CN)6 and at the Fe(CN)63‑/Pim/MWNT-modified GC electrode (red curve) in 0.10 M phosphate buffer (pH 7.0). Scan rate, 100 mV s−1.

The stronger adsorption of Fe(CN)63‑ than Fe(CN)64‑ onto electrode surface based on the stronger interaction of Pim toward Fe(CN)63‑ than Fe(CN)64‑ eventually leads to a negative shift of the redox potential, as could be clearly seen from Figure 4C. In 0.10 M phosphate buffer, the Fe(CN)63‑/ Pim/MWNT-modified electrode exhibits a well-defined redox peak at +0.16 V. This potential was more negative than the redox potential of Fe(CN)63‑/4‑ in the solution phase (i.e., +0.24 V). The negative shift of the redox potential of redox mediator remains very essential for selective in vivo neurochemical measurements with the as-prepared oxidase-based biosensors. This is because, according to the previous reports,9 most of electroactive neurochemicals endogenously existing in the cerebral systems such as uric acid, catecholamines, and their metabolites could be electrochemically oxidized at potentials higher than +0.20 V. Our strategy with careful controlling of different absorption ability of Fe(CN)63‑ and Fe(CN)64‑ onto electrode surface eventually enables good separation of the potential for in vivo neurochemical biosensing from those for the oxidation of endogenously existing electroactive species. Figure 5A displays consecutive CVs obtained at the Fe(CN)63‑/Pim/MWNT-modified GC electrode in 0.10 M phosphate buffer (pH 7.0). The peak current recorded for the surface-confined Fe(CN)63‑/4‑ remained almost unchanged upon consecutive potential cycling for 50 cycles, revealing that the use of Pim as a matrix could lead to stable surface confinement of Fe(CN)63‑ and Fe(CN)64‑. Moreover, the Fe(CN)63‑/Pim/MWNT-modified GC electrode shows the good linear relationship between the redox peak currents and the square root of the potential scan rate ranging from 5 to 500 mV s−1 (Figure 5B, inset) and less dependence of the redox potential on the scan rate employed. These results basically demonstrate a fast diffusion-controlled redox process of the surface-confined Fe(CN)63‑/4‑ presumably with an electronhopping mechanism in the Pim layer, as reported previously.20 The high stability and fast electron transfer rate of Fe(CN)63‑/4‑ adsorbed onto the Pim/MWNT-modified electrodes substantially enable them to serve well as efficient electron transfer in the oxidase-based biosensors, as typically depicted in Figure 5C. Upon coconfinement of GOD onto electrode surface to prepare glucose biosensor, the addition of glucose into the buffer clearly increases the oxidation peak current, while decreasing the reduction peak current of Fe(CN)63‑/4‑ adsorbed onto the Pim/MWNT-modified electrodes. This result demonstrates that the biosensors were responsive to glucose and further suggests that Fe(CN)63‑/4‑ stably adsorbed

Figure 5. (A) Consecutive CVs for 50 cycles obtained at the Fe(CN)63‑/Pim/MWNT-modified GC electrode in 0.10 M phosphate buffer (pH 7.0). Scan rate, 100 mV s−1. (B) CVs obtained at the Fe(CN)63‑/Pim/MWNT-modified GC electrode in 0.10 M phosphate buffer (pH 7.0) at different scan rates of (from inner to outer) 5, 20, 50, 100, 200, 300, and 500 mV s−1. Inset: plots of anodic and cathodic peak currents versus square root of scan rate. (C) CVs at the GOD/ Fe(CN)63‑/Pim/MWNT-modified GC electrode in 0.10 M phosphate buffer (pH 7.0) in the absence (black curve) and presence (red curve) of 5 mM glucose. Scan rate, 20 mV s−1. (D) Typical current−time responses obtained at the GOD/Fe(CN)63‑/Pim/MWNT-modified GC electrode fixed into thin-layer electrochemical flow cell toward glucose with different concentrations in a continuous-flow system. aCSF was used as the perfusion solution. Flow rate, 2 μL/min. Potential applied, +0.17 V. The concentrations of glucose were indicated in the figure. Inset, calibration curve for glucose biosensing.

onto the Pim/MWNT-modified electrodes, serving well as electron transfer mediator to efficiently transduce the GODbased biorecognition events into electronic signal output at a low potential. Upon being fixed into the thin-layer electrochemical flow cell, the biosensor shows a good response in a continuous-flow system, as displayed in Figure 5D. With aCSF as the perfusion solution at a perfusion rate of 2 μL/min, welldefined current responses were recorded for glucose and the current responses were linear with the concentration of glucose with a dynamic linear range from 300 to 1500 μM (I (nA) = 0.186 + 0.0109Cglucose (μM), γ = 0.9783). This dynamic linear 1904

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the microdialysate, suggesting that the in vivo measurements at this potential is virtually interference free. As shown in Figure 6D, the replacement of the Fe(CN)63‑/Pim/MWNT-modified GC electrode from the electrochemical cell with the glucose biosensor clearly produces current response for the microdialysate at +0.17 V, which was attributed to the selective oxidation of glucose in the microdialysate. The basal levels of microdialysate glucose in the striatum of guinea pig was measured to be ca. 600 μM, which was almost consistent with the values reported previously.21 These results substantially demonstrate that our strategy for redox potential modulating through rational controlling of different surface absorption ability of Fe(CN)63‑ and Fe(CN)64‑ successfully enables the well-studied ferricyanide-based second-generation biosensors for selective in vivo measurements of neurochemicals in the cerebral systems.

range well covers the physiological level of glucose in the microdialysate, further validating application of the electrochemical biosensors developed in this study for in vivo measurements of glucose in the cerebral systems. Selectivity and Online Measurements of Cerebral Glucose. To investigate the utility of the ferricyanide-based second-generation biosensors developed with our strategy for in vivo measurements of glucose in the brain of living guinea pigs, the biosensor was fixed into the thin-layer electrochemical flow cell and was used as the selective detector to integrate with in vivo microdialysis to finally form an online detecting system (Figure S5, Supporting Information). Figure 6 depicts amperometric responses obtained with the online detecting

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CONCLUSIONS Taking advantages of the fundamental electrochemical phenomena on regulation of redox potential of electrochemical species by carefully controlling different adsorption ability of reactants and products onto electrode surface, we have effectively developed a new strategy to enable the well-studied ferricyanide-based second-generation biosensors for selective in vivo measurements of neurochemicals based on the different interaction activity of synthetic imidazolium-based polymer toward Fe(CN)63‑ and Fe(CN)64‑. The stronger interaction activity of the imidazolium-based polymer with Fe(CN)63‑ than with Fe(CN)64‑ eventually enables the stable surface adsorption of both species to generate integrated biosensors and, more importantly, leads to a negative shift of the redox potential of the surface-confined redox mediator. These properties substantially validate the ferricyanide-based second-generation biosensors for selective in vivo neurochemical measurements in terms of the good differentiation of the potential of the biosensors from those for the oxidation of electroactive neurochemicals. This study actually offers an effective approach to developing new electrochemical methods that may be developed to be general for in vivo neurochemical measurements.

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Figure 6. Typical current−time responses obtained for the microdialysates continuously sampled from the striatum of guinea pig with the Fe(CN)63‑/Pim/MWNT (A, B, C)-modified electrode and glucose biosensor (D) fixed into the thin-layer electrochemical flow cell as the detector in the online detecting system with aCSF as the perfusion solution. Flow rate, 2 μL/min. Potential applied, +0.25 V (A), +0.20 V (B), +0.17 V (C), and +0.17 V (D).

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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system for the microdialysates continuously sampled from the striatum of guinea pigs. To study the selectivity for in vivo measurements, the Fe(CN)63‑/Pim/MWNT-modified GC electrode (i.e., no GOD) was fixed into the thin-layer electrochemical flow cell and the electrode was polarized at different potentials for the measurements of the microdialysates continuously sampled from the striatum of guinea pigs. When the electrode was polarized at +0.25 V (A) and +0.20 V (B), an obvious current response was observed for the microdialysates, indicating that some kinds of electroactive neurochemicals were oxidized at these potentials. In contrast, when the electrode was polarized at a lower potential of +0.17 V, a potential employed for glucose measurement with the biosensors developed in this study (Figure 5D); almost no current increase was observed for

AUTHOR INFORMATION

Corresponding Author

*Fax: +86-10-62559373. E-mail: [email protected] (L.M.); [email protected] (P.Y.); [email protected] (W.J.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the NSF of China (Grant Nos. 20975104, 20935005, 90813032, and 21127901 for L.M.; 20805050 and 91132708 for P.Y.; and 20905071 for Y.L.), the National Basic Research Program of China (973 program, 2010CB33502), and The Chinese Academy of Sciences (KJCX2-YW-W25 and Y2010015). 1905

dx.doi.org/10.1021/ac202748s | Anal. Chem. 2012, 84, 1900−1906

Analytical Chemistry

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Article

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dx.doi.org/10.1021/ac202748s | Anal. Chem. 2012, 84, 1900−1906