Phenylboronic Acid Monolayer-Modified Electrodes Sensitive to

Smartphone-based cyclic voltammetry system with graphene modified screen printed electrodes for .... Journal of Electroanalytical Chemistry 2016 761, ...
0 downloads 0 Views 133KB Size
Download PDF
5102

Langmuir 2005, 21, 5102-5107

Phenylboronic Acid Monolayer-Modified Electrodes Sensitive to Sugars Shigehiro Takahashi and Jun-ichi Anzai* Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan Received January 21, 2005. In Final Form: March 10, 2005 The surface of a gold (Au) electrode was modified with 4-mercaptophenylboronic acid (MPBA) and dithiobis(4-butyrylamino-m-phenylboronic acid) (DTBA-PBA) to prepare sugar-sensitive electrodes. MPBA and DTBA-PBA formed well-packed monomolecular layers on the Au electrode through a sulfur-Au bond. The MPBA- and DTBA-PBA-modified electrodes exhibited a nearly reversible cyclic voltammogram (CV) for the Fe(CN)63-/4- ion in acidic solution, while the CVs were significantly attenuated in alkaline media as a result of addition of OH- ion to the boron atom to generate the negatively charged surface. In other words, the negatively charged monolayers blocked the surface of the Au electrode from the access of the Fe(CN)63-/4- ion. The pKa values of the addition equilibrium of the OH- ion to the MPBA and DTBA-PBA monolayers were estimated to be 9.2 ( 0.1 and 8.0 ( 0.2, respectively, on the basis of the pH-dependent peak current (ip) in the CV of the Fe(CN)63-/4- ion. On the other hand, in the presence of sugars, the addition of the OH- ion was accelerated by forming the phenylboronate esters of sugars on the surface of the monolayers. The pKa values for the MPBA and DTBA-PBA monolayers were 8.3 ( 0.1 and 7.2 ( 0.1, respectively, in the presence of 50 mM D-fructose. The MPBA- and DTBA-PBA-modified electrodes can be used for determining sugars on the basis of the change in ip of the Fe(CN)63-/4- ion in the presence of sugars. The calibration curves useful for determining 1-100 mM D-glucose, D-mannose, and D-fructose were obtained.

1. Introduction Enzyme-based biosensors have been widely used for determining sugars in diagnostic analysis, process control of food industries, and so forth.1 The merit of enzyme biosensors is that a specific sugar can be determined selectively among the mixture of isomers and even in the biological fluids such as blood and urine. However, one of the significant drawbacks of the enzyme sensors comes from difficulty in the standardization of quality of enzymes (the catalytic activity often depends on the purity and origin of the sample and on the protocol of immobilization), resulting in the different performances of the sensors from sample to sample. Another demerit of enzyme sensors stems from the fact that the catalytic activity of enzymes usually degrades gradually day by day, requiring a frequent calibration before use. For these reasons, the development of nonenzymatic devices sensitive to sugars has been a focal subject in bioanalytical science and technology. Phenylboronic acid derivatives have been employed as an alternative to enzyme for developing sugar-sensitive systems based on UV-visible absorption,2 fluorescence,3 circular dichroism,4 and surface plasmon resonance.5 The systems based on UV-visible and fluorescence spectra * To whom correspondence should be addressed. E-mail: [email protected]. (1) (a) Maestre, E.; Katakis, I.; Dominguez, E. Biosens. Bioelectron. 2001, 16, 61-68. (b) Hoshi, T.; Saiki, H.; Kuwazawa, S.; Tsuchiya, C.; Chen. Q.; Anzai, J. Anal. Chem. 2001, 73, 5310-5315. (c) Wu, B.; Zhang, G.; Shuang, S.; Choi, M. M. F. Talanta 2004, 64, 546-553. (d) Lupu, A.; Compagnone, D.; Palleschi, G. Anal. Chim. Acta 2004, 513, 67-72. (2) (a) Davis, C. J.; Lewis, P. T.; McCarroll, M. E.; Read, M. W.; Cueto, R.; Strongin, R. M. Org. Lett. 1999, 1, 331-334. (b) Koumoto, K.; Shinkai, S. Chem. Lett. 2000, 2, 856-857. (c) DiCesare, N.; Lakowicz, J. R. Org. Lett. 2001, 3, 3891-3893. (3) (a) Gao, S.; Wang, W.; Wang, B. Bioorg. Chem. 2001, 29, 308320. (b) DiCesare, N.; Lakowicz, J. R. Tetrahedron Lett. 2002, 43, 26152618. (c) Camara, J. N.; Suri, J. T.; Cappuccio, F. E.; Wessling, R. A.; Singaram, B. Tetrahedron Lett. 2002, 43, 1139-1141.

have been studied most extensively among the detection modes, because the optical properties of phenylboronic acid derivatives are significantly modulated upon binding of sugars. For example, boronic acid-substituted azo dyes have been synthesized for the colorimetric detection of sugars, where the absorption maximum of the dyes exhibited a red shift upon sugar binding.2c The sugarinduced changes in UV-visible and fluorescence spectra stem from the formation of the anionic form of boronic acid. The anionic form of boronic acid is characterized by an electron-rich sp3 boron atom with a tetrahedral geometry, whereas the nonionic form is an electrondeficient Lewis acid with an sp2-hybridized boron atom. The change in the electronic properties of the boronic residue is an origin of the sugar-induced spectral changes of colorimetric and fluorometric determination of sugars. In addition, it has been reported that the electrochemical properties of boronic acid-substituted ferrocenes and triphenylamines are also modified by the formation of anionic forms upon binding of sugars.6 The phenylboronic acid derivatives described above can be used as soluble reagents for detecting sugars in solution. To develop an alternative to enzyme-based biosensors, the phenylboronic acid derivatives have to be immobilized on the surface of the transducers such as electrodes and optical devices. To this end, phenylboronic acid-modified electrodes have been prepared for the electrochemical detection of sugars. Okano and co-workers reported an (4) (a) Kimura, T.; Kakeuchi, M.; Nagasaki, T.; Shinkai, S. Tetrahedron Lett. 1995, 36, 559-562. (b) Takeuchi, M.; Mizuno, T.; Shinkai, S.; Shirakami, S.; Itoh, T. Tetrahedron: Asymmetry 2000, 11, 33113322. (5) Lee, M.; Kim, T.; Kim, K.; Kim, J.; Choi, M.; Choi, H.; Koh, K. Anal. Biochem. 2002, 310, 163-170. (6) (a) Dusemund, C.; Sandanayake, K. R. A. S.; Shinkai, S. J. Chem. Soc., Chem. Commun. 1995, 333-334. (b) Moore, A. N. J.; Wayner, D. D. M. Can. J. Chem. 1999, 77, 681-686. (c) Nicolas, M.; Fabre, B.; Chapuzet, J. M.; Lessard, J.; Simonet, J. J. Electroanal. Chem. 2000, 482, 211-216.

10.1021/la050171n CCC: $30.25 © 2005 American Chemical Society Published on Web 04/14/2005

Phenylboronic Acid Monolayer-Modified Electrodes

Figure 1. Equilibrium between the phenylboronic acid derivative and the OH- ion and/or sugar.

electrochemical sensing of D-glucose with platinum electrodes coated with a film of copolymers containing phenylboronic acid side chains.7 The electrochemical signal of the electrode depended on the D-glucose concentration in the sample solution due to the D-glucose-dependent swelling of the polymer film. A drawback of this electrode is its slow response to D-glucose (the response time was ca. 30-40 min), arising from a thick nature of the polymer film (ca. 6 µm in the dry state). A molecular-level modification of the surface of a gold (Au) electrode was carried out by Nakashima and co-workers by means of a self-assembled monolayer of phenylboronic acid-substituted viologen derivative.8 The redox potential of the viologen residue in the self-assembled film was found to be sensitive to sugars. Recently, a potentiometric sugar sensor has been constructed by Shoji and Freund by means of a poly(aniline boronic acid) film-coated glassy carbon (GC) electrode.9 The modified GC electrode exhibited a potentiometric response to sugars on the basis of the pKa changes of the poly(aniline boronic acid) film upon sugar binding. The mechanism by which sugars can be detected on the basis of the optical and electrochemical techniques originates from the fact that an anionic form of the boronic acid-sugar conjugate is produced accompanied by sugar binding, while the boronic acid residue contains no electric charge in the absence of sugars (Figure 1). This fact promoted us to construct a voltammetric sugar sensor by means of Au electrodes coated with a self-assembled monolayer film composed of thiol-modified phenylboronic acids.10 The surface density of the anionic charge of the boronic acid-modified electrode would increase upon sugar binding, which in turn may modify the voltammetric response of redox ions in the solution, as in the case of “ion channel sensors”.11 We report here the voltammetric behavior of Au electrodes modified with self-assembled monolayers of 4-mercaptophenylboronic acid (MPBA) and dithiobis(4-butyrylamino-m-phenylboronic acid) (DTBAPBA) (Figure 2). 2. Experimental Section 2.1. Materials. MPBA and 3-aminophenylboronic acid hemisulfate were purchased from Aldrich Chemical Co. (Milwaukee, WI) and Nakalai Tesque (Kyoto, Japan), respectively. 4,4′-Dithio-3-di(n-butyric acid) was obtained from Tokyo Kasei Co. (Tokyo, Japan). DTBA-PBA was synthesized from 3-aminophenylboronic acid hemisulfate and 4,4′-dithio-3-di(n-butyric acid) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (7) Kikuchi, A.; Suzuki, K.; Okabayashi, O.; Hoshino, H.; Kataoka, K.; Sakurai, Y.; Okano, T. Anal. Chem. 1996, 68, 823-828. (8) Murakami, H.; Akiyoshi, H.; Wakamatsu, T.; Sagara, T.; Nakashima, N. Chem. Lett. 2000, 940-941. (9) (a) Shoji, E.; Freund, M. S. J. Am. Chem. Soc. 2001, 123, 33833384. (b) Shoji, E.; Freund, M. S. J. Am. Chem. Soc. 2002, 124, 1248612493. (10) Takahashi, S.; Kashiwagi, Y.; Hoshi, T.; Anzai, J. Anal. Sci. 2004, 20, 757-759. (11) Umezawa, Y.; Aoki, H. Anal. Chem. 2004, 76, 321A-326A.

Langmuir, Vol. 21, No. 11, 2005 5103

Figure 2. Chemical structures of MPBA and DTBA-PBA. as a coupling agent according to the reported procedure.12 All other reagents used are of commercial products of highest grade available. 2.2. Apparatus. A Shimadzu UV3100 UV-visible absorption spectrophotometer (Kyoto, Japan) was employed for evaluating pKa of MPBA and DTBA-PBA in solution. A potentiostat of type NPGFZ 2501A (Atugi, Japan) was used for all electrochemical measurements. 2.3. Determination of pKa of MPBA and DTBA-PBA in Solution. The UV-visible absorption spectra of the MPBA and DTBA-PBA solutions in a water-methanol mixture (9:1 by volume) containing 100 mM NaCl were recorded as a function of pH. The pH values of the solutions were regulated by a small amount of HCl or NaOH. The absorbance of the spectra at 256 nm for MPBA and at 287 nm for DTBA-PBA was plotted over pH 4-11, and the pKa values were obtained from the titration curves. 2.4. Preparation of MPBA and DTBA-PBA Monolayer Films on the Surface of the Au Electrode. The surface of an Au disk electrode (3.0-mm diameter) was polished with alumina slurry and sonicated twice in distilled water for 5 min. The polished Au electrode was electrochemically treated in a 0.5 M H2SO4 solution by scanning the electrode potential from -0.2 V to 1.5 V at a scan rate of 0.1 V s-1 for 20 min. The clean Au electrode thus treated was modified with the MPBA or DTBAPBA by immersing the electrode in the MPBA or DTBA-PBA solution (0.5 mg mL-1 in tetrahydrofuran-methanol ) 9:1) for 8 h at room temperature (ca. 20 °C). 2.5. Electrochemical Measurements. A cyclic voltammogram (CV) was measured with a conventional three-electrode system using Ag/AgCl as a reference electrode and a Pt wire (1-mm diameter) as an auxiliary electrode. All measurements were carried out at room temperature (ca. 20 °C).

3. Results and Discussion 3.1. Modification of the Au Electrode with MPBA and DTBA-PBA. It is well-known that thiol and disulfide compounds form a self-assembled monolayer on the surface of Au through a strong Au-sulfur bond.8,13,14 We prepared phenylboronic acid monolayer-modified Au electrodes using MPBA and DTBA-PBA by immersing a Au electrode in the phenylboronic acid solutions (0.5 mg mL-1 in tetrahydrofuran-methanol ) 9:1) for 8 h. The surface density or surface coverage of MPBA and DTBAPBA in the monolayers was evaluated by an electrochemical reductive desorption of the monolayers, according to the reported procedure.8,13,14 The monolayer-modified (12) Kanayama, N.; Kitano, H. Langmuir 2000, 16, 577-583. (13) (a) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (b) Carrey, R. I.; Folkers, J. P.; Whitesides, G. M. Langmuir 1994, 10, 2228-2234. (c) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir 1996, 12, 3763-3772. (d) Higashi, N.; Takahashi, M.; Niwa, M. Langmuir 1999, 15, 111-115. (e) Su, L.; Sankar, C. G.; Sen, D.; Yu, H. Anal. Chem. 2004, 76, 5953-5959. (f) Rhee, C.; Kim, Y. Appl. Surf. Sci. 2004, 228, 313-319. (14) (a) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335-359. (b) Walczak, M. M.; Popenone, D. D.; Deinhammer, R. S.; Lamp, D.; Chung, C.; Porter, M. D. Langmuir 1991, 7, 26872639. (c) Zhong, C. J.; Porter, M. D. J. Am. Chem. Soc. 1994, 116, 11616. (d) Yang, W.; Gooding, J. J.; Hibbert, D. B. J. Electroanal. Chem. 2001, 516, 10-16. (f) El-Deab, M. S.; Ohsaka, T. Electrochim. Acta 2004, 49, 2189-2194.

5104

Langmuir, Vol. 21, No. 11, 2005

Figure 3. CVs of the Fe(CN)63- ion (5 mM) on the DTBA-PBA monolayer-modified electrode in the absence (a) and in the presence of 50 mM D-fructose (b) and on an unmodified Au electrode (c). The 10 mM phosphate buffer containing 100 mM KCl was used. The scan rate was 50 mV s-1.

Au electrodes exhibited reduction peaks from -0.7 to -1.1 V that can be ascribed to the reductive desorption of MPBA and DTBA-PBA. The surface densities of MPBA and DTBA-PBA were calculated to be 7.5 × 10-10 and 5.1 × 10-10 mol cm-2, respectively, from the amount of charge passed and taking the surface roughness of the Au electrode (3.6) into account.15 In other words, the occupied areas of MPBA and DTBA-PBA on the electrode surface are 0.23 and 0.33 nm2, respectively. Kitano et al.12 and Koh et al.5 have independently found that DTBA-PBA forms a well-packed monolayer film, whose occupied area is reported to be 0.24-0.28 nm2. Thus, it can be said that MPBA and DTBA-PBA monolayer films are formed on the Au electrode by the present procedure. 3.2. Voltammetric Response of Fe(CN)63-/4- on the MPBA and DTBA-PBA Monolayer-Modified Electrodes. Phenyboronic acids are known to assume an anionic form in alkaline solutions as a result of the addition of the OH- ion to the boron atom (Figure 1). Consequently, the surface density of the anionic charges on the MPBA and DTBA-PBA film-modified electrodes would be a function of pH of the solution in which the electrodes are immersed. Thus, the voltammetric behavior of the MPBA and DTBA-PBA film-modified electrodes toward anionic species in solution may depend on pH. Figure 3 shows CVs of the Fe(CN)63-/4- ion recorded on the DTBA-PBA film-modified and unmodified Au electrodes at pH 6 and 9 in the absence and in the presence of 50 mM D-fructose. In Figure 3a, a nearly reversible CV was observed at pH 6 in the absence of sugar (the difference between the anodic and cathodic peak potentials, ∆Ep, was ca. 80 mV), confirming that the redox reactions of the (15) The roughness factor of the electrode surface was determined from the charge passed upon forming an oxide monolayer on the surface, according to the reported procedure: Trasatti, S.; Petrii, O. A. Pure Appl. Chem. 1991, 63, 711-734.

Takahashi and Anzai

Fe(CN)63-/4- ion occurred rather smoothly across the DTBA-PBA monolayer on the electrode. In contrast, the CV was significantly attenuated at pH 9, ∆Ep being about 430 mV. This is due to the electrostatic repulsion between the Fe(CN)63-/4- ion and the negatively charged surface of the electrode, which originates from the addition of the OH- ion to the boron atom of the DTBA-PBA in the alkaline media (as in Figure 1). We have separately ascertained that CVs of the Fe(CN)63-/4- ion on a bare Au electrode are pH-independent over pH 4-10 (Figure 3c), confirming that the attenuated CV at pH 9 originates from the negatively charged DTBA-PBA monolayer on the electrode surface. Similar measurements were carried out in the presence of 50 mM D-fructose to estimate the effects of sugar on the voltammetric response (Figure 3b). The DTBA-PBA monolayer-modified electrode was incubated in a 50 mM D-fructose solution for 20 min before measuring the CVs to complete the binding of D-fructose to the DTBAPBA monolayer. We used here D-fructose as a prototype of sugars because D-fructose is known to be bound by phenylboronic acids rather strongly among monosaccharides.16 The CV recorded at pH 6 was almost the same as the CV obtained in the absence of D-fructose at the same pH, showing that the effects of sugar binding is negligible or D-fructose does not bind to the phenylboronic acid monolayer in the pH 6 solution. On the other hand, no peak was observed in the CV recorded at pH 9, suggesting that the electron transfer between the Fe(CN)63-/4- ion and the electrode was strongly disturbed. The suppressed electron transfer can be ascribed to the formation of a negatively charged surface of the electrode due to the addition of the OH- ion and D-fructose, as schematically shown in Figure 4. It should be noted here that the attenuation of CV obtained in the presence of D-fructose at pH 9 is much more significant than that observed in the absence of sugar at the same pH, suggesting that the formation of the DTBA-PBA-D-fructose adduct accelerated the accumulation of negative charges. To monitor the kinetics of sugar binding, we measured CVs of the Fe(CN)63-/4- ion on the DTBA-PBA monolayermodified electrode in 1 mM and 50 mM D-fructose solutions (pH 7.5), and the anodic peak current (ip) of the CV was plotted as a function of the reaction time (Figure 5). The ip reached a steady-state value after about 15 min in 1 mM D-fructose, while the response was slightly faster in the presence of 50 mM D-fructose. Judging from the results, the CV measurements were carried out after the 20-min incubation throughout the present study. To fully verify the effects of pH on the voltammetric response, the CV of the Fe(CN)63-/4- ion was measured with changing pH of the media in the presence and absence of D-fructose. Figure 6A plots the ip of the CVs measured on the DTBA-PBA monolayer-modified electrode as a function of pH. The ip values were higher in the acidic region than those in the solutions of alkaline pH. The transition of the curves was observed at a neutral pH in the absence of D-fructose and at a slightly acidic pH in the presence of 50 mM D-fructose. This is due to the fact that the pKa of the DTBA-PBA-D-fructose conjugate is smaller than that of the unconjugated DTBA-PBA, which is in line with the general trend for pKa values determined in solution; pKa values of sugar esters of phenylboronic acid are often more acidic than that of phenylboronic acid itself.16 The MPBA monolayer-modified electrode exhibited a similar behavior (Figure 6B). (16) (a) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, 5291-5300. (b) Yan, J.; Springsteen, G.; Deeter, S.; Wang, B. Tetrahedron 2004, 60, 11205-11209.

Phenylboronic Acid Monolayer-Modified Electrodes

Langmuir, Vol. 21, No. 11, 2005 5105

Figure 4. Schematic illustration of the binding of sugar onto the DTBA-PBA monolayer-modified electrode.

Figure 5. Peak current in the CV of the 5 mM Fe(CN)63- ion on the DTBA-PBA monolayer-modified electrode in the presence of 1 (a) and 50 mM D-fructose (b) as a function of the time. The 10 mM phosphate buffer containing 100 mM KCl (pH 7.6) was used. The scan rate was 50 mV s-1. The average values of two measurements are plotted (error, within 5%).

The pKa values of surface-immobilized compounds have been determined by many different techniques, including interfacial capacitance,17 gravimetry using a quartz crystal microbalance,18 contact angle,19 surface-enhanced Raman scattering,20 and cyclic voltammetry.21 We can estimate the apparent pKa of DTBA-PBA and MPBA and their sugar conjugates confined on the electrode surface by taking advantage of the titration curves in Figure 6, according to the reported procedure.21 The apparent pKa can be defined according to the acid-base equilibrium of DTBAPBA and MPBA as eq 1, where [B-] and [B] denote the concentrations of the anionic and neutral forms, respectively, of the surface-confined phenylboronic acid and its sugar ester.

pKa ) pH + log([B-]/[B])

(1)

The peak current of the CV at a given pH can be described as a sum of the currents originating from the redox reactions that occurred on the electrode surfaces covered with the neutral and negatively charged fractions (17) (a) Bryant, M. A.; Crooks, R. M. Langmuir 1993, 9, 385-387. (b) Kakiuchi, T.; Iida, M.; Imabayashi, M.; Niki, K. Langmuir 2000, 16, 5397-5401. (c) Kim, K.; Kwak, J. J. Electroanal. Chem. 2001, 512, 83-91. (18) (a) Shimazu, K.; Teranishi, K.; Sugihara, K.; Uosaki, K. Chem. Lett. 1998, 669. (b) Sugihara, K.; Shimazu, K.; Uosaki, K. Langmuir 2000, 16, 7101-7105. (19) (a) Chatelier, R. C.; Drummond, C. J.; Chan, D. Y. C.; Vasic, Z. R.; Gengenbach, T. R.; Griesser, H. J. Langmuir 1995, 11, 4122-4128. (b) Zhao, J.; Luo, L.; Yang, X.; Wang, E.; Dong, S. Electroanalysis 1999, 11, 1108-1111. (20) Hu, K.; Bard, A. J. Langmuir 1997, 13, 5114-5119. (21) (a) Cheng, Q.; Brajter-Toth, A. Anal. Chem. 1996, 68, 41804185. (b) Godines, L. A.; Castro, R.; Kaifer, A. E. Langmuir 1996, 12, 5087-5092. (c) Li, X.; Wan, L.; Sun, C. J. Electroanal. Chem. 2004, 569, 79-87.

Figure 6. Peak current in the CV of the 5 mM Fe(CN)63- ion on the DTBA-PBA monolayer- (A) and MPBA monolayermodified electrodes (B) in the absence (a) and in the presence of 50 mM D-fructose (b). The 10 mM phosphate buffer containing 100 mM KCl was used. The scan rate was 50 mV s-1. The average values of three measurements are plotted (error, within 5%).

of the monolayer (eq 2), where i is the peak current in the CV recorded at a given pH, and iB and iB- are the peak currents observed for the neutral and fully negatively charged monolayer-modified electrodes, respectively.

i ) iB[B]/([B] + [B-]) + iB-[B-]/([B] + [B-])

(2)

Consequently, from eqs 1 and 2, one can obtain eq 3.

pKa ) pH + log[(iB - i)/(i - iB-)]

(3)

Thus, using the data in Figure 6, we obtained the pKa values of the monolayers in the absence and presence of 50 and 100 mM D-fructose. The results are collected in Table 1 together with the pKa values obtained in solution. The pKa of the DTBA-PBA monolayer was found to be 8.0 ( 0.2 in the absence of sugar while the values were shifted to 7.2 ( 0.1 and 6.4 ( 0.1 in the presence of 50 and 100 mM D-fructose, respectively. Thus, the pKa depends on

5106

Langmuir, Vol. 21, No. 11, 2005

Takahashi and Anzai

Table 1. pKa Values of MPBA and DTBA-PBA in the Self-Assembled Monolayer and in Solution D-fructose,

mM

in self-assembled monolayera

in solutionb

MPBA

0 50 100

9.2 ( 0.1 8.3 ( 0.1 8.3 ( 0.1

10.1 ( 0.1 7.0-8.0c

DTBA-PBA

0 50 100

8.0 ( 0.2 7.1 ( 0.1 6.4 ( 0.1

8.6 ( 0.1 6.0 ( 0.1 5.7 ( 0.1

a Determined by cyclic voltammetry. b Determined by UV-visible spectrometry in a methanol-water (1:9 by volume) mixture containing 100 mM NaCl. c A roughly estimated value (see text).

Figure 7. CVs of the Fe(CN)63- ion on the DTBA-PBA monolayer-modified electrode in the presence of D-fructose. The 10 mM phosphate buffer containing 100 mM KCl (pH 7.6) was used. The scan rate was 50 mV s-1.

the concentration of sugar, confirming that the shift in pKa is associated with the formation of the phenylboronate ester of sugar on the surface of the monolayer. The pKa for the MPBA monolayer was 9.2 ( 0.1 in the absence of sugar and 8.3 ( 0.1 in the presence of 50 and 100 mM D-fructose. We have separately determined the pKa of DTBA-PBA and MPBA dissolved in an aqueous solution by taking advantage of the fact that the neutral and ionic forms of the phenylboronic acids exhibit different UV-visible absorption spectra from each other.15a Thus, we prepared absorbance-pH titration curves for both compounds to obtain the pKa values. The pKa of DTBA-PBA was determined to be 8.6 ( 0.1 in the absence of sugar, while the values were 6.0 ( 0.1 and 5.7 ( 0.1 in the presence of 50 and 100 mM D-fructose, respectively. The pKa of MPBA in solution was obtained to be 10.1 ( 0.1 in the absence of sugar. However, unfortunately, the value cannot be precisely obtained in the presence of sugar because the deprotonation from the -SH group took place in pH 4.0-7.0, where the addition of the OH- ion to the MPBA-D-fructose adduct also occurred, resulting in a partial overlapping of the two titration curves.22 Despite the partially overlapped titration curves, we may be able to roughly estimate the pKa in the presence of 50 mM D-fructose to be 7.0-8.0. Figure 7 shows CVs of 5 mM K3[Fe(CN)6] on the DTBAPBA monolayer-modified electrode in the 10 mM phosphate buffer containing 100 mM KCl (pH 7.6). In the absence of sugar, the CV exhibited clear redox peaks originating from a nearly reversible redox reaction of the [Fe(CN)6]3- ion, ∆Ep being 125 mV. The slight broadening of the CV is due the fact that a part of the monolayer (22) The absorption maxima of MPBA in the presence of 50 mM were found at 256 nm for neutral MPBA at pH 3.5, at 288 nm for deprotonated MPBA at pH 6.6, and at 264 nm for dianionic form of MPBA at pH 11.6. However, no isosbestic point was observed probably due to the overlapping of the deprotonation and the addition of the OHion. D-fructose

Figure 8. Calibration graphs of the DTBA-PBA monolayer(A) and MPBA monolayer-modified electrode (B) to D-glucose (a), D-mannose (b), and D-fructose (c). The I/I0 shows the relative value of the peak current in the presence of sugar to that observed in the absence of sugar. The 10 mM phosphate buffer containing 100 mM KCl (pH 7.6 for A and pH 8.9 for B) was used. The scan rate was 50 mV s-1. The average values of three measurements are plotted (error, within 5%).

surface is negatively charged at pH 7.6 (see Figure 6A). On the other hand, when the measurements were carried out in the presence of D-fructose, attenuated CVs were observed; the ip in the CV decreased and the ∆Ep increased. The shape of the CVs significantly depended on the concentration of D-fructose. The D-fructose-dependent voltammetric behavior can be rationalized based on the binding of D-fructose on the surface of the DTBA-PBA monolayer-modified electrode. It is reasonable to assume that, at pH 7.6, the DTBA-PBA moieties on the electrode bind D-fructose to form the negatively charged surface as a result of the addition of the OH- ion from the solution. The negatively charged DTBA-PBA would limit the access of the [Fe(CN)6]3-/4- ion to the electrode surface due to the electrostatic repulsion. This is rationalized from the titration curves in Figure 6A, in which the ip in the 50 mM D-fructose solution is about 20 µA at pH 7.6 while that observed in the absence of D-fructose is about 45 µA. Thus, it is clear that the ip decreases with the increasing concentration of sugar in the solution. Figure 8 plots the ip values for the modified electrodes as a function of the concentrations of D-fructose, Dmannose, and D-glucose. The monolayer-modified electrodes exhibited a response to all sugars tested. The response to D-fructose is more sensitive than those to D-mannose and D-glucose for both electrodes, which is in line with the selectivity observed for phenylboronic acidbased optical sensors for sugar detection.2,3 This is reasonable because the chemical structures of the binding site of phenylboronic acid compounds used in the optical

Phenylboronic Acid Monolayer-Modified Electrodes

Langmuir, Vol. 21, No. 11, 2005 5107

Table 2. Comparison of Detection Range of Phenylboronic Acid-Based Sugar Sensors sensor system voltammetry MPBA

sugar D-fructose D-glucose D-fructose D-mannose D-glucose

potentiometrya

D-fructose D-glucose

voltammetryb

D-fructose D-mannose D-glucose

sprc

D-fructose D-mannose D-glucose

colorimetryd

D-fructose D-glucose

fluorometrye

reference present work

D-mannose

DTBA-PBA

detection range, mM

D-fructose D-glucose

3-100 10-300 30-300 0.3-30 3-300 3-300 3.4-40.8 3.4-40.8 0.001-1 0.001-1 0.01-1 10-9-0.1 10-7-0.1 10-9-0.001 1-50 1-180 1-20 1-50

9 10 5 2c 3b

a Phenylboronic acid (PBA)-substituted polyaniline-coated electrode. b PBA-substituted viologen monolayer-modified electrode. c DTBA-PBA monolayer-coated SPR. d PBA-substituted azobenzene dye. e Chalcone fluorescent dye.

sensors and in the present study are basically the same. For improving selectivity of the electrodes, the chemical structure of the phenylboronic acids have to be suitably designed to bind a specific sugar. The D-fructose is known to be bound by phenylboronic acid more strongly than D-mannose and D-glucose.16 These results suggest that the modified electrodes may be useful for determining about a millimolar level of these sugars. Table 2 lists the detection range of typical sugar sensors based on phenylboronic acid derivatives. It is clearly seen that the detection range is highly dependent on the detection mode. Among the sensors listed, the electrochemical sensor using phenylboronic acid-substituted viologen and the surface plasmon resonance-based optical sensor are suitable for detecting a micromolar level of sugars, while other optical and electrochemical sensors including the sensors developed in the present work can be used for determining a millimolar level of sugars. The latter sensors may be useful for detemining D-glucose in blood or serum in view of the fact that the normal D-glucose level in blood is about 5 mM but 10 mM or higher for diabetic patients. Reusability of the monolayer-modified electrode was evaluated. Figure 9 shows CVs of the [Fe(CN)6]3-/4- ion recorded on the DTBA-PBA-modified electrode before and after regeneration of the original surface. After the CV measurement in 50 mM D-fructose solution (CV b), the electrode was rinsed in a 10 mM acetate buffer (pH 4.5) for 10 min to regenerate the sugar-free surface. The original CV was recovered by this treatment (CV c), as a result of the dissociation of D-fructose in the acidic buffer. Thus, the sugar ester of DTBA-PBA on the electrode was completely decomposed in the acidic buffer due to the

Figure 9. CVs of 5 mM Fe(CN)63- ion on the DTBA-PBA monolayer-modified electrode before and after regeneration of the surface. The CV a is in the absence of sugar, and CV b is in the presence of 50 mM D-fructose. The CV c is measured in the absence of sugar after regeneration of the surface by rinsing the used electrode in 10 mM acetate buffer (pH 4.5) for 10 min.

intrinsic low stability in the acidic medium. Thus, the phenylboronic acid monolayer-modified electrode can be used repeatedly for determining sugars. From the viewpoint of selective determination of sugars, the present system is still immature because the MPBA and DTBA-PBA monolayer-modified electrodes exhibit low selectivity to a specific sugar. Instead, the electrodes exhibit a group-sensitive nature to sugar derivatives as in the case for sugar-sensitive colorimetric and fluorometric reagents based on phenylboronic acids.2,3 Another challenging goal is to develop reagentless sensors that can be used without adding redox ions such as Fe(CN)63-/4in the sample solution. The reagentless sensors may be developed using self-assembled monolayers composed of phenylboronic acid derivatives bearing a redox-active moiety. 4. Conclusion Self-assembled monolayers composed of MPBA and DTBA-PBA have proved to be useful for constructing sugar-sensitive electrodes that can be employed for voltammetric determination of sugars. The present work has demonstrated a novel strategy based on the phenylboronic acid derivatives for developing protein-free electrochemical sugar sensors. Acknowledgment. The present work was supported in part by a Grant-in-Aid (No. 16390013) from Japan Society for the Promotion of Sciences (JSPS). LA050171N