Monolayer modification of glassy carbon electrode by using propionylcholine for selective detection of uric acid

Electrochimica Acta 50 (2005) 3210–3216

Monolayer modification of glassy carbon electrode by using propionylcholine for selective detection of uric acid Xiang-Qin Lin∗ , Guan-Ping Jin Department of Chemistry, University of Science and Technology of China, 96 Jinzhai, Hefei 230026, China Received 29 October 2004; accepted 24 November 2004 Available online 4 January 2005

Abstract A monolayer modified glassy carbon electrode by using propionylcholine (denoted as PCh/GCE) was fabricated via cyclic voltammetry. A strong electrochemical catalytic activity toward the oxidation of uric acid (UA) was observed. The peak potential separation of differential pulse voltammetry for UA and ascorbic acid (AA) was about 293 mV, allowing the determination of UA in the presence of high concentration of AA. X-ray photoelectron spectroscopy (XPS), in situ UV–visible spetroelectrochemistry (UV–vis) and electrochemical impedance spectrum techniques were used for characterization. It was demonstrated that the PCh was hydrolyzed to choline (Ch) and propionic acid as soon as the Ch covalently bounded on the carbon surface accompanying with an imbedding of the propionic acid into the Ch modified layer, forming the novel monolayer modification of choline residue and propionic acid interdigitated assembling. In optimal condition, a linear range of 0.07–70 ␮M, a detection limit of 0.02 ␮M (3σ) in the presence of 0.1 mM AA were achieved. Also, choline, acetylcholine and butylcholine modified GCEs were similarly prepared for comparison. Utility of the PCh/GCE was demonstrated by the measurement of UA in human urine without any pretreatment. © 2004 Elsevier Ltd. All rights reserved. Keywords: Propionylcholine; Monolayer; Modified electrode; Uric acid; Ascorbic acid

1. Introduction Uric acid (UA) is the primary end product of purine metabolism. Abnormal levels of UA are symptoms of several diseases, such as gout, hyperuricemia, and Lesch-Nyan disease [1]. Since UA and AA are electrochemically active at carbon-based electrodes, their electrochemical detection becomes one of the feasible methods. However, since they coexist in biological fluids such as blood and urine [2], earlier electrochemical procedures based on the oxidation of UA at carbon-based electrodes suffered from the interference from AA that can also be oxidized at close potentials. Various methods, such as enzyme-based techniques [4], chemically modified electrode [5–12] were developed to solve the UA detection problem. Until now, sensitive and selective methods still needed to be developed for the detection. ∗

Corresponding author. Tel.: +86 551 3606646; fax: +86 551 3601592. E-mail address: [email protected] (X.-Q. Lin).

0013-4686/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2004.11.057

Propionylcholine (PCh) is a choline derivative; which can be hydrolyzed to generate propionic acid and choline (Ch), a precursor of acetylcholine (ACh), an important neurotransmitter, synthesis. Considerable interest is dealing with the developing biosensors for Ch and acetylcholine detections by using cholinesterase [13,14]. However, the usage of Ch and its deritives, such as PCh, for surface modification for fabrication of biosensors had never been reported until our recent work. Recently in our laboratory, it has been revealed that Ch, PCh could be planted on the surface of carbon electrodes forming a monolayer modification. The mechanism of their modification was cafully studied by various characterization techniques. We found that the Ch and its derivatives can be planted on the surface of carbon electrodes forming such a monolayer-modified electrode with specific activities. The significance of Ch modification is to provide a positively charged N+ (CH3 )3 polar head groups in the monolayer, which may play an important inherence for bio-active interactions. Propionic acid can also be modified on the

X.-Q. Lin, G.-P. Jin / Electrochimica Acta 50 (2005) 3210–3216

electrode surface by using PCh as a modifier. The imbedded propionic acid plays tiny modulate effect on the activity of the monolayer. Certainly, we found an interesting catalytic activity of PCh modified glassy carbon electrode toward UA and AA oxidations, which resolves their voltammetric responses into two well-defined voltammeter peaks. In this report, the PCh modified glassy carbon electrode was discussed in comparison with Ch modified electrode for catalytic activities toward UA and AA oxidation reactions. And the experimental conditions were optimized for determination of UA.

2. Experimental 2.1. Materials Choline (Ch), acetylcholine (ACh) were purchased from Chemical Regent Factory of Beijing (Beijing China). Propionylcholine (PCh) and Butylcholine (BuCh) were obtained from Sigma (USA). Uric acid (UA) and ascorbic acid (AA) were obtained from Chemical Regent Company of Shanghai (Shanghai, China). All other regents used were analytical grade. Solutions of Ch, ACh, PCh and BuCh were prepared in 10 mM LiClO4 . Phosphate-buffered saline (PBS) solutions of different pH were prepared by mixing four stock solutions of 0.1 M H3 PO4 , KH2 PO4 , K2 HPO4 and K3 PO4 . Solutions of UA and AA were prepared in 0.1 M PBS (pH 7.0). Double distilled water and high purity N2 were used. 2.2. Apparatus and procedure Electrochemical experiments including cyclic voltammetry (CV), differential pulse voltammetry (DPV) were performed on a model CHI 832 electrochemical analyzer (Chen-Hua, Shanghai, China). Electrochemical impedance spectroscopy (EIS) was carried out at CHI 660A workstation (Chen-Hua, Shanghai, China). A conventional three-electrode electrochemical system was used for all electrochemical experiments, which consisted of a working electrode, a twisted platinum wire counter electrode and a saturated calomel reference electrode (SCE). A glassy carbon disk electrode (GCE) of formal surface area of 0.125 cm2 was used as the basal working electrode. All potentials reported are versus SCE. EIS measurements were carried out before and after the surface modification in 0.1 M Fe(CN)6 4−/3− + 0.1 M PBS (pH 7.0) in the range from 100 kHz to 0.05 Hz at 0.215 V, the formal potential of the redox couple. UV–vis absorption spectra were measured at UV-2401PC spectrophotometer (Shimadzu, Japan). A homemade versatile long path-length thin-layer electrochemical cell was used for in situ spectroelectrochemical measurements as described previously [15]. A 1 ml volume of PCh solution was injected into the cell through its reference electrode chamber into the thin-layer compartment. In this way, any air bubbles in the

3211

thin-layer compartment should be removed. A similar cell was set in the reference beam of the spectrophotometer for background subtraction during the measurements. Each spectrum was measured after the thin-layer electrolysis at a potential for 2 min. XPS was obtained by using an ESCALAB MK2 spectrometer (VG, UK) with an Mak-Alpha radiation source. Surface analysis of the electrode was performed with an ESCA lab MK2 (VG, UK) with a Mak-Alpha radiation source at 50 eV, 0.05 eV per step. The elemental nitrogen-tocarbon ratio (N/C) was used to assess the extent of modifier coverage, which was calculated by dividing the total number of counts under the N (1s) band by that under C (1s) band. Surface analysis of the electrode was performed with an ESCA lab MK2 (VG, UK) with a Mo K␣ radiation source at 50 and 0.05 eV per step. The elemental nitrogen-to-carbon ratio (N/C) was used to assess the extent of modifier coverage was calculated by dividing the total number of counts under the N (1s) band by that under C (1s) band. 2.3. Preparation of monolayer modified electrodes The GCE was prepared by polishing to a mirror-like finish with fine wet emery paper (grain size 4000). After sonication cleaning in water for 15 min, it was resurfaced using alumina slurry. After cleaning, the electrode was electrochemically pre-treated by potential cycling in the range from −1.7 to 1.8 V at 20 mV s−1 for six scans in 0.01 M LiClO4 containing 1.0 × 10−3 M Ch, ACh, PCh or BuCh. The electrode was then rinsed with ethanol and sonicated for 15 min in water to remove any physisorbed unreacted materials from the surface. The Ch, ACh, PCh or BuCh modified electrodes, denoted as Ch/GCE, ACh/GC, PCh/GCE or BuCh/GCE, respectively. These modified electrodes were stored at 4 ◦ C in 0.1 M PBS (pH 7.0) after use, except that the Ch/GCE was stored in double distilled water. All experiments were performed at room temperature. The electrochemical solutions were thoroughly deoxygenated by bubbling with nitrogen before sampling, and maintaining a nitrogen atmosphere through out experiment.

3. Results and discussion 3.1. Electrochemical modification of Ch and PCh on GCE The Ch and PCh modified GCEs, denoted as Ch/GCE and PCh/GCE respectively, were fabricated by cyclic voltammetry (CV) in LiClO4 solution in a potential scan range from −1.7 to 1.8 V. Fig. 1A shows the modification process of PCh/GCE. It can be seen that a broad and irreversible anodic wave was growing up at about 1.38 V (versus SCE) during the six scans and finally reached to a steady response, indicating a saturation of the surface planting. This oxidation peak

3212

X.-Q. Lin, G.-P. Jin / Electrochimica Acta 50 (2005) 3210–3216

Fig. 3. UV–vis spectroelectrochemical spectra in the process of PCh modification before (1) and after the 3rd (2) and 6th (3) potential cycling in 2.0 mM PCh + 10 mM LiClO4 . The dashed line (4): spectrum of 25.0 ␮M propionic acid in 10 mM LiClO4 .

Fig. 1. Multi-circle CVs for the GCE modification in 5.0 mM PCh + 10 mM LiClO4 (A); CVs of PCh/GCE (B) and Ch/GCE (C). Supporting electrolyte: ACN/0.1 M NBu4 BF4 .

should attribute to both the oxidation of PCh at the GCE and the oxidation of surface modified monolayer. After cleaning with water, the PCh/GCE was transferred to a blank solution for CV scans, the oxidation peak at about 1.38 V was reproducibly appeared, as shown in Fig. 1B, showing a surface wave of the PCh modified monolayer. Similar phenomena were observed on Ch modification on GCE, as shown in Fig. 1C. XPS data were obtained as shown in Fig. 2. The N (1s) is appeared at 399.95 eV at PCh/GCE (a) and Ch/GCE (b) and 400.20 eV (c) at bare GCE. Calculation shows the N/C ratio was increased to 152 and 188% in comparison with that for bare GCE, for the PCh/GCE and Ch/GCE, respectively. These

Fig. 2. XPS of N (1s) region of PCh/GCE (a), Ch/GCE (b) and fresh prepared GCE (c).

data clearly indicate that the nitrogen containing residues of PCh and Ch have been immobilized on the GCE surfaces. Thin-layer spectroelectrochemistry was carried out for in situ monitoring the surface modification process, as shown in Fig. 3. The UV–vis absorption spectra of the PCh system were collected as the adsorption of the 1 cm path-length thin-layer solution. It can be seen that an absorption peak at 195 nm was existed before electrolysis (1) and moved to 197 nm after sixth cycling (3), and a broad absorption in the range of 190–230 nm was increased during the cycling. Evidently, propionic acid has the same broad absorption at about 204 nm (4). Thin-layer spectroelectrochemistry was also carried out for Ch modification. It showed only an absorption peak at about 197 nm before and after the potential cycling and without the broad absorption band to appear. We propose that PCh was hydrolyzed to Ch and propionic acid during the surface modification. Thus, the major difference of PCh/GCE to Ch/GCE is obviously at the existence of the component, propionic acid that incorporated into the modified layer. This will be discussed further in the later sections. The CV shown in Fig. 1B further demonstrated the modification of PCh at GCE. The electrode was carefully rinsed in an ultrasonic cleaner and transferred to a black solution containing only 0.1 M NBu4 BF4 for CV scans. The surface re-oxidation peak appeared at 1.32 V, which is very close to the 1.38 V observed during the modification. With continuous CV scanning for 60 min, only 8% decrease of the peak height was observed. Whereas, a pair of reversible reduction/reoxidation waves with Ep of 0.023 V appeared at −0.90 and −0.67 V in the negative scan, which can be attributed to the reduction and re-oxidation of carboxylic functionalities in the modified layer on the PCh/GCE by a four-electron process [10,16,17]. Thus the number of molecules of the modified layer can be calculated from the peak areas obtained in the first run. Knowing the apparent surface area of the GCE is probably smaller than the real area, surface concentration (Γ ) of the modified Ch and propionic acid were obtained as 1.3 × 10−10 and 2.2 × 10−10 mol cm−2 , respectively. The CV in Fig. 1C for the prepared Ch/GCE shows a similar irreversible oxidation peak at about 1.32 V, and a Γ value of 4.8 × 10−10 mol cm−2 for Ch was calculated. Differently,

X.-Q. Lin, G.-P. Jin / Electrochimica Acta 50 (2005) 3210–3216

Fig. 4. EI spectra at GCE (a), Ch/GCE (b) and PCh/GCE (c) in 1.0 mM Fe(CN)6 3−/4− /0.1 M PBS (pH 7.0) at 0.215 V vs. SCE. Signal: from 100 kHz to 0.05 Hz.

only a minimal irreversible reduction peak appeared at about −0.90 V with no re-oxidation peak was observed, which can be attributed to the reduction of carboxy sites generated after the oxidation of carbon surface [18]. The obvious difference from the PCh/GCE is due to a lack of propionic acid modification. This was further demonstrated by the modification of GCE in Ch + propanol (1:1) solution under CV scans. The resulting modified electrode had the same CV as the Ch/GCE in blank solution. However, if the prepared Ch/GCE was immersing in propionic acid for 6 h or the electrode was prepared in Ch + acetate (1:1) solution, a pair of reversible redox peaks at about −0.90 and −0.67 V for carboxylic functionalities were observed. It is apparent that the carboxylic acid had been bonded to the modified layer at the PCh/GCE. It propose that the PCh is hydrolyzed at the electrode surface during the CV scans forming Ch and propionic acid before and/or during the surface modification; the Ch is covalently bonded on the carbon surface and the propionic acid is adsorbing and embedding into the Ch modified layer for charge balancing. The Ch and propionic acid should be alternatively assembling on GCE shows an interesting two-dimensional structure of surface modification. EIS is an effective method to probe the features of a surface-modified electrode [19,20]. Fig. 4 shows the EI

3213

spectra of PCh/GCE, Ch/GCE and GCE in Fe(CN)6 3−/4− solution. An equivalent circuit shown in Fig. 4 (inset) comprising the solution resistance (Rsol ), film resistance (Rm ), film capacitor (Cm ), charge-transfer resistance (Rct ), Warburg impedance (ZW ) and the double layer capacitance (Cdl ) was used for data simulation. The Rct values were obtained as 1967.5  for PCh/GCE), 876.4  for GCE, and 192.5  for Ch/GCE. These indicates the existence of block effect on Fe(CN)6 4−/3− redox reaction at the PCh modified layer, however, acceleration effect at the Ch modified layer. It is indicative that the negatively charged propionic acid existed in the PCh/GCE but not in Ch/GCE. A mechanism was proposed as shown in Scheme 1. As an alkanol, the chemistry of its electrochemical modification can be related to the reaction of Ch with the oxidized carbon surface forming covalent bindings [21–24], which is expressed in Scheme 1. The glassy carbon electrode can be oxidized forming cation radicals on the surface (reaction 1), which then react with Ch through nucleophilic attack forming O ether bound linkages (reaction 2). The PCh molecules may be hydrolyzed to choline (Ch) and propionic acid at the electrode surface before the binding, accompanying propionic acid embedding in the Ch modified layer for charge balance forming a novel choline–propionic acid interdigitated assembling (reaction 3).

3.2. Electrochemical oxidation of UA and AA Fig. 5(a) shows the CVs of UA at a bare GCE and the prepared PCh/GCE. The voltammetric peak of UA in the neutral pH 7.0 solution appeared at about 0.47 V at the bare electrode, which is in close agreement with the previous report [8]. The peak was rather broad, indicating a slow electron transfer kinetic. However, a sharp oxidation peak at 0.32 V and a small re-reduction peak at 0.28 V were obtained at PCh/GCE. The 150 mV negative shift with much enhanced peak current indicates a strong catalytic effect of

Scheme 1.

3214

X.-Q. Lin, G.-P. Jin / Electrochimica Acta 50 (2005) 3210–3216

Fig. 6. DPVs of UA at PCh/GCE in 0.1 mM AA + UA (3.0, 5.0, 7.0, 10.0, 30.0, 50.0, 70.0 and 90.0 ␮M, indicated by the arrow). Dashed line: DPV of 50 ␮M UA + 0.1 mM AA at GCE. Supporting electrolyte: 0.1 M PBS (pH 7.0).

Fig. 5. CVs of 0.2 ␮M UA (a) and 0.1 mM AA (b) in 0.1 M PBS (pH 7.0) at PCh/GCE (1) and GCE (2) at 50 mV s−1 .

the PCh modified layer. The 40 mV peak separation is closely in agreement with a two electrons and two protons process of UA oxidation. Noted that the appearance of the re-reduction peak of UA oxidation has not been reported in the literature [3–12], showing increased reaction reversibility and reduced rate of following reactions at the PCh/GCE. The anodic peak currents of UA was proportional to the scan rate in the range of 10–40 mV s−1 , however, proportional to the square root of scan rate in the range of 50–100 mV s−1 , showing certainly a diffusion-controlled surface adsorption kinetics. AA shows a broad and irreversible oxidation peak at 0.36 V at the bare GCE, while the oxidation peak shifted to 0.02 V with well-defined peak shape and 10-fold enhanced peak current at the PCh/GCE, as shown in Fig. 5(b). Interestingly, a small re-reduction peak at about −0.05 V could be seen. The 340 mV negative shift and enhanced current of the anodic peak indicates that the PCh modified

layer also plays a strong catalytic effect on the AA oxidation. The anodic peak current was proportional to the scan rate in the range of 10–50 mV s−1 and proportional to the square root of scan rate in the range of 60–200 mV s−1 , showing also a diffusion-controlled surface adsorption kinetics. The significant adsorption of UA and AA on the PCh modified monolayer layer could even show similar CVs after switching the electrode from UA or AA solution to a blank solution for the first potential scan. Since both UA and AA exist as anions at pH 7.0 [6] (pK␣,UA = 5.75, pK␣,AA = 4.17), it is possible that a favorable electrostatic attraction between the anionic species and the [ N+ (CH3 )3 ] head groups plays an important role for the adsorption and the catalytic activities. Furthermore, AA is more hydrophilic and more soluble in water than UA, a favorable electrostatic attraction with the cationic quaternary ammonium functions [ N+ (CH3 )3 ] for AA could be expected. Fig. 6 shows DPVs in AA and UA mixtures. It shows no resolvable AA or UA peaks at a bare GCE (the dashed line). However, two well separated DPV peaks at −0.02 V for AA and 0.25 V for UA. With an increase of UA concentration, the peak potentials of UA and AA shifted to a positive direction for only about 10–20 mV. The current sensitivity of UA was about three times larger than that for AA. About 270–290 mV peak separation allows to determine UA in the

Table 1 DPV peak parameters of UA and AA at different pH at the PCh/GCEa pH

UA

2.80 4.14 5.04 5.89 7.00 7.94 8.81 a b

AA

ipa (␮A)

Epa (mV)

ipa (␮A)

Epa (mV)

2.44 3.50 1.90 1.17 0.74 0.26 0.24

496 432 376 324 273 220 164

2.32 6.19 5.86 4.14 3.04 1.18 1.26

−4 96 48 28 −20 −108 −136

Solution: 20 ␮M UA + 50 ␮M AA + 0.1 M PBS. Ep =Ep(UA) − Ep(AA).

Epa b

ipa,UA /ipa,

500 336 328 296 293 328 300

0.95 1.77 3.08 3.55 4.09 4.30 5.28

AA

X.-Q. Lin, G.-P. Jin / Electrochimica Acta 50 (2005) 3210–3216

3215

presence of large amount of AA, or to determine UA and AA simultaneously. 3.3. Analytical application for the selective determination of UA Table 1 summarizes the peak potential (Epa ) and peak current (ipa ) of AA and UA at the PCh/GCE at pH from 2.8 to 8.8. It shows a maximal ipa of UA is appeared at pH 4.14, and a slow decrease for pH > 5. The Epa of UA decreases linearly shifted to the negative direction at slope of about 59 mV per pH unit. Similar peak potential shift was observed for AA. However, the current ratio of ipa,UA /ipa,AA was increased with increase of pH, while the Epa (Epa separations) was minimal of 293 mV at pH 7.0. It is obvious that lower pH value is favorable to UA determination. However, for practical consideration for UA determination in bio-fluid system, pH 7 is selected. Time effect for the adsorption accumulation of UA at the PCh/GCE was investigated, studied in PBS (pH 7.0). The result showed that the peak current increased rapidly with the time and reached to a plateau at 1 min. Hence, 1 min of open circuit accumulation was used for the determination. The DPVs at different concentrations of UA in the presence of 0.10 mM AA on the above mentioned optimum conditions showed a linear relationship between the peak current and the UA concentration in a range of 0.07–70 ␮M with a correlation coefficient (r) of 0.993 and a detection limit (3σ) of 0.02 ␮M (Fig. 6, inset). The peaks were reproducible for subsequent DPV sweeps, indicating a good stability and antifouling ability in the presence of AA. This electrode can be used for UA determination in present of high concentration AA or for a simultaneous determination of UA and AA. The detection limit of 20 nM UA in 0.1 mM AA solution means that 5 × 103 -fold concentration of AA did not interfere with the detection of UA, which is large enough for application in physiological conditions. 3.4. Interference The major interference of AA is discussed in the above sections. Other common co-existing substances were also investigated for determination of 1.0 ␮M UA. The tolerance ratio (shown in the blanket) for those substances is: citric (1000), cytosine (100), glucose (40), purine (50) and urea (50).

Fig. 7. DPVs of 50.0 ␮M AA and 20.0 ␮M UA at Ch/GCE (a), ACh/GCE (b), PCh/GCE (c) and BuCh/GCE (d) in 0.1 M PBS (pH 7.0).

3.5. Sample analysis As an example of the assay using the PCh/GCE, a direct analysis of urine was performed. Three human urine samples were determined. All the samples were directly used without any pretreatment. To fit into the linear range, 2 ␮l potions of the urine sample were added into 5.0 ml flasks and diluted to the volume with PBS (pH 7.0). The results are listed in Table 2. To ascertain the correctness of the results, the samples were spiked with about the same amount of existing UA. The recovery rates of the spiked samples were in the ranged of 98.8–102.7%. 3.6. Comparisons of oxidation results of UA and AA at the Ch/GCE, ACh/GCE, PCh/GCE, BuCh/GCE In order to demonstrate the existence and functions of the accompanying carboxylate molecules in the choline modified layer, Ch/GCE, ACh/GCE and BuCh/GCE were also prepared and tested in the UA and AA mixture, as shown in Fig. 7. It can be seen that all these modified electrodes facilitated the oxidation process of UA and AA. Although the UA peak appeared at almost the same potential at these electrodes, it showed a maximal height with sharp DPV peak at the PCh/GCE. AA peak appeared at most negative potential at the PCh/GCE. The Epa of AA and UA is the largest at PCh/GCE (293 mV) in comparison with 200 mV at the Ch/GCE, 200 mV at the ACh/GCE and, 213 mV at the BuCh/GCE. It documents that the PCh/GCE is an excellent for the determination of UA. The N+ (CH3 )3 groups on the monolayer should play major roles for facilitating the oxidation process of UA and AA by attracting negatively charged UA and AA species as mentioned above. However, the large CH3 moiety on the

Table 2 Determination of UA in urine samples using PCh/GCEa Urine sample

Detected (␮M)

Spike (␮M)

After spike (␮M)

Recovery (%)

Total valueb (mg/l)

1 2 3

2.81 2.44 2.21

2.00 2.00 2.00

4.94 4.42 4.16

100.6 99.5 98.8

473 411 372

a b

Number of samples assayed: 5. Total value was calculated from the detected value.

3216

X.-Q. Lin, G.-P. Jin / Electrochimica Acta 50 (2005) 3210–3216

N+ (CH3 )3 group may also play an important role on dissolving these organic species by hydrophobic interactions. Moreover, the existence of the N+ (CH3 )3 groups on the covalently assembled Ch monolayer should introduce an extra electronic positive field on the double layer structure, accelerating the discharge of the attached anions. On the other hand, the accompanying oganic acid molecules imbedded modulated the catalytic activity by charge balancing, which provide also a self-satisfactory and stable monolayer for enhanced stability and reproducibility. However, the imbedding molecules should also play some delicate molecular interactions with the assembled Ch moieties, which allow not only the organic residues imbedded firmly but also molecular recognition abilities. For example, the PCh monolayer is favorable to UA adsorption generating larger DPV peak (Fig. 7c), the Ach monolayer is favorable to AA adsorption generating larger DPV peak (Fig. 7b), while both Ch and BuCh monolayer are much less favorable for both UA and AA accumulation. The detailed mechanism needs further investigation.

4. Conclusions Electrochemical modification of glassy carbon electrode by propionylcholine generates propionic acid imbedded choline covalently bonded monolayer modified electrode PCh/GCE. In comparison with similar fabricated Ch/GCE, Ach/GCE and BuCh/GCE, significant reduction of the overpotentials, enhancement of electrochemical reversibility of AA and UA oxidation, and significant surface accumulation of UA and AA were achieved at the PCh/GCE. This PCh/GCE can be used for DPV determination of UA as low as 20 nM in the presence of 0.1 mM AA without interaction and surface fouling in a linear range from 70 nM to 70 ␮M. This electrode is sensitive, stable, quick response and good in resisting interferences for UA determination. Together with low cost and ease of preparation, the monolayer choline derivatives modified electrode is hopefully to be excellent for in vivo and in vitro sensor development.

Acknowledgement The authors gratefully acknowledge the financial support from National Natural Science Foundation of China (no. 20075025) and Research Foundation of MEC (no. 20040358021). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24]

V.S.E. Dutt, H.A. Mottola, Anal. Chem. 46 (1974) 1777. J. Zen, J. Jou, G. Ilangovan, Analyst 123 (1998) 1345. G. Park, R.N. Adams, W.R. White, Anal. Lett. 5 (1972) 887. R.N. Goyal, N.K. Singhal, Bioelectrochem. Bioenerg. 44 (1998) 201. M. Fumio, S. Yukari, H. Yoshiki, I. Seiichiro, Anal. Chim. Acta 41 (2001) 175. T. Hoshi, H. Saiki, J.I. Anzai, Talanta 61 (2003) 363. J.S. Ye, Y. Wen, W.D. Zhang, L.M. Gan, G.Q. Xu, F.S. Sheu, Electroanalysis 15 (2003) 1693. Z. Wang, Y. Wang, G. Luo, Analyst 127 (2002) 1353. Y. Sun, J. Fei, K. Wu, S.G. Hu, Anal. Bioanal. Chem. 375 (2003) 544. L.Z. Zheng, S.G. Wu, X.Q. Lin, N. Lei, L. Rui, Electroanalysis 13 (2001) 1351. C.R. Raj, R. Kitamura, T. Ohsaka, Analyst 127 (2002) 1155. A. Yu, H. Zhang, H.Y. Chen, Analyst 122 (1997) 839. P.C. Pandet, S.H. Upadhyay, G.C. Pathak, M.D. Pandey, I. Tiwri, Sens. Actuators B 62 (2001) 109. M.G. Gagruilo, A.C. Michael, Biosens. Bioelectron. 10 (1995) 9. H. Cui, L.S. Wu, J. Chen, X.Q. Lin, J. Electroanal. Chem. 504 (2001) 195. H. Dai, K.K. Shiu, J. Electroanal. Chem. 419 (1996) 7. M.M. Baizer, H. Lund, Organic Electrochemistry, Marcel Dekker, Inc., New York, 1983, p. 379. P. Bianco, J. Haladjian, J. Electroanal. Chem. 293 (1990) 151. C. Saby, B. Ortiz, G.Y. Champagne, D. B´elanger, Langmuir 13 (1997) 6805. N. Tsutomu, T. Yoshino, Anal. Chem. 58 (1986) 1037. H. Maeda, Y. Yamauchi, M. Hosoe, T. Li, E. Yamaguchi, M. Kasamatsu, H. Ohmori, Chem. Pharm. Bull. 42 (1994) 1870. H. Maeda, T. Li, M. Hosoe, M. Itami, Y. Yamauchi, H. Ohmori, Anal. Sci. 10 (1994) 963. H. Maeda, M. Itami, Y. Yamauchi, H. Ohmori, Chem. Pharm. Bull. 44 (1996) 2294. O. Hammeich, B. Svensmark, in: H. Lund, M.M. Baizer (Eds.), Organic Electrochemistry, third ed., Marcel Dekker Inc., New York, 1991, p. 628.