Elsevier

Analytica Chimica Acta

Volume 659, Issues 1–2, 5 February 2010, Pages 144-150
Analytica Chimica Acta

Amperometric biosensor based on tyrosinase immobilized onto multiwalled carbon nanotubes-cobalt phthalocyanine-silk fibroin film and its application to determine bisphenol A

https://doi.org/10.1016/j.aca.2009.11.051Get rights and content

Abstract

An amperometric bisphenol A (BPA) biosensor was fabricated by immobilizing tyrosinase on multiwalled carbon nanotubes (MWNTs)-cobalt phthalocyanine (CoPc)-silk fibroin (SF) composite modified glassy carbon electrode (GCE). In MWNTs-CoPc-SF composite film, SF provided a biocompatible microenvironment for the tyrosinase to retain its bioactivity, MWNTs possessed excellent inherent conductivity to enhance the electron transfer rate and CoPc showed good electrocatalytic activity to electrooxidation of BPA. The cyclic voltammogram of BPA at this biosensor exhibited a well defined anodic peak at 0.625 V. Compared with bare GCE, the oxidation signal of BPA significantly increased; therefore, this oxidation signal was used to determine BPA. The effect factors were optimized and the electrochemical parameters were calculated. The possible oxidation mechanism was also discussed. Under optimum conditions, the oxidation current was proportional to BPA concentration in the range from 5.0 × 10−8 to 3.0 × 10−6 M with correlation coefficient of 0.9979 and detection limit of 3.0 × 10−8 M (S/N = 3). The proposed method was successfully applied to determine BPA in plastic products and the recovery was in the range from 95.36% to 104.39%.

Introduction

At present, pollutants and related diseases are of great concern, and bisphenol A (BPA, 2,2′-bis(4-hydroxyphenyl)propane) is among these pollutants. It has been demonstrated that BPA exhibits estrogenic activity, which mimics the action of the hormone estrogen [1]. Its blood levels in men and women are associated with reproduction dysfunctions, endometrial hyperplasia, recurrent miscarriages, abnormal karyotypes and polycystic ovarian syndrome [2]. However, BPA is a major component in the production of polycarbonate (PC) and epoxy resin (EP) which are widely used as plastic food containers, inner surface coating of food, beverage cans, water bottles, baby bottles, and sealants in dentistry. Because of the incomplete reaction, the unreacted BPA will be present in plastic products. For example, the residual levels of BPA in PC baby bottles ranged from 1 to 599 ppm, with average levels from 9.9 to 177 ppm [3]. Nevertheless, what is more serious is that the migration of BPA from these materials into food has been reported [4], [5], [6], [7], which could threaten the health of humans because humans may routinely ingest trace amounts of BPA. In addition, BPA can also be released into the environment during the manufacturing process or the degradation products of plastics [8]. A recent report has indicated that health risks can result from exposure to doses much lower than the limit of 0.05 mg kg−1 body weight day−1[9]. In order to evaluate actual human health risks caused by BPA exposure, it is necessary to achieve accurate data on their levels in the environment, even at very low levels. Therefore, it is desirable to develop an easy and sensitive analytical method for the determination of trace amounts of BPA.

Until now, the widely used methods for the determination of BPA are liquid chromatography (LC) [10] and gas chromatography (GC) [11]. These techniques have the high sensitivity and low detection limit. However, they also require time-consuming pre-treatment steps, and thus do not allow for rapid processing of multiple samples. Moreover, these instrumentations are rather complicated, expensive, and are hardly employed for on-site measurement. Hence, there is a demand for new analytical technique to determine the low concentration of BPA.

In recent years, electrochemical biosensors based on tyrosinase have attracted many interests for the advantages of good reliability, fast response, inexpensive instrument, low energy consumption, simple operation, time saving and high sensitivity. Thus, they are widely investigated on determining some phenolic environmental pollutants, such as phenol [12], [13], catechol [14], [15], 17β-estradiol [16], etc., and some satisfactory results have been obtained. In the presence of dissolved oxygen, tyrosinase could catalyze the oxidation of phenolic compounds to o-diphenols. The products of the first catalytic process could be further catalytically oxidized to the corresponding o-quinones, which will be reduced at the electrode surface, reforming the original o-diphenols, thus forming a bio-electrocatalytic amplification cycle [17]. The reduction signal of o-quinone is normally used to determine phenolic compound for the purpose of preventing interference [12], [14], [16], [18], [19], [20]. However, the content of dissolved oxygen will influence the catalytic oxidation of phenolic compounds to o-quinones by tyrosinase, and this will further influence the reduction of o-quinones. In order to decrease or eliminate this influence factor, the base solution is frequently saturated with air or pure oxygen, but it will increase the operation complexity. Therefore, based on the oxidation signal of phenolic compounds at tyrosinase-based biosensor for determination of phenolic compounds would be a promising alternative method.

However, to the best of our knowledge, determination of phenolic compounds using the oxidation signal at tyrosinase-based biosensor has not yet been reported. In order to achieve it, in this work, tyrosinase was immobilized onto the composite film of silk fibroin (SF), multiwalled carbon nanotubes (MWNTs) and cobalt phthalocyanine (CoPc) at glassy carbon electrode. MWNTs and CoPc have been widely used to fabricate sensors and biosensors. Recently, immobilization of unsubstituted metal phthalocyanine (MPc) at the surface of carbon nanotubes (CNTs) has been achieved by means of noncovalent adsorption [21]. The resulting MPc-CNTs complexes possess the catalytic properties of MPc without any destruction of electronic properties and structures of CNTs [22], [23]. SF is a natural protein with carboxyl and amino groups. It is a kind of excellent matrix for enzyme immobilization with attractive properties, including excellent biocompatibility, thermal stability, nontoxicity, hygroscopicity, low cost [24]. With the synergistic effect of SF-MWNTs-CoPc incorporated with tyrosinase modification materials, the oxidation response of BPA enhanced greatly at Tyr-SF-MWNTs-CoPc/GCE in contrast to those at Tyr/GCE, Tyr-SF/GCE, CoPc/GCE, MWNTs/GCE, MWNTs-CoPc/GCE and Tyr-MWNTs-CoPc/GCE. Accordingly, a novel biosensor based on the oxidation signal of BPA was fabricated for the first time. In order to assess the selectivity of the proposed biosensor, various main interferents were investigated for their effects on the assay of BPA. The effect factors were optimized and the kinetic parameters were calculated. As a preliminary application in environmental monitoring, the new biosensor was used to evaluate the levels of BPA in plastic products.

Section snippets

Reagents

Tyrosinase (EC 1.14.18.1, 5370 unit mg−1 from mushroom) was purchased from Sigma (USA). CoPc was obtained from Tokyo Chemical Industry Co., Ltd. (Japan). MWNTs were from Nachen S&T Ltd. (China) and treated according to the reported procedure [25]. BPA was obtained from Beijing Chemical Technology Co., Ltd. (China). Silkworm cocoon was obtained from a local market and treated as reported to obtain SF (3%, w/w) [26], [27]. Phosphate buffer solution (PBS) was prepared by mixing the stock solutions

Cyclic voltammetric behaviors of BPA

Fig. 1 showed cyclic voltammograms of BPA at GCE (a), Tyr/GCE (b), Tyr-SF/GCE (c), MWNTs-CoPc/GCE (d), Tyr-MWNTs-CoPc/GCE (e) and Tyr-SF-MWNTs-CoPc/GCE (f). Compared with other electrodes, the oxidation peak current of BPA was small at GCE, Tyr/GCE and Tyr-SF/GCE, indicating a weak oxidation of BPA. Among them, the oxidation peak current of BPA at Tyr/GCE was smaller than that at GCE, which could be attributed to the nonconductivity film of tyrosinase with low bioactivity on GCE surface,

Conclusion

An amperometric BPA biosensor was fabricated based on tyrosinase immobilized onto SF-MWNTs-CoPc composite matrix and a novel sensitive and reliable electrochemical method was developed to determine trace amounts of BPA in plastic products using the oxidation signal. With the synergistic effect of the Tyr-SF-MWNTs-CoPc, the oxidation peak current increased remarkably compared with Tyr/GCE, Tyr-SF/GCE, CoPc/GCE, MWNTs/GCE, MWNTs-CoPc/GCE and Tyr-MWNTs-CoPc/GCE. Moreover, this is the first time to

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 20775044) and the Natural Science Foundation of Shandong province, China (Y2006B20).

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