Enzymatically induced formation of neodymium hexacyanoferrate nanoparticles on the glucose oxidase/chitosan modified glass carbon electrode for the detection of glucose

Abstract

The formation of neodymium hexacyanoferrate (NdHCF) nanoparticles (NPs) on the surface of glucose oxidase/chitosan (GOx/CHIT) modified glass carbon electrode induced by enzymatic reaction was described and characterized. CHIT can be used not only as enzyme immobilizer, but also to provide active sites for NPs growth. Results showed that the optimized conditions of the GOx/CHIT film induced NdHCF NPs for the biosensing of glucose were 1.0 mM Nd³⁺ and 20.0 mM Fe(CN)₆³⁻. The biocatalyzed generation of NdHCF NPs enabled the development of an electrochemical biosensor for glucose. The calculated apparent Michaelis–Menten constant was 7.5 mM. The linear range for glucose detection was 0.01–10.0 mM with the correlation coefficient of 0.9946, and the detection limit was 5 μM (S/N = 3). Furthermore, this system avoids the interferences of other species during the biosensing process and can be used for the determination of glucose in human plasma samples.

Introduction

In recent years, the application of biocatalysts is becoming more and more attractive for the synthesis and the enlargement of metallic NPs, especially the integration of NPs into biocatalytic reaction which opened a new way for designing simple and sensitive electrochemical and optical sensors (Katz and Willner, 2004, Medintz et al., 2005, Möller et al., 2005). Such new field that conjugates biomolecules to metal nanoparticles (MNPs) has recently attracted substantial interest in the rapidly developing area of nanobiotechnology (Katz et al., 2004, Niemeyer, 2001).

The unique catalytic properties of MNPs have been exploited by Willner and co-workers for amplified optical enzymatic assays in connection with biocatalytically induced particles-growth processes (Shlyahovsky et al., 2005, Xiao et al., 2004, Zayats et al., 2005a, Baron et al., 2005, Xiao et al., 2005a, Xiao et al., 2005b). Hwang et al. (2005) have reported the metal deposition by the enzymatically produced reducing agent for the amplified electrochemical detection of DNA. Zhou et al. (2006) also reported the design of a self-assembly monolayer modified electrode with the biological catalytic reaction for the biosensing of cholesterol. Such technique involves the reduction and deposition of metal onto the NPs by an enzymatically generates reducing agent, has been exploited for the quantitative optical monitoring of several enzymatic reactions. Further application studies, based on the enzyme-stimulated catalytic deposition of metals on MNP seeds, have been extended to other biocatalysts that yield products capable of reducing metal salts. For example, the NADH-stimulated growth of Au NPs under special conditions led to shaped NPs consisting of dipods, tripods, and tetrapods (Xiao et al., 2005a, Xiao et al., 2005b). The alkaline phosphatase hydrolysis of p-aminophenol phosphate yields p-aminophenol that catalyzes the reduction of Ag⁺ on Au NP seeds (Basnar et al., 2006). Most recently, a kind of metal–NP–enzyme hybrid system have been employed as biocatalytic templates (or “biocatalytic inks”) for the generation of enlarged metal NPs or metallic nanowires (Willner et al., 2006, Xiao et al., 2003, Zayats et al., 2005b). It is expected that the metal–NP–enzyme conjugates may act as biocatalytic labels for amplifying a variety of biorecognition events, such as antigen–antibody or nucleic acid–DNA, and enabling the optical, electrochemical, or microgravimetric readout of the recognition processes (Möller et al., 2005).

However, the present study has emphasized the biocatalyzed synthesis of metallic NPs. In fact, the biocatalyzed synthesis of other materials such as semiconductor NPs (e.g., CdS, PbS) or magnetic NPs is a very attractive field (Willner et al., 2006). It is expected that once such biocatalytic transformations are materialized, the development of new optical or electrochemical biosensing systems can be accomplished. Generally, the biocatalytic precipitation of an insoluble product on electronic transducers was used as a general amplification route for sensing and recognition events by layered biomaterial-sensing interface (Patolsky et al., 1999, Abad et al., 1998), such as enzyme-based electrodes, immunosensors and DNA sensors. Recently, Wang and Arribas (2006) represented the first example of deliberately exploiting enzymatic reactions for generating cupric ferrocyanide (CuHCF) NPs and exploiting their formation for monitoring biocatalytic reactions. However, since such protocol was performed in solution, only a small part of Fe(CN)₆⁴⁻ formed by enzymatic reaction will react with Cu²⁺ and deposited on the carbon-paste electrode surface, while a large portion diffuse into bulk solution. Obviously, this limited the application of the protocol, especially for real sample analysis. So, it is desirable to develop novel method to solve the problem. Entrapment or encapsulation within a biocompatible material by simple procedures has been widely used for enzyme immobilization. Among various biocompatible materials, chitosan (CHIT) was widely used as an immobilization matrix for biosensors and biocatalysis (Huang et al., 2002, Bindhu and Abraham, 2003).

Here, we wish to develop a novel method for the improvement of biosensor design and the application of metal hexacyanoferrate (MHCF) NPs from biocatalyzed synthesis. A kind of real-earth MHCF (neodymium ferrocyanide, NdHCF) was chosen as an example to illustrate the protocol. The use of rare-earth metal ion (Nd³⁺) avoided the formation of unpredicted precipitate, which extended the application of enzymatic reactions for generating MHCFs. The amine function of CHIT have the ability to chelate metal ions, which can provide better nucleation sites for the growth of NdHCF NPs. After optimized, the proposed biosensor was used for the determination of glucose. The combination of cooperative performance of polymer, highly efficient enzyme catalysis, and attractive electrochemical properties of MHCFs provides a general platform for the synthesis of nanowires and nanocircuits, the construction of bioelectronic devices, and the design of novel biosensors.