Electrodeposition of gold–platinum alloy nanoparticles on ionic liquid–chitosan composite film and its application in fabricating an amperometric cholesterol biosensor

Abstract

An electrodeposition method was applied to form gold–platinum (AuPt) alloy nanoparticles on the glassy carbon electrode (GCE) modified with a mixture of an ionic liquid (IL) and chitosan (Ch) (AuPt–Ch–IL/GCE). AuPt nanoparticles were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and electrochemical methods. AuPt–Ch–IL/GCE electrocatalyzed the reduction of H₂O₂ and thus was suitable for the preparation of biosensors. Cholesterol oxidase (ChOx) was then, immobilized on the surface of the electrode by cross-linking ChOx and chitosan through addition of glutaraldehyde (ChOx/AuPt–Ch–IL/GCE). The fabricated biosensor exhibited two wide linear ranges of responses to cholesterol in the concentration ranges of 0.05–6.2 mM and 6.2–11.2 mM. The sensitivity of the biosensor was 90.7 μA mM⁻¹ cm⁻² and the limit of detection was 10 μM of cholesterol. The response time was less than 7 s. The Michaelis–Menten constant (K_m) was found as 0.24 mM. The effect of the addition of 1 mM ascorbic acid and glucose was tested on the amperometric response of 0.5 mM cholesterol and no change in response current of cholesterol was observed.

Introduction

Cholesterol has recently attracted much interest because its level in blood is an important parameter in the diagnosis and prevention of disease. Ideally, the total cholesterol concentration in a healthy person's blood should be less than 200 mg/dL (<5.17 mM). The borderline high is defined as 200–239 mg/dL (5.17–6.18 mM), and the high value is defined as above 240 mg/dL (≥6.21 mM) (Shen and Liu, 2007). The measurement of blood cholesterol concentration is a routine practice in medical screening or diagnosis. Enzymatic procedures for the determination of cholesterol have practically replaced the chemical methods because the traditional method of determination of cholesterol by spectrometry suffered from poor specificity, instability of color forming agent and standardization difficulties (Gopalana et al., 2009).

Biosensors using immobilized enzymes as the bio-recognition element are among the most widely investigated devices for both fundamental research and applications point of view (Weetall, 1974, Emr and Yacynych, 1995, Hall, 1991). Amperometric biosensors are usually chosen for biochemical analysis because of their good selectivity, rapid response, and low cost (Karube et al., 1982).

In the fabrication of a cholesterol biosensor, cholesterol oxidase (ChOx) is most commonly used as the biosensing element. Cholesterol oxidase catalyzes the oxidation of cholesterol to H₂O₂ and cholest-4-en-3-one in the presence of oxygen (Ahn and Sampson, 2004).

The enzymatic reaction in the use of cholesterol oxidase (ChOx) as a receptor can be described as follows:

$$\text{Cholesterol}+{\text{O}}_2\stackrel{\text{ChOx}}{\to }\text{Cholest-}4-\text{en}-3-\text{one}+{\text{H}}_2{\text{O}}_2$$

The electrooxidation current of hydrogen peroxide is detected after application of a suitable potential to the system. The major problem for amperometric detection is the overestimation of the response current due to interferences such as ascorbic acid. This problem can be overcome by using a combination of two or three enzymes, which are more selective for the analyte of interest (Bongiovanni et al., 2001) or by devising techniques to eliminate or reduce the interference. Many researchers have reported the inclusion of metal nanoparticles with a catalytic effect in polymer modified electrodes to decrease the overpotential applied to the amperometric biosensors (Safavi et al., 2009, Hrapovic et al., 2004, Ren et al., 2005, Huang et al., 2004). Amperometric cholesterol biosensors based on carbon nanotube–chitosan–platinum–cholesterol oxidase nanobiocomposite was fabricated for cholesterol determination at an applied potential of 0.4 V (Tsai et al., 2008). To improve the selectivity of the biosensor, Gopalana et al. (2009) reported the construction of a cholesterol biosensor by monitoring the reduction current of H₂O₂ at −0.05 V.

Bimetallic alloys are widely used in catalysis and sensing fields. Owing to the interaction between two components in bimetallic alloys, they generally show many favorable properties in comparison with the corresponding monometallic counterparts, which include high catalytic activity, catalytic selectivity, and better resistance to deactivation. Among various bimetallic alloys, gold–platinum (AuPt) alloy is very attractive. It has excellent catalysis and resistance to deactivation due to the high synergistic action between gold and platinum (Xiao et al., 2009). Owing to these advantages of bimetallic nanoparticles, it becomes significant to develop AuPt nanoparticles for application in electrochemical sensors with appropriate characteristics such as high sensitivity, fast response time, wide linear range, better selectivity, and reproducibility.

Chitosan is an abundant natural biopolymer with excellent film forming ability, biocompatibility, nontoxicity, good water permeability and high mechanical strength (Zhang et al., 2004, Lu et al., 2006a, Lin et al., 2007). Chitosan can accumulate metal ions through various mechanisms, such as chelation, electrostatic attraction, and ion exchange, depending on the nature of the metal ion and pH of the solution. To improve the properties of chitosan, the combination of chitosan with other materials such as ethylene diamine (Katarina et al., 2006), thiourea (Mokhodoeva et al., 2007), carbon naotubes (Kang et al., 2007) and functionalized ionic liquids (Xiao et al., 2009) were reported. Kang et al. (2007) constructed a glucose biosensor on a glassy carbon electrode which was modified with gold–platinum alloy nanoparticles (AuPt NPs) by electrodeposition on multiwalled carbon nanotubes in chitosan film. Recently, AuPt NPs were fabricated on chitosan-ionic liquid (i.e., trihexyltetradecylphosphonium bis(trifluoromethylsulfonyl)imide, [P(C₆)₃C₁₄][Tf₂N]) film by using an ultrasonic electrodeposition method which displayed high catalytic activity towards the reduction of H₂O₂ (Xiao et al., 2009).

Ionic liquids (ILs), a new class of environmentally benign solvents which are composed entirely of ions, have been used in numerous applications. Because of their unique properties such as wide electrochemical window, high ionic conductivity, and good thermal stability, they are applied in different electrochemical applications such as in lithium batteries, capacitors, and solar cells (Howlett et al., 2004, McEwen et al., 1999, Wang et al., 2003). They are also widely applied as the binder in carbon ionic liquid electrodes (Maleki et al., 2006, Liu et al., 2005, Safavi et al., 2007) or as the electrode modifier especially for construction of different biosensors (Safavi and Farjami, 2010, Safavi et al., 2008, Zhu et al., 2009, Lu et al., 2006b). In this work, we have utilized the unique properties of AuPt bimetallic NPs for fabrication of a cholesterol biosensor. This system was developed by electrodeposition of AuPt nanoparticles on the GCE modified with chitosan and IL, 1-butyl-3-methylimidazolium chloride [bmim]Cl. Then cholesterol oxidase was immobilized on the surface of the electrode by cross-linking ChOx and chitosan through glutaraldehyde and the biosensor is designated as ChOx/AuPt–Ch–IL/GCE. The ChOx/AuPt–Ch–IL/GCE biosensor exhibited an excellent linear response to cholesterol for a wide concentration range with good selectivity and sensitivity.