Fabrication and application of amperometric glucose biosensor based on a novel PtPd bimetallic nanoparticle decorated multi-walled carbon nanotube catalyst

Abstract

A sensitive, selective and stable amperometric glucose biosensor employing novel PtPd bimetallic nanoparticles decorated on multi-walled carbon nanotubes (PtPd-MWCNTs) was investigated. PtPd-MWCNTs were prepared by a modified Watanabe method, and characterized by XRD and TEM. The biosensor was constructed by immobilizing the PtPd-MWCNTs catalysts in a Nafion film on a glassy carbon electrode. An inner Nafion film coating was used to eliminate common interferents such as uric acid, ascorbic acid and fructose. Finally, a highly porous surface with an orderly three-dimensional network enzyme layer (CS-GA-GOx) was fabricated by electrodeposition. The resulting biosensor exhibited a good response to glucose with a wide linear range (0.062–14.07 mM) and a low detection limit 0.031 mM. The biosensor also showed a short response time (within 5 s), and a high sensitivity (112 μA mM⁻¹ cm⁻²). The Michaelis–Menten constant (K_m) was determined as 3.3 mM. In addition, the biosensor exhibited high reproducibility, good storage stability and satisfactory anti-interference ability. The applicability of the biosensor to actual serum sample analysis was also evaluated.

Introduction

A biosensor contains a bioreceptor (such as an enzyme) and a suitable transducer, this combination can be chosen to have unique advantages in terms of: specificity, low-cost, portability, and a fast response time (Malhotra and Chaubey, 2003). A large group of biosensors are based on various oxidases, e.g. glucose oxidase and cholesterol oxidase. From the specific oxidase mediated reaction, hydrogen peroxide (H₂O₂) is produced (1).

$$\text{Analyte}+{\text{O}}_2\stackrel{\text{Oxides}}{⟶}\text{Byproduct}+{\text{H}}_2{\text{O}}_2$$

The H₂O₂ can be detected by chemiluminescence (Hanaoka et al., 2001), spectral techniques (He et al., 2006), and electrochemical methods (Hrapovic et al., 2004). Among these, the electrochemical methods are preferred because of their specificity, user-friendliness, high sensitivity, fast response time, and the general need for only low-cost equipment.

Electrochemical biosensors based upon nanomaterials have recently attracted considerable attention. Some examples are Ir, Rh, Pt, Pd, Ag nanoparticles (NPs) assembled on different forms of carbon supports, e.g. E-TEK carbon, graphite, carbon nanofiber, and carbon nanotubes. The fascinating physical and chemical properties of carbon nanotubes such as their electrical conductance, high mechanical stiffness, light weight, electron-spin resonance, field emission, electrochemical actuation, transistor behavior, piezoresistance, contact resistance, coulomb drag power generation, thermal conductivity, luminescence, electrochemical bond expansion, opto-mechanical actuation, and the possibility of introducing functionalization to change their intrinsic properties are reasons for the increasing interest in their use in novel biosensors (Iijima, 1991, Wang, 2004, Merkoci, 2006, Gong et al., 2005, Veetil and Ye, 2007). Pt-based metal NPs-based hybrid nanocomposites have been found to exhibit good catalytic activity towards the redox reduction of H₂O₂ (Wang et al., 1993, Wang et al., 1994, Wang et al., 1996, Ming et al., 2006, Huang et al., 2008, Guo and Dong, 2009, Zhang et al., 2011). Bimetallic nanoparticle-assemblies are of recent origin and are of particular importance in catalysis, since the addition of the second metal brings about variations in particle size, shape, surface morphology, chemical and physical properties including catalytic activity and chemical selectivity. With the advent of new synthetic routes, surface analyzing techniques and surface science modeling facilities, it has become possible to design and prepare tailor-made bimetallic NPs with desired properties. Specifically, Pt-based bimetallic systems with NPs of different structures (alloyed NPs, mixed monometallic NPs, core-shell NPs) have proved themselves as novel architectures with remarkably improved catalytic and electrocatalytic activity; thus, they are now used extensively for both methanol oxidation and the oxygen reduction reaction in fuel cells (Lim et al., 2009, Chang et al., 2010, Kim et al., 2010, Tao et al., 2008, Stamenkovic et al., 2007).

Immobilization of an enzyme onto a substrate (matrix) is a crucial step in the construction of an enzyme based biosensor. Chitosan (CS) is a biocompatible polymer and has remained a focus of study as one of the most promising materials for enzyme immobilization, due to its hydrophilicity, remarkable biocompatibility and low cost (Kaushik et al., 2008, Li et al., 2008a, Li et al., 2008b, Li et al., 2006). In addition, CS is a pH shift polymer because it possesses amino groups with a pKa of about 6.3. Consequently, the solubility and the net charge of CS are pH-dependent. CS has many primary amino groups, and a pKa value of ∼6.3. At pH values sufficiently below the pKa, CS exists as a water-soluble cationic polyelectrolyte, since most of the amino groups are protonated. When the solution pH is raised to near or above the pKa, many of the amino groups are deprotonated, and CS becomes insoluble. Usually, electrochemical deposition of CS is performed via water reduction to yield a hydrogel that can be tightly attached to the electrode, thereby allowing it to retain its natural properties (Wu et al., 2002, Wu et al., 2003, Wu et al., 2005, Xi et al., 2008, Luo et al., 2004). At reducing potentials, H⁺ in the solution is consumed at the cathode. Using the locally generated H⁺ gradient, the acidic side chains of CS can be titrated, thereby changing the solubility of CS, leading to the controlled deposition of a CS film.

Here the bimetallic nanoparticles decorated on multi-walled carbon nanotubes (PtPd-MWCNTs) catalyst was prepared by a modified Watanabe process employing microwave heating. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) results show the presence of Pt-based bimetallic nanoparticles PtPd with mean diameters of 3.2 nm. The catalyst layer was fabricated by drop coating method. The resulting PtPd-MWCNTs nanocomposite film offers new capabilities for electrochemical devices resulting from the synergistic action of PtPd bimetallic nanaoparticles and MWCNTs. The anti-interference layer (Nafion) was coated on the PtPd-MWCNT nanocomposite film surface. The biosensor was fabricated by immobilizing the CS-GA-glucose oxidase (GOx) enzyme layer onto the electrode's surface by electrodeposition. The electrochemical behavior of the modified electrode was been investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and amperometry. The resulting biosensor exhibits high sensitivity, good reproducibility, long-term stability with notable freedom from interference from co-existing electroactive species.