An efficient colorimetric biosensor for glucose based on peroxidase-like protein-Fe₃O₄ and glucose oxidase nanocomposites

Highlights

• Protein-magnetite nanoparticles possess enhanced peroxidase-like activity.

• Glucose biosensor based on biocompatible protein-Fe₃O₄ nanocomposite is developed.

• The biosensor exhibits an excellent performance toward glucose detection in a colorimetric way.

Abstract

An efficient colorimetric biosensor for glucose based on peroxidase-like protein-Fe₃O₄ and glucose oxidase nanocomposites is reported in this work. Compared with bare MNPs, peroxidase-like casein-MNPs exhibit good catalytic properties, stability, dispersibility. Casein incorporated on MNPs notably improves the affinity toward both H₂O₂ and TMB, proved by variation in the determined kinetic parameters. As low as 0.2 μM H₂O₂ can be detected with a linear range from 0.5 μM to 200 μM H₂O₂. More importantly, the casein/MNP nanocomposite was further used to immobilize GO_x and to construct a glucose biosensor for the one-step determination of glucose. This method is simple, inexpensive, highly sensitive, and selective for glucose detection, with a detection limit of 1.0 μM over a linear range from 3 μM to 1000 μM.

Introduction

Magnetite (Fe₃O₄) particles have long been considered as one of the most attractive nanomaterials for applications in magnetic resonance imaging, biological separation, drug delivery, and biological catalysis, particularly due to their favorable magnetic properties and low toxicity (Wang et al., 2008, Laurent et al., 2008). Recently, it was reported that Fe₃O₄ magnetic nanoparticles (MNPs) exhibited an intrinsic enzyme mimetic activity similar to that found in natural peroxidases (Gao and Yan, 2007, Wei and Wang, 2008, Liu et al., 2011). Compared with a native enzyme-horseradish protein (HRP), the enzyme-like nanomaterials display potential excellent properties, such as greater resistance to extremes of pH and temperature. Furthermore, their preparation, storage, and separation are relatively simple and cheaply. However, bare MNPs often display aqueous instability, due to their magnetic attractive forces and the long-rang van der Waals attractive forces (Bonacchi et al., 2004, Lee et al., 2007). In addition, magnetic nanoparticles display much weaker affinity toward substrates than HPR (Gao and Yan, 2007), which results in low catalytic activities and restricted applications.

Detection of glucose has been paid much attention in biomedical fields and plays an increasingly important role in the improvement of life quality (Mizutani and Yabuki, 1997, Radhakumary and Sreenivasan, 2011). H₂O₂ is the main product of the glucose oxidase (GO_x)-catalyzed reaction, and the enzyme-like nanomaterials based sensors were developed for colorimetric detection of glucose (Wei and Wang, 2008, Yu et al., 2010). However, due to the different optimal pH for enzyme-like nanomaterials and GO_x, the detection of glucose by using enzyme-like nanomaterials usually involved two-step analytical processes in these studies. First, GO_x was used to catalyze the oxidation of glucose in neutral buffer. Then, the H₂O₂ produced served as a substrate for MNPs to catalyze the oxidation of the peroxidase substrate to the colored radical in acidic buffer. Obviously, the method used was a multiple-step analysis, and GO_x cannot be recycled in those studies. This limits the application of those enzyme-like nanomaterials in practice.

Considering the above limitations of the enzyme-like nanomaterials, a suitable surface functionalization plays a crucial role (Yu et al., 2009, Liu and Yu, 2011). A suitable surface modification should provide good colloidal stability of MNPs, high affinity towards the substrates of the MNPs, and functional groups to immobilize enzyme on MNPs. Recently, Yu et al. reported that SH–NH₂–MION, by virtue of its features of both sulfhydryl groups and amines, displayed elevated affinity toward substrates, and high storage stability (Liu and Yu, 2011). There is still a much requirement for searching for new functionalized materials to realize the highly sensitive sensing of hydrogen peroxide and other oxidase-based reactions giving H₂O₂ as a product (such as glucose).

Proteins with carboxyl and hydroxyl functional groups have the potential to coat magnetic nanoparticles. The conjugation of proteins with nanocrystals not only affords stabilization of the system, but also introduces biocompatible functionalities onto these nanoparticles for further biological application (Gole et al., 2001, Jiang et al., 2005). Casein with an isoelectric point of about 4.5 has a highly content of acid amino acid residues (such as glutamic acid and aspartic acid residues), and is negatively charged at neutral pH. The casein constituents, α_s1-, α_s2-, β-, and κ-casein, exist in proportions of approximately 4:1:4:1 by weight, and can be thought of as block copolymers consisting of blocks with high levels of hydrophobic or hydrophilic amino acid residues. Therefore, caseins exhibit a strong tendency to self-assemble into spherical casein micelles (Liu and Guo, 2007).

Herein, we report a facile synthesis of aqueous casein stabilized, monodisperse iron oxide nanoparticles via a coprecipitation method. The as-prepared casein-MNPs were then used to catalyze the oxidation of a peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂ to the oxidized colored product which provides a colorimetric detection of H₂O₂. As low as 0.2 μM H₂O₂ could be detected with a linear range from 0.5 to 200 μM via our method. More importantly, a one-step, sensitive and selective method for glucose detection was developed using the composite of casein-MNPs and GO_x. At pH 5.5, the casein-MNP/GO_x nanocomposites catalyze the oxidation of glucose to generate H₂O₂. At the same pH buffer, the resulting H₂O₂ oxidizes TMB in the presence of casein-MNP/GO_x nanocomposites. The detection platforms for H₂O₂ and glucose developed in the present work confirmed that the casein-MNPs not only possess highly effective peroxidase-like activity but also provide functional surfaces suitable for immobilizing biomolecules and preserving their biological activity.