A new self-assembled layer-by-layer glucose biosensor based on chitosan biopolymer entrapped enzyme with nitrogen doped graphene

Highlights

• New, very stable self-assembled layer by layer (LbL) enzyme biosensor

• LbL of N-doped graphene/glucose oxidase/chitosan and poly(styrene sulfonate)

• Gravimetric quartz crystal microbalance monitoring of multilayer film formation

• Characterization by cyclic voltammetry and electrochemical impedance spectroscopy

• The biosensor works at low potential with high sensitivity and operational stability.

Abstract

The layer-by-layer (LbL) technique has been used for the construction of a new enzyme biosensor. Multilayer films containing glucose oxidase, GOx, and nitrogen-doped graphene (NG) dispersed in the biocompatible positively-charged polymer chitosan (chit⁺(NG + GOx)), together with the negatively charged polymer poly(styrene sulfonate), PSS⁻, were assembled by alternately immersing a gold electrode substrate in chit⁺(NG + GOx) and PSS⁻ solutions. Gravimetric monitoring during LbL assembly by an electrochemical quartz microbalance enabled investigation of the adsorption mechanism and deposited mass for each monolayer. Cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the LbL modified electrodes, in order to establish the contribution of each monolayer to the overall electrochemical properties of the biosensor. The importance of NG in the biosensor architecture was evaluated by undertaking a comparative study without NG in the chit layer. The GOx biosensor's analytical properties were evaluated by fixed potential chronoamperometry and compared with similar reported biosensors. The biosensor operates at a low potential of − 0.2 V vs., Ag/AgCl, exhibiting a high sensitivity of 10.5 μA cm^− 2 mM^− 1, and a detection limit of 64 μM. This study shows a simple approach in developing new biosensor architectures, combining the advantages of nitrogen-doped graphene with the LbL technique for enzyme immobilization.

Introduction

One of the key issues in developing new biosensors with improved sensitivity and stability is effective immobilization of the recognition element, e.g. the enzyme. Among the enzyme immobilization methods, such as covalent linkage [1], [2], sol–gel entrapment [3], [4], adsorption [5], etc., layer-by-layer (LbL) self-assembly is a simple and powerful method, efficient because protein denaturation is minimized since the films are produced under mild conditions, based on the adsorption of macromolecules from aqueous solution onto solid supports [6]. LbL films have unique mechanical properties, uniformity and stability [7], the technique having the advantage of allowing the construction of thin multilayer films, based mainly on electrostatic interactions in between layers, which require a very small amount of material, therefore being a cost-effective preparation method for enzyme biosensors.

One of the problems to be overcome in an enzyme biosensor is the slow electron transfer between the enzyme redox center, which is usually buried in a hydrophobic cavity formed by polypeptide, and the electrode surface. Nanomaterials, such as graphene, carbon nanotubes (CNTs), metal nanoparticles, etc., are advantageous in increasing the possibility of direct electron transfer between the enzyme active sites and the electrode, acting as electrical bridges [8], [9], [10]; however, direct electron transfer between enzymes and carbon nanomaterials is not always the mechanistic basis of the substrate detection [11]. Nanomaterials can also bring benefits for immobilizing enzymes since they maintain enzyme bioactivity due to their microenvironment [12], [13]. Among the above-mentioned materials, graphene is a 2D plane sheet with an open structure and both sides of graphene could be utilized for enzyme immobilization, unlike 1-D CNTs, which are more difficult to be controllably assembled [14].

LbL formation of multilayer films with incorporation of graphene in one of the components of the self-assembly process combines the excellent electrochemical properties of graphene and the versatility of LbL assembly, showing great promise for highly efficient sensors and advanced biosensing systems. The excellent conductivity and small band gap of graphene are favorable for conducting electrons from the biomolecules [15]. Although, by using different types of intermolecular interactions, LbL structures are able to incorporate diverse molecules as building blocks, it is still a challenge to include certain kinds of molecules, especially hydrophobic species, into LbL films [16].

Graphene and nitrogen-doped graphene (NG) have been successfully dispersed in chitosan and used as substrate for the immobilization of enzymes [9], [17]. The N-doping of graphene has been done by thermal annealing in the presence of ammonia, the nitrogen atom in the graphene framework existing in “graphitic”, pyridinic or pyrrolic forms, which are beneficial for the electric conductivity of the material [18]. The biopolymer chitosan (chit) is often employed for enzyme immobilization, through covalent linkage, when the polymer is chemically modified to allow crosslinking with enzyme amino acids [19], and by electrostatic interaction in LbL films [20], in this case usually combined with carbon nanotubes, redox mediators or metal nanoparticles, due to its relatively poor conductivity [10], [21].

In the LbL enzyme immobilization study presented here, the positively-charged chitosan layer contains the enzyme glucose oxidase (GOx) together with dispersed NG, chit⁺(GOx) or chit⁺(NG + GOx), and the negatively charged layer is poly(styrene sulfonate), PSS⁻. Self-assembled adsorption of the multilayers on Au substrates was monitored by using an electrochemical quartz crystal microbalance (EQCM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The influence of NG and of each monolayer on the electrochemical properties of the LbL biosensor was analyzed, together with biosensor sensitivity after addition of each enzyme layer, in order to determine the best biosensor architecture.