Elsevier

Biosensors and Bioelectronics

Volume 87, 15 January 2017, Pages 764-771
Biosensors and Bioelectronics

Electroactive and biocompatible functionalization of graphene for the development of biosensing platforms

https://doi.org/10.1016/j.bios.2016.09.030Get rights and content

Highlights

  • An electroactive graphene supported composite is synthesized and characterized.

  • This hybrid material is biocompatible to accommodate different redox enzymes.

  • Nano-bio-hybrid materials are tested for constructing glucose and cholesterol biosensors.

Abstract

Design and synthesis of low-cost, highly stable, electroactive and biocompatible material is one of the key steps for the advancement of electrochemical biosensing systems. To this end, we have explored a facile way for the successful synthesis of redox active and bioengineering of reduced graphene oxide (RGO) for the development of versatile biosensing platform. A highly branched polymer (PEI) is used for reduction and simultaneous derivation of graphene oxide (GO) to form a biocompatible polymeric matrix on RGO nanosheet. Ferrocene redox moieties are then wired onto RGO nanosheets through the polymer matrix. The as-prepared functional composite is electrochemically active and enables to accommodate enzymes stably. For proof-of-concept studies, two crucial redox enzymes for biosensors (i.e. cholesterol oxidase and glucose oxidase) are targeted. The enzyme integrated and RGO supported biosensing hybrid systems show high stability, excellent selectivity, good reproducibility and fast sensing response. As measured, the detection limit of the biosensors for glucose and cholesterol is 5 µM and 0.5 µM (S/N=3), respectively. The linear response range of the biosensor is from 0.1 to 15.5 mM for glucose and from 2.5 to 25 µM for cholesterol. Furthermore, this biosensing platform shows good anti-interference ability and reasonable stability. The nanohybrid biosensing materials can be combined with screen-printed electrodes, which are successfully used for measuring the glucose and cholesterol level of real human serum samples.

Introduction

In the 21st century, highly modernized society and our fast-pace lifestyle have increased the probability of suffering from different so-called ‘lifestyle diseases’, such as type-2 diabetes, obesity and various cardiovascular problems (Health and Information, 2013). Therefore, regular-basis monitoring and control of these diseases are of high priority in our daily life. For these reasons, biosensors are becoming an essential part of our everyday life (Turner, 2013). Determination of clinically important human metabolites in whole blood is the most important for early stage disease diagnostics. An imbalance of these analytes in blood can cause serious illness and life threatening diseases. Among various important analytes in blood, glucose and cholesterol are considered as the most crucial (Zhang et al., 2007). The abnormality in the blood glucose level is the main cause of global epidemic diabetes and nearly 80 million people are suffering for this in today's world (Herman and Zimmet, 2012). On the other side, irregularity in blood cholesterol level is closely associated with a high risk of cardiovascular diseases, which can cause coronary heart disease, stroke, and peripheral vascular diseases (Goldstein and Brown, 2001). High level of cholesterol is also linked to diabetes and high blood pressure. For all these reasons, diagnosis and management of these analytes in blood require highly sensitive, affordable and reliable biosensors.

With this high demand, the biosensor technology is going through a rapid development phase in the last few decades. According to WHO (World Health Organization) guidelines, practical biosensing devices should be simple, affordable, user-friendly, sensitive and specific to a particular analyte (Peeling et al., 2006). Recent advances in nanotechnology and materials chemistry could offer new promises for this purpose (Sekretaryova et al., 2015). Several factors are very crucial for the design of an ideal biosensing platform, including (1) electroactive and biocompatible support material, (2) stable immobilization of selected biological recognition elements on support with intact activity, and (3) efficient electronic communication between the redox active area of biomolecules and the electrode surface (Walcarius et al., 2013). A number of redox enzymes associated with nanostructured materials have been extensively studied to design various biosensors or biofuel cells (Willner et al., 2007).

Owing to their extraordinary physicochemical properties (Kostarelos and Novoselov, 2014, Liao et al., 2014), graphene based nanomaterials could offer a unique solution for the fabrication of biosensors. Although pristine graphene is chemically inert and electrochemically inactive, tunable functionalization and reduction of graphene oxide (GO) is very promising for development of a wide range of biosensors (Halder et al., 2016). Solution processed (or wet-chemical) synthesis is a very promising approach for low-cost production and facile functionalization of graphene based materials and their further use for the development of biosensing materials. In the last decades, several approaches have been used for the development of graphene and other nanomaterials based biosensors (Walcarius et al., 2013). Among them redox mediator based biosensors have received huge interest due to their low redox potential, high stability, solubility and efficient electron transport properties (Chaubey and Malhotra, 2002). Ferrocene is one of the mostly used electron-transfer mediators for fabrication of electrochemical biosensors due to its facile physicochemical properties (Rabti et al., 2016b). Ferrocene mediator based biosensor was introduced for the first time by the Turner's group (D’Costa et al., 1986). Among others, the Schmidtke group has used ferrocene functionalized linear polymers for glucose and H2O2 biosensors (Merchant et al., 2009, Merchant et al., 2010). The Niu group used ferrocene functionalized graphene for H2O2 biosensors (Fan et al., 2012). Heller and co-workers used the osmium based redox complexes for the fabrication of commercial glucose biosensor (Heller and Feldman, 2008). Dey et al. used ferrocene functionalized GO architecture for the development of versatile biosensing platforms (Dey and Raj, 2013). More recently, Merkoci and co-workers have used ferrocene functionalized graphene for the development of glucose biosensor (Rabti et al., 2016a). Although several groups have already attempted to develop ferrocene-modified graphene materials for biosensing applications, most reports lack to provide a specific biocompatible matrix for environmentally-friendly accommodation of particular bio-recognition elements such as enzymes.

Herein we describe a simple way for synthesis of redox active functionalization of polymer linked reduced graphene oxide (RGO) as well as the development of biocompatible matrix for accommodation of different bio-recognition elements. Ferrocene redox moieties were anchored onto RGO nanosheets via a branched polymer (PEI). The polymer acts as both a reducing agent and a molecular spacer for RGO. The functionalized RGO is electrochemically active and can offer a biocompatible microenvironment for immobilization of different enzymes (e.g. cholesterol oxidase and glucose oxidase). The as-constructed electroactive matrix was further used for the development of integrated biosensing platforms for cholesterol and glucose sensing. The biosensing process is dependent on bioelectrocatalytic oxidation of bio-analytes through redox active functionalized graphene-enzyme nanohybrid architecture. This nano-bio-hybrid material was further combined with an integrated screen-printed electrode and used for the glucose and free cholesterol level measurements of real human serum samples.

Section snippets

Reagents and materials

Graphite flakes (< 20 µm), D-(+)-glucose (≥ 99%), N-(3-dimethylaminopropyl)-N-ethylcarbodii-mide hydrochloride (EDC), 1-hydroxy benzotriazole (HOBt), Ferrocene carboxylic acid (≥ 97.0% (Fe)), poly (ethyleneimine) solution (50% (w/v) in water, Mw=750,000), glucose oxidase (from Aspergillus Niger, 100,000–250,000 units/g solid), Cholesterol oxidase from Streptomyces sp. (lyophilized powder, ≥ 20 units/mg) were purchased from Sigma-Aldrich. K2HPO4 and KH2PO4 were purchased from Fluka. 10 mM phosphate

Synthesis of Fc-RGOP nanohybrid material

Tunable derivation can enrich graphene with new physicochemical properties and functionalities. Fig. 1 represents the procedure for reduction and electrochemically active functionalization of graphene as well as enzyme loading. High-quality GO was synthesized and used as a starting material for the synthesis of Fc-RGO nanohybrids. The AFM topography imaging of GO clearly shows that the average thickness of single-layer GO nanosheets is 0.8–1.2 nm and the lateral diameters are in the size range

Conclusion

We have demonstrated the wiring of ferrocene electron transfer mediator and redox enzyme to graphene nanosheets through a highly branched polymeric matrix to synthesize a hybrid nanomaterials for fabrication of biosensors. The PEI matrix can offer linking groups (e.g. amine groups) for covalent attachment of ferrocene units to the whole planes of graphene nanosheets, not limited to the nanosheet edges as shown by most of previous reports. The PEI matrix can also provide a biocompatible

Acknowledgements

This work is supported by DFF_FTP, the Danish Research Council for Technology and Product Science (to Q.C., Project No. 12-127447). M.Z. acknowledges the CSC PhD scholarship (No. 201306170047). The authors are grateful to Glostrup hospital, Denmark for providing human serum samples and their standard analysis by clinical laboratory method.

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