Elsevier

Carbon

Volume 61, September 2013, Pages 357-366
Carbon

Assembled gold nanoparticles on nitrogen-doped graphene for ultrasensitive electrochemical detection of matrix metalloproteinase-2

https://doi.org/10.1016/j.carbon.2013.05.016Get rights and content

Abstract

A new electrochemical immunosensor was developed for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), which is one of the key biomarkers in blood. In our approach, an effective assembly of well-defined gold nanoparticles on nitrogen-doped graphene sheets was demonstrated. The composite facilitated robust immobilization of antibodies, promoted electron transfer and exhibited excellent electrochemical activity, which are suitable for biosensing. The design of the immunosensor also involved a polydopamine functionalized graphene oxide hybrid conjugated to horseradish peroxidase-secondary antibodies by covalent bonds as a multi-labeled and biocompatible probe to increase the electrochemical response. This novel signal amplification strategy with a sandwich-type immunoreaction significantly enhanced the sensitivity of detection of biomarkers. The proposed immunosensor displayed excellent analytical performance in the detection of MMP-2 ranging from 0.0005 to 50 ng mL−1, with a detection limit of 0.11 pg mL−1. Furthermore, it not only exhibited good stability with adequate reproducibility and accuracy, but also demonstrated efficiency in the detection of MMP-2 in real samples.

Introduction

Matrix metalloproteinases (MMPs) are a family of secreted zinc-dependent endopeptidases which are crucial for the regulated degradation and processing of extracellular matrices, and are up-regulated in almost every type of human cancer [1], [2]. In particular, MMP-2 (also known as gelatinase A), is one of the crucial MMPs in tumor growth, invasion and metastasis, and plays a key role in physiological and pathological states including morphogenesis, reproduction and tissue remodeling due to its ability to degrade type VI collagen [3], [4]. Therefore, sensitive detection of MMP-2 is of great importance in the reliable early detection of cancer and disease. Due to the low level of MMP-2 in human blood and the complexity of clinical samples such as serum, plasma and whole blood, it is imperative, although challenging, to develop new analytical methods with high sensitivity and specificity to circumvent the interferences arising from complex biological sample matrices. Thus, improved detection approaches are continually required to meet increasing demands [5]. Immunoassays based on highly specific antibody-antigen recognition have been widely used in the sensitive and quantitative detection of disease-related proteins which are critical in biomedical research and diagnostics [6]. Among these assays, the electrochemical immunoassay has attracted considerable interest due to its intrinsic advantages including good portability, low cost and precise current measurement, and has been developed and extensively applied in the determination of biomarkers [7], [8].

In order to improve the sensitivity and selectivity of biosensors, several methods have been developed using signal amplification or the use of different detection technologies, and nanomaterial-based systems have attracted particular interest in immunoassays due to their abundant properties [9], [10]. Recently, graphene, a single-atom-thick sheet of sp2-bonded carbon atoms, has attracted immense interest due to its unique properties and potential applications [11], [12], [13], [14], [15], [16]. Considerable efforts are now being made to dope graphene with nitrogen to further tune its electrical properties and geometric parameters [17], [18]. Compared with pristine graphene (G), nitrogen-doped graphene (NG) is more commonly used in supercapacitors, fuel cells and field-effect transistors, etc., as a result of its much larger functional surface area, higher ratio of surface active groups to volume, more biocompatible C–N microenvironment, higher electrical conductivity and more chemically active sites for further functionalization such as anchoring of metal nanoparticles [19], [20], [21], [22]. Moreover, it has been demonstrated that the Au/nitrogen-doped carbon nanotube composite enhanced the biocompatibility and sensitivity in biosensing applications [23]. Consequently, it is crucial to explore the potential bioapplications of NG-based composites.

In addition, graphene oxide (GO) has been used to detect target molecules through different analytical principles owing to its large specific surface area and high water solubility [24], [25]. To improve the stability and practicality of GO, it is generally functionalized with biomolecules, inorganic materials and polymers [26], [27]. Dopamine can undergo self-polymerization to form polydopamine (PD), which has been reported to be an efficient binding agent for coating graphene and other substrates, thus exhibiting versatile applications [28], [29]. Dopamine may play a dual-functional role as both the reductant and surface functionalization agent for GO. Therefore, a PD-functionalized GO hybrid (PD–GO) with good biocompatibility and conductivity may have potential applications in drug delivery and bioassay systems.

Herein, we report an approach to use the exceptional properties of the NG-based composite for the fabrication of an ultrasensitive electrochemical immunosensor based on a signal amplification strategy for the detection of MMP-2 (Fig. 1). Due to the strong binding between metal nanoparticles and NG, an effective chemical route was developed to directly attach gold nanoparticles (AuNPs) uniformly with high density onto NG which served as an immobilization scaffold for antibodies. The obtained Au–NG composite not only increased the surface area to capture large amounts of proteins and promoted electron transport, but also displayed efficient electrochemical activity for biosensing. In order to further facilitate signal amplification, the PD–GO architecture was used to provide a favorable surface to load multiple horseradish peroxidase-labeled anti-MMP-2 (HRP-Ab2), resulting in an ideal probe for a sandwich electrochemical immunoassay. The electrochemical response of the fabricated biosensor was greatly enhanced and ultrasensitive detection of MMP-2 was achieved with good precision, acceptable stability and reproducibility. Therefore, these results should generate interest in utilizing this electrochemical immunoassay for the detection of other biomolecules in order to develop efficient clinical biomedical applications.

Section snippets

Materials and apparatus

Graphite powder (KS-10), bovine serum albumin (BSA) and dopamine were obtained from Sigma–Aldrich. Thionine (Th), H2O2 (30%), chloroauric acid (HAuCl4 4H2O), 2-amino-2-hydroxymethylpropane-1,3-diol (Tris), trisodium citrate and glutaraldehyde (GLU, 25% aqueous solution) were from Shanghai reagent Co. Inc. (Shanghai, China). Human MMP-2 standard (Ag), the MMP-2 capture antibody (Ab1), HRP-Ab2, the enzyme-immunoassay kit for human MMP-2, Bicinchoninic Acid (BCA) protein assay kit and Tween-20

Characterization of Au–NG and the sensing probe

Doping graphene with nitrogen atoms is regarded as an effective strategy to introduce defective sites on the graphene surface for improving electrocatalytic effects, however, NG-based composites have rarely been used in electrochemical immunosensors. Combined with metallic nanoparticles, the remarkable conductivity and large surface area of Au–NG could provide a feasible pathway for electron transfer, and enhance intrinsic electrocatalytic activity of NG through nanoparticle electronic

Conclusions

An effective approach was developed to directly attach well-defined AuNPs onto the surface of NG, which promoted faster nucleation and growth kinetics of AuNPs resulting in their small size and uniform dispersion with high density. Therefore, a promising electrochemical immunosensor was successfully designed for the determination of MMP-2 by a novel signal amplification strategy. The Au–NG modified GCE not only accelerated electron transfer, but also exhibited high electrochemical activity to

Acknowledgments

We gratefully appreciate the support from the International S&T Cooperation Projects of China (2010DFA42060), the National Basic Research Program of China (2011CB933502) and the National Natural Science Foundation (21121091).

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