Elsevier

Biomaterials

Volume 32, Issue 33, November 2011, Pages 8584-8592
Biomaterials

An integrated sensing system for detection of DNA using new parallel-motif DNA triplex system and graphene–mesoporous silica–gold nanoparticle hybrids

https://doi.org/10.1016/j.biomaterials.2011.07.091Get rights and content

Abstract

In this article, we demonstrate the use of graphene–mesoporous silica–gold NP hybrids (GSGHs) as an enhanced element of the integrated sensing platform for the ultra-sensitive and selective detection of DNA by using strand-displacement DNA polymerization and parallel-motif DNA triplex system as dual amplifications. We find that the present new sensing strategy based on GSGHs is able to detect target DNA with a fairly high detection sensitivity of 10 fm through the hybridization of duplex DNA to the acceptor DNA for the formation of parallel-motif DNA triplex on the multilayer film (containing GSGHs and redox probe) modified functional interface, and even has a good capability to investigate the single nucleotide polymorphisms (SNPs). The detection limit for target DNA is about two orders of magnitude lower than that of graphene-based DNA electrochemical impedance spectroscopy (EIS) sensor (6.6 pm), four orders of magnitude lower than those of graphene-based DNA sensors coupled with fluorescent assay (100 pm and 1 nm) and five orders of magnitude lower than those of field effect transistor (FET)-based assays (1 nm and 2 nm). Most importantly, our present sensing system can also be facilely achieved in the ITO electrode array, which is of paramount importance for possible multiplex analysis in lab-on-chip.

Introduction

Graphene, Nobel Prize material, has stimulated intense research interest because of its unique physical and chemical properties, such as high surface area, high electrical conductivity, good chemical stability and strong mechanical strength [1]. These unique characteristics enable it to hold great promise for application in many technological fields, e.g. in particular used as enhanced materials for developing high-performance sensors [2], [3], [4], [5]. Recent significant advances also reveal that the clever combination of graphene with metal nanoparticles (NPs), leading to the development of a multicomponent nanoassembly system, opens a new avenue for utilizing graphene-based hybrid nanomaterials as enhanced elements for constructing higher-performance electrochemical sensing platform [6], [7]. Chen et al. reported that graphene/gold NP hybrids were important nanostructured electrode materials for achieving a highly sensitive and selective field effect transistor (FET) biosensor [6]. Our previous work showed that platinum NPs ensemble-on-graphene hybrid nanosheets had more favorable electron transfer kinetics and much enhanced electrochemical reactivity than graphene itself, which provide a kind of more robust and advanced hybrid electrode material for sensing small molecules [7]. These contributions indicate that graphene-based hybrid nanomaterials play an important role on acting as magnified materials for constructing high-performance analytical sensing device. However, the design of new routes for controllable synthesis of new graphene-based nanocomposites with desirable surface characteristics, electron and biocompatible properties for achieving high-sensitivity analytical sensors is still a great challenge and continues to be a hot research topic.

On the other hand, among the various analytical applications with graphene, the graphene-based DNA sensors are more attractive, which are of great importance in the fields of diagnosis of genetic disease, environmental monitoring and antibioterrorism, etc. Consequently, various analytical techniques, fluorescent sensors in particular, have been facilely employed for the development of high-performance graphene-based DNA sensors [8], [9], [10], [11], [12]. Although the as-prepared graphene-based fluorescent sensors hold great promise for the detection of different targets based on “turn-on” or “turn-off” mode, such strategies usually require the fluorophore and quencher labeling at the ends of the DNA molecules, which often suffers from some problems such as high cost, low yield and complex purification steps. As an attractive alternative, the electrical sensing techniques can allow the detection of targets in a label-free, sensitive and convenient manner, which have been well realized using graphene-based FETs [6], [13]. However, it remains a great challenge for exploring the graphene–Au NPs hybrids-based new nanobioelectronic devices in a lab-on-chip mode to achieve the ultra-sensitivity detection of DNA. Furthermore, almost all the present reports were based on the use of the duplex DNA sensing design. Recently, triplex DNA, one of the most useful recognition motifs, has received more attention on analytical sensors. The DNA triple helix is a structure of DNA in which three oligonucleotides wind around each other. In this structure, one strand binds to a B-form DNA double helix through Hoogsteen or reversed Hoogsteen hydrogen bonds [14]. For example, an N-3 protonated cytosine, represented as C+, can form a base-triplet with a C–G pair through the Hoogsteen base-pairing of a GxC+ (x represents a Hoogsteen base pair). Thus, the triple-helical DNAs using these Hoogsteen pairings consist of two homopyrimidines and one homopurine, and one of the homopyrimidine strands is parallel to the homopurine strand. Due to such special characteristics, the triplex DNA has been employed to design novel biosensors. For example, Zhang et al. reported a kind of triplex DNA molecular beacons as signaling probes and achieved the detection of cellular ATP for cancer cells based on the chemiluminescence resonance energy transfer (CRET) and a structure-switching aptamer [15]. However, the exploration of triplex DNA with biosensing application is still at an early stage and has not been demonstrated on the aspect of electrochemical sensors.

In this article, we developed a multi-step chemical approach to synthesize graphene–mesoporous silica–gold NP hybrids (GSGHs) through combining sol–gel and self-assembly techniques. Such GSGHs have several advantages for the design of DNA-based electrochemical analytical devices. (1) The Au NPs on the surface of GSGHs have good surface chemical property, which are easily functionalized by SH-DNA. (2) Mesoporous silica itself has good biocompatibility, and also can reduce the nonspecific adsorption of the DNA. (3) GSGHs may have electronic conductivity, which facilitate the electronic transfer of probe molecule. (4) GSGHs can enlarge the electrode area, which is important for enhancing the detection performance. Taking these nice properties into consideration, GSGHs were used as an enhanced element of the integrated sensing platform for the ultra-sensitive and selective detection of DNA through the use of strand-displacement DNA polymerization and parallel-motif DNA triplex system as dual amplifications. And, the present sensing system was also explored to be achieved in the indium tin oxide (ITO) electrode array, which was of paramount importance for possible multiplex analysis in lab-on-chip.

Section snippets

Materials

Graphite, tetraethylorthosilicate (TEOS), 3-aminopropyltriethoxysilane (APTES) and cetyltrimethylammonium bromide (CTAB) were obtained from Alfa Aster. K3Fe(CN)6 and K4Fe(CN)6 were purchased from Aldrich. Graphene oxide (GO) was synthesized according to our previous papers [16], [17], [18]. Thirteen-nanometer Au nanoparticles were obtained through employing the typical citrate reduction method [19]. In a typical synthesis, 4.13 mL 1% HAuCl4 was added into 100 mL water and heated to boiling.

Sensing procedure

The schematic procedure for preparing the GSGHs-based integrated DNA sensing platform is shown in Scheme 1. A simple wet-chemical process was employed to synthesize GSGHs (building block) through combining self-assembly technique (Scheme 1A). For the GSGHs-based LBL sensing interface (Scheme 1B), the positively charged Fc-PEI (Fc, redox probe) was firstly assembled on the negatively charged patterned ITO electrode through the electrostatic interaction, and then followed by the assembly of

Conclusion

In summary, we have developed a wet-chemical approach to synthesize graphene–mesoporous silica–Au NPs hybrids through combining self-assembly technique. The as-prepared GSGHs could be used as an advanced building block to construct new Fc-PEI/GSGHs LBL functional interface with tunable thickness, which could be used as sensitive electrochemical sensing platform for the detection of DNA hybridization and SNPs. Most importantly, we demonstrated the use of triplex DNA system for amplifying the

Acknowledgments

This research was supported by the National Natural Science Foundation of China (Nos. 21075116, 20935003 and 20820103037) and 973 Project (2009CB930100, 2011CB911002 and 2010CB933600).

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    Yan Du and Shaojun Guo contributed equally to this work.

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