Target triggered self-assembly of Au nanoparticles for amplified detection of Bacillus thuringiensis transgenic sequence using SERS

Highlights

• A universal SERS sensing platform was developed for sensitive detection of DNA.

• DNA–Au NPs amplification units were adopted to obtain high sensitivity.

• The method required fewer steps and lower experimental requirements.

• The biosensors strategy could be extended to electrochemical detection for DNA.

Abstract

The research methods for DNA detection have been widely extended since the application of nanotechnology, but it remains a challenge to detect specific DNA sequences or low abundance genes in the biological samples with accuracy and sensitivity. Here we developed a SERS biosensing platform by target DNA (tDNA) triggered self-assembly of Au nanoparticles (Au NPs) probes on DNA nanowires for signal amplification in DNA analysis. Based on the hybridization chain reactions (HCR) and surface enhanced Raman scattering (SERS) technology, the SERS intensity reveals a good linearity with tDNA ranging from 50 pM to 500 pM under optimal conditions. The specific detection of tDNA sequence was realized with a detection limit of 50 pM (S/N=3). To demonstrate the specificity and universality of the strategy, the single-base mismatches in DNA and the Bacillus thuringiensis (Bt) transgenic sequence were successively applied in the SERS assay. The results showed that the sensitivity and accuracy of the SERS-based assay were comparable with real-time PCR. Besides, the method would provide precise and ultra-sensitive detection of tDNA but also informative supplement to the SERS biosensing platform.

Introduction

As a specific DNA sequence, Bacillus thuringiensis (Bt) transgene has aroused attention worldwide since its introduction into crops for insect control, which brings benefits to the farmers but also raises people's great concern in food security and bio-security (Hutchison et al., 2010, Quist and Chapela, 2001). Therefore, it is imperative to develop certain accurate and rapid methods for testing transgenes in food and feed. Recently, many important technological advances have been made in the development of multiple sensors for monitoring the DNA interactions and recognition events (Morisset et al., 2008, Hiroshi et al., 2005). Wherein, the detection methods are mostly based on DNA hybridization pattern recognition, which possess high sensitivity and specificity but usually require amplification by polymerase chain reaction (PCR). Despite its high sensitivity and stability, the PCR method typically suffers from the disadvantages such as high cost, high complexity and time consuming (Zhu et al., 2008, Qiu et al., 2011). Moreover, it still remains a challenge to detect specific DNA sequences or low abundance genes in the biological samples with accuracy and sensitivity (Wang et al., 2011). Hence, it is of great importance to establish a time-saving, reliable and sensitive detection method for detecting specific DNA sequence.

To develop newly amplified detection methods for the analysis of DNA, various sensing strategies have been employed in bioassay field based on electrochemical and optical technology (Wang et al., 2014, Hsu and Huang, 2004). These methods often adopt an enzymatic reaction (Wan et al., 2013) or rolling circle amplification (RCA) (Bi et al., 2010) for signal amplification in ultra-sensitive detection of DNA. However, these approaches are confronted with the difficulties of harsh reaction conditions and long amplification period (Zhang et al., 2012). At present, new strategies for the detection of trace DNA have aroused broad interest (Tang et al., 2012). One of the impressive arts is designed upon the recognition of target DNA (tDNA) triggering hybridization chain reaction (HCR) to construct self-assembly DNA nanowires (Dirks and Pierce, 2004). Based on the self-assembly amplification system, the biosensing platform enables sensitive DNA analyses without the assistance of enzymes and can be implemented at room-temperature. The method for constructing DNA biosensors via HCR makes it an attractive protein-free, room-temperature alternative to PCR and RCA for signal amplification in DNA analysis (Huang et al., 2011, Ren et al., 2011).

Because of its high sensitivity and abundant structural information content for molecules, surface enhanced Raman scattering (SERS) technology has become a well-established analytical tool for chemical and biological sensing by combining with Ag or Au nanoparticles (Au NPs) (Gao et al., 2013, Lim et al., 2010). Herein, on the basis of HCR, we present a universal biosensing platform for DNA sequences assay via SERS technology coupled with Au NPs probes. Fig. 1 illustrates the target triggered self-assembly of Au NPs on the biosensing platform for the ultra-sensitive detection of tDNA amplified by HCR. First, HS-hairpin 1 (HS-H1) was fixed on the substrate followed by a blocking procedure of 6-mercapto-1-hexanol (MCH). Second, tDNA was injected and hybridized with HS-H1, which was unfolded and triggered HCR by alternative cross-hybridization of biotinylated hairpin 1 (bio-H1) and hairpin 2 (bio-H2) forming a long DNA nanowire. Third, the Au NPs probes grew along the DNA nanowires after the addition of streptavidin (SA). Finally, under laser exposure, the sensing platform could provide SERS signals originated from Raman active molecules (X-rhodamine: ROX) on the surface of Au NPs. The results demonstrated that the proposed strategy possesses many advantages such as simple procedures, low experimental requirements and high sensitivity (50 pM).