Enzyme-free fluorescence microarray for determination of hepatitis B virus DNA based on silver nanoparticle aggregates-assisted signal amplification

Highlights

• Fluorescent intensity was extremely enlarged by silver nanoparticles aggregation without involving any enzymes.

• The fluorescent microarray achieved a detection limit of 50 fM and a broad linear range over 5 orders of magnitude.

• This method exhibited high specificity against single-base mismatched sequences.

• This method had a good applicability under the interference of genomic DNA.

Abstract

In this study, we designed a fluorescence enhancement strategy based on silver nanoparticle (AgNP) aggregates for the detection of hepatitis B virus DNA sequences. AgNPs were functioned with recognition probes (Cy3-probe) and hybrid probes (Oligomer-A and Oligomer-B). The presence of target DNA mediated the formation of sandwich complexes between the immobilized capture probes and the functionalized AgNPs, which was followed by hybridization-induced formation of AgNP aggregates. The fluorescent intensity could be extremely amplified by both the increasing number of fluorophores and metal enhanced fluorescence (MEF) effect. Under optimal conditions, this method achieved a detection limit of 50 fM which was 1560-fold lower than that of un-enhanced fluorescent assays. It was illustrated that the HBV DNA concentrations ranging from 100 fM to 10 nM had a good log-linear correlation with the corresponding fluorescent intensity (R = 0.991). Moreover, this method had high specificity both for distinguishing single-base mismatches and identifying target DNA under the interference of genomic DNA. This fluorescent microarray had high-throughput analytical potential and could apply to many other disease diagnoses.

Introduction

Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV) infection in liver cells [1]. The World Health Organization reported that there are 240 million people chronically infected with HBV around the world, with about 600,000 deaths per year [2]. The HBV DNA testing is an examination that patients with hepatitis B need to check regularly because HBV DNA testing can evaluate the virus in patients to ensure the effectiveness of diagnosis and treatment. It is important to have a diagnosis in early stages of diseases, because the fragments of HBV DNA presenting in blood are usually too few to be detected in many cases [3]. So far, a large amount of approaches for the determination of HBV DNA have been reported, including fluorescence [[4], [5], [6], [7], [8], [9], [10]], electrochemistry [[11], [12], [13], [14], [15], [16], [17]], chemiluminescence [[18], [19], [20], [21]], colorimetric assay [[22], [23], [24], [25], [26], [27]] and surface enhanced Raman scattering (SERS) [28,29]. However, some of these approaches are lack of the efficiency that only one sample can be quantified per assay. Microarray chips are a high-throughput technology that allow the simultaneous quantification of multiplexed samples on a single chip. Recently, various methods based on microarray have been developed to detect the DNA sequences of HBV, especially electrochemical methods and fluorescent methods. Electrochemical methods based on chips have advantages of high sensitivity and low-cost requirements [[30], [31], [32], [33], [34]], the only disadvantage is the poor repeatability of electrode arrays. Although microarrays with fluorescence detection are a powerful tool for DNA detection, it is still a challenge to perform sensitive DNA assay because the signal is undetectable at low concentration.

Metal enhanced fluorescence (MEF) is a phenomenon that the interaction for a metallic surface with proximal fluorophores can considerably enhance the intensity of the fluorescent signal [35]. Gold (Au) and silver (Ag) nanoparticles are mostly used for MEF because they can exhibit strong surface plasmon resonance in the visible wavelength range. Although AgNPs showed higher toxicity to protein than AuNPs in some studies [36], AgNPs have shown greater promise for the signal amplification than AuNPs. For AgNPs, the absorption peaks of the surface plasmon span almost the entire visible spectrum. However, for AuNPs, the absorption peak lies in the range of 510–570 nm [37]. The silver substrate platform has been extensively studied on fluorescence microarray. Mei et al. [38] developed an ordered gold nanorod array biochip and observed 1-fold fluorescence enhancement factor for DNA detection. Ji et al. [39] made silver zigzag nanorod arrays by oblique angle deposition and obtained a 14-fold enhancement factor for biotin-neutravidin detection. Kannegulla et al. [40] built a plasmon-enhanced fluorescence nanoarray on plane silver surface and achieved a 40-fold enhancement factor for DNA detection. These silver substrates improved the sensitivity by MEF but suffered from complicated fabrication of ordered nanostructural arrays and uncontrollable features of nanostructures formed from various deposition methods. Comparablely, metallic colloids MEF platforms has the superiority of both the controllable preparation of nanoparticle colloids and the high affinity of self-assembly nanoparticle probes. Several researches have reported that the significant fluorescence enhancement via the coupling between silver nanoparticles [41,42]. Our group further amplificated the signal of fluorescence microarrays by employing two kinds of nucleic acid-modified AgNP probes, each of which functionalized with aptamers specific to target proteins [43,44] and oligonucleotides complementary to those on the other AgNPs. Herein, it was the first time that we performed the AgNPs aggregation-based fluorescence enhancement strategy for sensitive, specific and quantitative detection of HBV DNA sequences.

In this work, we used Oligomer-A/Cy3-functioned AgNPs (Tag-A) and Oligomer-B/Cy3-functioned AgNPs (Tag-B) to form AgNP aggregates. Thus, the fluorescent intensity could be extremely enlarged by both the increasing number of fluorophores and metal enhanced fluorescence (MEF) effect. Compared to the assay without AgNPs, this AgNPs aggregation-based assay demonstrated up to 120-fold enhancement factor. Our assay showed a sensitive analytical performance for HBV DNA detection over the range from 100 fM to 10 nM with a detection limit of 50 fM and a high specificity for distinguishing single-base mismatches. Additionally, the detection signal of 1 pM target DNA could be identified against the interference of genomic DNA, which suggested the clinical application potential of this assay.