Visual Detection of Single-Nucleotide Polymorphism with Hairpin Oligonucleotide-Functionalized Gold Nanoparticles

Abstract

We report a simple, fast, and sensitive approach for visual detection of single-nucleotide polymorphism (SNP) based on hairpin oligonucleotide-functionalized gold nanoparticle (HO-Au-NP) and lateral flow strip biosensor (LFSB). The results presented here expand on prior work ( Mao , X. , Xu , H. , Zeng , Q. , Zeng , L. , and Liu , G. Chem. Commun. 2009 , 3065-3067 .) by providing new approach to prepare HO-Au-NP conjugates with a deoxyadenosine triphosphate (dATP) blocker, which shortens the preparation time of the conjugates from 50 to 8 h and lowers the detection limit 500 times. A hairpin oligonucleotide modified with a thiol at the 5'-end and a biotin at the 3'-end was conjugated with Au-NP through a self-assembling process. Following a blocking step with dATP, the hairpin structure of HO and dATP embed the biotin groups, and make the biotin groups in close proximity to the Au-NP surface, leading to the biotins being "inactive". The strategy of detecting SNP depends on the unique molecular recognition properties of HO to the perfect-matched DNA and single-base-mismatched DNA to generate different quantities of "active" biotin groups on the Au-NP surface. After hybridization reactions, the Au-NPs associated with the activated biotins are captured on the test zone of LFSB via the specific reaction between the activated biotin and preimmobilized streptavidin. Accumulation of Au-NPs produces the characteristic red bands, enabling visual detection of SNP. The preparations of HO-Au-NP conjugates with dATP and the parameters of assay were optimized systematically, and the abilities of detecting SNP were examined in details. The current approach is capable of discriminating as low as 10 pM of perfect-matched DNA and single-base-mismatched DNA within 25 min without instrumentation. Moreover, the approach provides a lower background and higher selectivity compared to the current molecular beacon-based SNP detection. The protocol should facilitate the simple, fast, and cost-effective screening of important SNPs and could readily find wide applications in molecular diagnosis laboratories and in point-of-care testing (field testing).

Recent trends in molecular beacon design and applications

“…Another application of AuNP-based MBs is as lateral flow strip biosensors; these were developed by Mao et al [58] and He at al. [66] for rapid detection of nucleic acid sequences with high sensitivity and low cost.…”

A SERS-based lateral flow assay biosensor for highly sensitive detection of HIV-1 DNA

“…A paper-based lateral flow (LF) strip, which combines nanoparticles with conventional chromatographic separation, has attracted significant interest because nanoparticles are favorable for signal amplification, which is used to achieve high sensitivity and selectivity for target analysis (He et al, 2010;Mao et al, 2009a;Xu et al, 2009). LF strips are considered to be one of the most promising techniques due to their simplicity, rapid analysis, relatively low interference due to chromatographic separation, low costs, long-term stability over a wide range of climates, and their ability to be easily used with no requirements for skilled technicians (Chen et al, 2013;Mao et al, 2009b;Shen et al, 2013).…”

“…LF strips are considered to be one of the most promising techniques due to their simplicity, rapid analysis, relatively low interference due to chromatographic separation, low costs, long-term stability over a wide range of climates, and their ability to be easily used with no requirements for skilled technicians (Chen et al, 2013;Mao et al, 2009b;Shen et al, 2013). Until now, several different types of LF strip have been developed to detect nucleic acids including DNA or miRNA (Fang et al, 2014;Gao et al, 2014;He et al, 2010;Hu et al, 2013;Mao et al, 2009b). For example, a dry-reagent strip biosensor based on a molecular beaconfunctionalized gold nanoparticle probe has been developed for rapid and quantitative detection of specific nucleic acids (Mao et al, 2009b).…”

Visual and On-site Detection of Mercury(II) Ions on Lateral Flow Strips Using DNA-functionalized Gold Nanoparticles

“…This colorimetric method can also be used to detect other targets through replacing the thymine-rich DNA by other special DNA bases. 29,[34][35][36] However, the method still requires sophistic instruments and professional skills, restricting its use as a common assay tool. Furthermore, it is often difficult to distinguish the blue color of aggregates against the strong red background even by special instruments, particularly at low Hg 2+ concentrations.…”