Chip-based visual detection of microRNA using DNA-functionalized gold nanoparticles.

Ping Wang,Qinghui Jin,Hongju Mao,Zule Cheng,Yanan Bai,Huiying Liu,Sanqiang Li,Jianlong Zhao,Bin Hu

doi:10.1007/s11427-015-4987-0

Abstract

In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle (AuNP) probes and a gene chip to detect microRNAs (miRNAs). Specific oligonucleotides were attached to the glass surface as capture probes for the target miRNAs, which were then detected via hybridization to the AuNP probes. The signal was amplified via the reduction of HAuCl4 by H2O2. The use of a single AuNP probe detected 10 pmol L(-1) of target miRNA. The recovery rate for miR-126 from fetal bovine serum was 81.5%-109.1%. The biosensor detection of miR-126 in total RNA extracted from lung cancer tissues was consistent with the quantitative PCR (qPCR) results. The use of two AuNP probes further improved the detection sensitivity such that even 1 fmol L(-1) of target miR-125a-5p was detectable. This assay takes less than 1 h to complete and the results can be observed by the naked eye. The platform simultaneously detected lung cancer related miR-126 and miR-125a-5p. Therefore, this low cost, rapid, and convenient technology could be used for ultrasensitive and robust visual miRNA detection.

Highlights

MicroRNAs are endogenous, highly conversed, noncoding small (21-25 nucleotides in length) RNAs
We developed a highly sensitive and convenient biosensor consisting of gold nanoparticle (AuNP) probes and a gene chip to detect microRNAs
Target miRNAs, reporter probes, and AuNP probes were added to the system and allowed to conjugate to the chip via base pairing

Summary

INTRODUCTION

MicroRNAs (miRNAs) are endogenous, highly conversed, noncoding small (21-25 nucleotides in length) RNAs. It is necessary to develop a new method for the detection of miRNAs with high sensitivity, easy operation, and low cost. AuNP probes and Raman-active dyes have been used to detect target nucleic acids (Cao et al, 2002). The Raman tag can be used as a narrow-band spectroscopic fingerprint and the detection limit is 20 fmol L−1. Based on these studies, the combination of AuNPs and microarray allows detection of nucleic acids with high sensitivity. We developed a highly sensitive and convenient miRNA detection biosensor consisting of AuNP probes and a gene chip. Target miRNAs, reporter probes, and AuNP probes were added to the system and allowed to conjugate to the chip via base pairing. The enhancement proceeds as an autocatalytic reaction: the AuNPs serve as nucleation sites to catalyze the reduction of Au ions to metallic Au, the reduction product is deposited on the chip, and the results are observed with the naked eye

RESULTS AND DISCUSSION

CONCLUSION

MATERIALS AND METHODS