Research For MicroRNA Detection Based On Au Nanoparticles And Surface Plasmon Resonance

MicroRNAs(mi RNAs) play vital roles in important processes of life course. Thus sensitive and specific detection of mi RNAs expression levels is very important. However, most sensitive detection methods for mi RNA involve the use of enzymes. Since the enzymatic activity is susceptible to be influenced by the environmental matrix, these enzyme-based methods require relatively clean samples for mi RNAs detection, which greatly limits their applications in complicated biological samples. In this paper, to solve this problem, we developed the following enzyme-free and sensitive methods for mi RNA detection:1. Combining Au nanoparticles(Au NPs) with cationic polymer, we developed a new method for mi RNA-21 detection. At low salt conditions, both single-stranded DNA and double-stranded DNA could prevent the aggregation of Au NPs. The colloid solution appeared as red color associated with dispersed nanoparticles. In the absence of mi RNA-21, the cationic polymer sequestered single-stranded DNA, so the single-stranded DNA was unable to stabilize the Au NPs against aggregation and meanwhile a red-to-blue color variation was observed in the colloid solution. In the presence of mi RNA-21, the complex of DNA-RNA was formed, which binded weakly binded to the cationic polymer. Thus the complex of DNA-RNA was still absorbed on the Au NPs to stabilize the nanoparticles against aggregation. The colloid solution remained the red color associated with dispersed nanoparticles. This method was label-free and simple in design, avoided the use of sophisticated instrumentations and the enzymes, etc. This method provided a colorimetric detection limit of 100 p M for instrument detection and 1 n M for naked eye observation.2. Based on Au NPs and DNA supersandwich, an enzyme-free and sensitive surface plasmon resonance(SPR) biosensor was developed for mi RNA-21 detection. Due to the electronic coupling between localized plasmon of Au NPs and the surface plasmon wave associated with Au film, as well as the enhancement of the refractive index of the medium next to the metal film caused by DNA supersandwich products, the shift of resonance angle was enhanced obviously. By employing the enzyme-free amplification strategy, as low as ca. 8 f M mi RNA-21 could be detected. Moreover, this assay also showed high selectivity toward single-base mismatch, and demonstrated its applicability for the target detection in human serum. This work may provide great potential applications in future clinical analysis of mi RNA.3. Combining Au NPs, DNA supersandwich and Ag nanoparticles(Ag NPs), a highly sensitive surface plasmon resonance(SPR) biosensor for the detection of mi RNA-21 and SMMC-7721 cell was developed. On the basis of the above work, SPR signal was further enchanced by embeding the positively charged Ag NPs into the DNA supersandwich structure. It could detect as low as 0.6 f M mi RNA-21. It could be used for the detection of mi RNA-21 in serum sample. By introducing the magnetic beads and aptamer, the method was also applied to the detection of SMMC-7721 cell.