Combination Of The Gold Nanoparticles-based Enzyme-free Amplification And Chemiluminescence For The Detection Of MicroRNA

As a new type of biomarker,microRNAs?miRNAs?are not only involved in gene regulation in organisms,but also closely related to the emergence of major diseases such as cancer.Therefore,it is vital to develop new methods for the accurate and sensitive detection of miRNAs.In this paper,we develop two new methods for the detection of miRNA by combining catalytic hairpin DNA assembly?CHA?and hybridization chain reaction?HCR?with chemiluminescence detection.The main studies are as follows:1.Combination of the Au nanoparticles?AuNPs?-based CHA and luminol chemiluminescence,we developed a novel method for miRNA detection.Firstly,two hairpin DNA probe?P1?modified with sulfhydryl group?-SH?and the P2 labeled with biotin were designed.Afterwards,P1 was modified on the surface of AuNPs.When the target miRNA was present,it can hybrid with the part sequences of P1,generating the P1-miRNA intermediates that can open the hairpin P2.As a result,the double strand P1-P2 was formed.Meanwhile,the miRNA was released and subsequently hybrided with the other P1 probes on the surface of AuNPs to further trigger the formation of P1-P2,leading to the CHA and the enrichment of lots of biotin-labeled P2 on the surface of AuNPs.In terms of the interaction between streptavidin?STV?and biotin,horseradish peroxidase conjugated STV?STV-HRP?can aggregate on the AuNPs.After centrifugalization,the HRP on the AuNPs can further catalyze the chemiluminescence of H₂O₂-luminol for carrying out the miRNA detection.The method showed a good linear relationship with the concentration of miRNA from 5 pM to 100 pM.The detection limit was estimated as 1.7 pM.The method is simple,low cost,and sensitive,and provides a new CHA-based strategy for the detection of miRNA.2.By integration of the HCR and chemluminescence detection with the AuNP as a carrier,a novel and sensitive method for miRNA let-7a detection was developed.Briefly,three hairpin probes H1,H2 and H3 were designed and synthesized.The hairpin probe H1 was modified with-SH.The hairpin probe H2 was labeled with biotin.H1 was first modified on the surface of AuNPs through the Au-S bond.In the presence of target let-7a,it can hybridize with H1 modified on the AuNPs and make the hairpin structure of H1 opened.Then the opened H1 with the initiator sequence for H2 can trigger a cascade of HCR events between H2 and H3.Subsequently,the STV-HRP can bind to the HCR products through the specific interaction between biotin and STV.Ultimately,with the chemiluminescence reaction of HRP-luminol-H₂O₂,the target miRNA can be detected by the change of the chemluminescence intensity.This assay provided a sensitive detection of miRNA from 5 pM to 100 pM with a relatively low detection limit of 0.13 pM.