Colorimetric And Electrochemical Methods Based On Isothermal Nucleic Acid Amplification For The Detection Of MiRNA

Liquid biopsy,a non-invasive approach in early cancer diagnose,which could catch disease signs before conventional biopsy.Many efforts have been made in the understanding of miRNA as a promising biomarker for liquid biopsy.Currently,many new technologies,such as optical and electrochemical methods,have been widely exploited for the miRNA detection.In this article,with the in-depth study of isothermal nucleic acid amplification,several colorimetric and electrochemical detection strategies have been developed for the analysis of cancerous miRNA.This article includes two chapters.The first chapter introduces a colorimetric sensor for miRNA-21 detection.The second chapter constructs an electrochemical biosensor with dual signal amplification for the sensitive detection of miRNA-21.Chpater one:In this chapter,the primer exchange reaction cascades?PERC?is utilized to construct a G-quadruplex-hemin DNAzyme sensor for the miRNA-21detection.The principle is as follows.Befroe reaction,Pro was used to prevent the non-specific amplification by forming Pro/HA.When the target is added,miRNA-21could bind onto the toehold of Pro strand and branch migrate so that,the Pro spontaneously dissociate from HA,exposing the primer binding site.Then,with the help of Bst DNA polymerase,primer combines with HA and append the a domain onto the 3?end of P,forming P+a,the DNA sequence of a domain is TTAGGG.Because of the a domain of new primer P+a corresponds to the binding site?a*?of HB,which will initiate continuous addition of the a domain to the end of P+a,forming long chain ssDNA?P+a_n?.Finally,under proper conditions,synthetic sequences fold into G-quadruplex structure that the Hemin intercalates and becomes G-quadruplex-hemin DNAzyme.The G-quadruplex-hemin DNAzyme has horseradish peroxidase-like activity,could catalyze the oxidation of ABTS by H₂O₂.The consequence can be determinated by naked eyes.By integrating PER amplification and G-quadruplex DNAzyme together,this sensor enables specific and reliable detection of miRNA-21.The linear dynamic range was from1 pM to 5 nM with a LOD of 0.52 pM.In addition,the sensor is expected to provide a new road for miRNA research and cancer diagnosis.Chpater two:In this chapter,an double-cycle amplification electrochemical biosensor based on catalytic hairpin assembly?CHA?and PERC was developed for the miRNA-21 detection.The deteciton principle is:Firstly,hairpin DNA SH-H1 was assembled to the gold electrode by Au-S bond.In present of the target,the CHA reaction between SH-H1 and p-H2 was initiated via toehold-mediated strand displacement,forming a large number of p-H2/SH-H1 on the GE interface.Then,through PERC,the primer on the 3'end of p-H2 was extended to long ssDNA strand.Thus,the miRNA concentration could be determined by the number of DNA molecules at electrode surface.Meanwhile,Chronocoulometry?CC?was used to detect the charge signal by immersing modified electrode into RuHex buffer.Under the optimum conditions,the biosensor displayed a good linear range in the range of 10 fM¹00 pM,with the detection limit as low as 4.95 fM.In conclusion,this work provides a sensitive,reliable and specific method for the analysis of cancer related target.