Visualization Detection Of MiRNA By Isothermal Amplification Coupled With Cationic Conjugated Polymers

Micro RNA(miRNA)plays an important regulatory role in the life activities,and its expression in organisms is related to the occurrence and development of diseases.The development of simple and sensitive miRNA detection methods is vital to early diagnosis and treatment of diseases.In this thesis,catalytic hairpin assembly(CHA)and hyperbranched rolling circle amplification(HRCA)technology were used to develop two new methods for visualization detection of miR-221 through the design of different fluorescence quenching probes in combination with cationic conjugated polymer(CCP)homogeneous biosensor technology.The details are as follows:1.Fluorescence visualization detection of miR-221 by combining CHA technology with CCP homogeneous biosensing.Firstly,hairpin probes P1 and P2 are designed for the target miR-221 sequence.The 5’ partial sequence of P1 probe is complementary to the target miRRNA sequence.The two ends of the P2 probe were labeled with Black Hole Quencher(BHQ1)and Fluorophores(FAM),which were close to each other,and the fluorescence of FAM was quenched by BHQ1.When there is a target miRNA in the reaction solution,the double-stranded structure of P1 will be opened and P1 and P2 probe will be hybridized to form a P1P2 double-stranded DNA product while replacing the miRNA sequence.After being replaced,miR-221 will trigger the hybridization reaction between P1 and P2,form CHA amplification,and produce a large number of P1P2 double-stranded DNA products.BHQ1 and FAM groups in product P2 are far away,resulting in fluorescence release.When being added to the solution,CCP will combine with double-stranded P1P2 through electrostatic interaction,and a high efficient fluorescence resonance energy transfer(FRET)occurs from CCP to FAM.Irradiated by ultraviolet lamp,the solution shows green fluorescence.In comparison,although CCP can also combine with P2,effective FRET cannot occur between CCP and FAM because of the quenching state of P2 itself.The scheme of the detection range is 5-100 pM and the detection limit(3s)is 4.5 pM.In combination with CHA amplification and CCP fluorescence biosensing,the sensitivity of miR-221 detection was improved,and the signal can be read out via visualization,which was easy to operate at a low cost so as to provide a new idea for the research of real-time detection technology.2.Fluorescence visualization detection of miR-221 by combining hyperbranched rolling circle amplification(HRCA)with CCP.First of all,we design the forward and reverse primers in HRCA reaction.The reverse primer is a double-stranded fluorescence quenching switch probe,whose 5’(BHQ1-labeled)partial sequence is complementary to a short-stranded DNA labeled with FAM at 3’;And a padlock probe(PLP)is designed to be completely complementary to the target miR-221 sequence.When the target miR-221 exists,PLP is ligated into a ring under the action of RNA ligase.By adding forward and reverse primers,HRCA is triggered.In the reaction,the FAM-labeled DNA strands in the reverse primers will be continuously replaced and released into the solution.After the HRCA reaction,CCP is added and combined with the released FAM-labelled DNA to produce high efficiency FRET,and emit green fluorescence under UV light.According to the fluorescence transfer efficiency and the visualization green fluorescence signal,miR-221 can be detected simply and sensitively.The detection range is 25 f M-1 pM and the detection limit(3s)is 18 f M.In this method,double-stranded fluorescence quenching switch probe is designed as reverse primer to trigger HRCA,in combination with CCP fluorescence biosensing,the blue light background can be reduced,which makes it easier for visualization detection.