Preliminary Study On The Construction Of MicroRNA Nano-biosensor Based On Isothermal Amplification Reaction

The disordered regulation of some miRNAs is closely related to the development of various diseases.Therefore,the analysis of miRNAs would help early diagnosis and treatment of some diseases.Complex operations and expensive equipment are essential for miRNA traditional detection methods.These defects of the traditional methods are not suitable for early diagnosis of miRNA in underserved communities.Therefore,this research constructed the nano-biosensor platforms to realize the simple operation,high sensitivity,and strong specificity detection of miRNA based on nucleic acid isothermal amplification technology.This research realized quantitative detection of miRNA markers in vitro for cancer or degenerative diseases in underserved communities.The contents of the research were shown as follows:1.A bioluminescent magnetic nano-biosensor for ultra-sensitive miRNA detection based on Poly（A）polymerase isothermal tailing amplification:This chapter applied magnetic nanoparticles（MNBs）to couple stem-loop DNA（h DNA）through the reaction between streptavidin and biotin.The MNB-h DNA probes were prepared to achieve specific and rapid isolation of target miRNAs in the complex samples.Then,the 3′-hydroxyl terminus of the target miRNA was extended by a Poly（A）polymerase isothermal tailing amplification reaction to product the repeat adenine ribonucleotide sequence,and then the cycle bioluminescence generated by the AMP pyrophosphorylation-ATP dephosphorylation conversion reaction was collected.Bioluminescence signals for ultra-sensitive quantitative detection of target miRNAs.The results show that the sensitivity of the detection of miRNA-21 is as high as2.26×10^-17mol/L,and the detection time is 120 min.In addition,the sensor can realize reproducible and reusable detection in the same microwell to reduce analysis costs,but also directly detect miRNA-21 in serum without complicated extraction procedures.The detection results of miRNA-21 in whole blood samples of cancer patients were excellent consistency with that the results of q RT-PCR,which indicating that the sensor has potential for clinical application in miRNA detection.2.The AuNP rLFTS for rapid and portable detection of miRNA based on strand displacement isothermal amplification:In this chapter,gold nanoparticles（AuNP）of different sizes were prepared for the experiment.Stem-loop DNA labeled with black hole quencher 2（SH-h DNA-BHQ2）was modified to the surface of AuNP by gold-sulfur bonds for the preparation of AuNP@h DNA-BHQ2 probes,which can be complementary paired with the corresponding miRNA target.Then,the designed"Invading Stack Primer"（IS-Primer）isothermal chain displacement amplification reaction（ISAR）was activated after stem-loop structure of h DNA was destroyed by the hybridization of h DNA with miRNA.The activation of ISAR would release target miRNAs to achieve target circulation and signal amplification.Finally,a large number of AuNP@ds DNA-BHQ2 products labeled with biotin or digoxin were generated,which could react with the degsined test strips（rLFTS）.The change of the color intensity provided naked-eye detection of the target miRNA.At the same time,the fluorescent molecules Cy5/Cy3 coated on the test line were quenched by AuNP,which was achieved of quantitative detection of the miRNA target based on the change of fluorescence intensity.The results showed that the sensitivity of the rLFTS for miRNA detection was 3.42×10^-15mol/L,and the detection time was shortened to35 min.In addition,two neurodegenerative diseases miRNA-5010 and miRNA-331were detected successfully in whole blood samples of the patients with Parkinson’s disease by labeling different fluorescent molecules（Cy5/Cy3）to different T-lines.The quantitation results were basically consistent with those of q RT-PCR,which would be useful for the diagnosis and treatment of Parkinson’s disease.3.The rLFTS for sensitive and portable detection of miRNA based on ISAR coupled with CRISPR-Cas12a:In this chapter,AuNP with different sizes were prepared in Chapter 2.These AuNP were applied to label anti-digoxigenin antibodies.Then the AuNP@Digoxin-Abs probes were prepared for the fabrication of the strips.The corresponding h DNA and invading stack primer isothermal chain displacement amplification reaction（ISAR）were designed.When the target miRNA existed in the samples,the ISAR reaction would initiate and release the target miRNAs,and thus achieving the target cycle and signal amplification.This reaction generated a large number of double-stranded DNA products（ds DNA）.The ds DNA was recognized by CRISPR-Cas12a system,and then the"crazy-cut"mode of the Cas12a system was activated.It cut the single-stranded DNA sequences labeled with biotin and digoxin（Digoxin-ss DNA-Biotin）efficiently in the experimental system.The reaction liquid was reacted with the rLFTS.The reaction of the rLFTS generated the corresponding change of visible signals,and in the meantime,the fluorescence intensity of T-line was changed to generate the corresponding quantitative fluorescence signals.Dual signal amplification was achieved by the coupling of ISAR with CRISPR-Cas12a.The results showed that the sensitivity of the rLFTS of miRNA was as high as3.84×10^-17mol/L,and the detection time was only 90 min.Finally,this method was applied to detect miRNA-31 in saliva samples of oral cancer patients,and the detection results were in consistent with those of q RT-PCR,which indicating that this method could achieve non-invasive detection of miRNA in salivary samples in underserved communities.We also evaluated the stability of the CRISPR-rLFTS.The results showed that the CRISPR-rLFTS can be stored for more than 6 months.