Research On The Pneumatic Real-time Detection Method For Sensitive Detection Of MicroRNA

microRNAs(miRNAs)are a class of endogenous non-coding small-molecule single-stranded RNAs containing approximately 22 nucleotides(nt)and are ubiquitous in eukaryotic cells.miRNAs can regulate the activity of more than 50%of human coding genes.It plays a vital role in the biological processes,including cell differentiation,apoptosis,proliferation and immunity.As a potential target for disease diagnosis and clinical prediction,up to date,most of the methods used routine analytical instruments to detect miRNAs.However,the requirement of expensive equipment and professional made them hard to achieve point-of-care testing(POCT).POCT is a technology that can detect sample on site without professional by using portable equipment.Therefore,it is of great importance to develop a simple,fast,sensitive and reliable POCT method to detect miRNAs.The gas pressure is a common physical parameter.The portable pressure meter is easy to use and can be used without professional person.Therefore,gas pressure based bioanalytical methods has great advantages for developing novel POCT method.Herein,we have developed two gas pressure based POCT methods for the detection of miRNAs,which are aimed at realizing the simple,sensitive and reliable detection of miRNAs.The main contents of this paper can be summarized as follows:1.Strand displacement reaction based signal amplification for ultrasensitive POC detection of microRNA via gas pressure as readoutAn efficient,rapid and sensitive POCT method for microRNA21(miR-21)detection was developed based on portable pressure meter.Two hairpin probes,H1 and H2 were designed and modified with magnetic beads(MBs)and platinum nanoparticles(PtNPs)to form MBs-H1 and PtNPs-H2 complexes,respectively.In the presence of miR-21,the cyclic strand displacement reaction(SDR)between H1 and H2 is triggered,and the double strands of MBs-Hl/PtNPs-H2 will continue to form,then many PtNPs are enriched on the surface of MBs.Finally,MBs-H1/PtNPs-H2 complexes are added into the sealed wells of 96 well plate containing H2O2.PtNPs catalyze the decomposition of H2O2 to produce O2,resulting in a significant gas pressure in the sealed wells.The portable and digital pressure meter is used to detect the pressure for realizing the quantitative detection of miR-21.There is a good linear relationship between the gas pressure value and the logarithmic value of miR-21 concentration in the range of 10 fM to 10 pM,and the detection limit is 7.6 fM.Moreover,the specificity and reliability of the method are ensured by the hairpin DNA probes and magnetic separation.At the same time,single base mutation is easy to identify,and miR-21 in serum samples can be detected sensitively.Therefore,this method is simple,rapid,reliable and super-sensitive.It has important theoretical and practical significance for the development of miRNA POCT.2.Point-of-care and simultaneous detection of multiple miRNAs via gas pressure readoutCompared with single biomarker detection,multi-biomarker detection not only improves the accuracy of cancer diagnosis,but also has potential advantages in improving detection efficiency,simplifying analysis procedure and reducing cost.Therefore,reliable detection of multiple biomarkers is of great importance for early diagnosis of cancer.Herein,a gas pressure based POCT method was developed for simultaneous detection of multiple miRNAs by using multichannel paper chips to immobilize capture DNA probes and ring-oven washing to reduce the washing and separation.Firstly,four kinds of miRNAs capture probes which associated with lung cancer were immobilized in different regions of the paper chips.In the presence of target miRNAs,the capture probe and miRNAs were partially hybridized by base complementary pairing reaction.At the same time,the PtNPs modified cDNA hybridized with the other part of miRNAs to form a stable sandwich structure,which enriched a large amount of PtNPs on the surface of the paper chip.Then unreacted sample and PtNPs-cDNA were washed by ring-oven washing to avoid non-specific adsorption.Finally,the different regions of paper chips were cut and added into the sealed wells of 96 well plate containing H2O2.PtNPs catalyzes the decomposition of H2O2 to produce a large amount of O2.Different miRNAs were detected simultaneously by using portable pressure meter.The ring-oven washing greatly simplifies the washing operation and realized simple and fast multi-channel simultaneous washing.Therefore,it provides a simple and novel gas pressure based POCT for the simultaneous detection of miRNAs.