MicroRNA Analysis Based On Nanoparticle Tracking Analysis Technology

As a family of critical biomarker,microRNA(miRNA)widely exists in living bodies.Research shows that the abnormal expression of miRNA is closely related to various diseases.Therefore,the expression level of miRNA can be employed as a reliable index for disease diagnosis.Regarding this,researchers have devoted into the area of miRNA analysis.In recent years,due to the rapid development of nanotechnology,nanoparticlebased miRNA detection has become a research hotspot.However,the current nanoparticle-based detection methods generally depend on the characteristic of optical/electrochemical/magnetic properties for the nanoparticles.In addtion,a part of nanoparticles requires not only professional skills for the preparation of nanoprobes but also complicated surface modification,which greatly hampers the versatility of the nanoparticle-based detection.Nanoparticle tracking analysis(NTA)is an emerging technology for the direct and real-time visualization analysis of nanoparticles in the homogeneous solution.This technology is capable of providing accurate number-based concentration information of the nanoparticles without depending on specific characteristics of nanoparticles.In this thesis,for the first time,we proposed the NTAbased miRNA detection method.By establishing the quantitative relationships between the content of miRNA and the concentration of nanoparticles,the miRNA sensing strategy with high-versatility,convenience,and practicability is developed.The detailed information of the research work in this thesis is as follows:In the second chapter of the thesis,we made a series of explorations on establishing the NTA-based miRNA assay,and optimized several key parameters that affect the experimental design.First of all,we designed a miRNA analysis strategy by building up a quantitative relationship between the concentration of gold nanoparticles(GNPs)that is functionalized by a single nucleic acid and the miRNA content,in which the signal of the target miRNA is to be amplified by cycling enzyme cleavage reaction.Then we explored the on-nanoparticle single nucleic acid labeling technique,and proposed the scheme of adjusting the proportion of functional nucleic acids and blocking DNA to realize the single nucleic acid molecule label of the GNPs.Nevertheless,the experimental results showed that the scheme cannot be realized.Based on a series of experimental verification,we concluded that the failure of the scheme is mainly caused by the low concentration of functional nucleic acid probe,which makes the reaction difficult to be carried out in kinetics and it is difficult to ensure that all of the gold nanoparticles are modified.Thus,we adjusted the scheme to functionalizing the GNPs by multiple nucleic acid molecules.Afterward,we further explored the cycling enzyme cleavage involved in the experiment.The experimental results showed that DSN cannot perform cycling enzyme cleavage on the surface of GNPs.According to experimental analysis,we concluded that the reasons include the difficulty of DSN approaching functional nucleic acids,the spatial hindrance of blocking DNA,the detachment of nucleic acids from GNPs induced by heating,and the inhibition of enzyme activity of GNPs.In order to solve this problem,we adjusted the experimental strategy,and made the product hybridize with the nucleic acid probe on the functional GNPs after the cycling enzyme cleavage reaction in the homogeneous solution,and then made the follow-up reaction and quantitative analysis of miRNA.The verification experiment showed that the new design can achieve the quantitative analysis of miRNA.Finally,a feasible method for detecting miRNA based on NTA has been proposed.In the third chapter,a multiplexed miRNA detection method based on the integration of NTA and signal amplification by cycling enzyme cleavage is proposed according to the explorations in the second chapter.In this work,two kinds of DNA probes,Probe 1 and Probe 2 are rationally designed.Probe 1 is with biotin modification at the 5' end.Its 5' segment can hybridize with the target miRNA,and its 3' segment can hybridize with Probe 2 that is bound on the surface of GNPs.After the hybridization between Probe1 and the target microRNA,DSN recognizes and specifically cleaves the 5' segment of Probe 1 in the microRNA/Probe 1 duplex,releasing miRNA for the next-round hybridization and cleavage reaction.The residual 3'segment of Probe 1 will hybridize with Probe 2 on the surface of GNPs.In this way,the biotin molecule modified on the 5'end of Probe 1 will not be introduced to the surface of GNPs.On the contrary,in the absence of the target miRNA,DSN cannot recognize and cleave Probe 1,leading to the attachment of biotin to the surface of GNPs after the hybridization of the 3' segment of Probe 1 with Probe 2.After adding streptavidin-functionalized magnetic beads(STVMB s)to the system,the GNPs that have biotin molecules on the surface will be captured and separated.According to the design principle,with more miRNA added to the reaction,the number of residual GNPs in the reaction solution will show an increasing trend,which means the number variation of GNPs relative to the blank control sample is in positive correlation with target miRNA concentration.Furthermore,NTA can cluster nanoparticles of different materials or sizes with the help of the build-in software,enabling the method to detect multiple miRNAs at the same time.In this thesis,we have established a precise miRNA analysis strategy based on the quantitative relationship between the count of nanoparticles and the content of miRNA by using NTA.Unlike the previously reported methods,this strategy depending on no specific properties of the nanoparticles.This thesis provides new opportunities to establishing universal nucleic acid analysis methods as well as the clinical application of NTA.