A New Method For Quantitative Detection Of Nucleic Acid Based On Single Particle Analysis

Nucleic acids,including deoxyribonucleic acid（DNA）and ribonucleic acid（RNA）,play critical roles in the storage,transmission and encoding of genetic information in live systems.Abnormal expression of nucleic acids is closely associated with various diseases,suggesting that nucleic acids might be important biomarkers for disease diagnosis and pathological analysis.However,the quantitative analysis of nucleic acid markers is challenging due to their extremely low concentration in biofluids and the interference of the complex environment in the organism.The commonly used methods for nucleic acid markers include quantitative polymerase chain reaction（qPCR）and nucleic acid microarray.Nevertheless,poor repeatability have been reported in qPCR analysis previously.The nucleic acid microarray analysis usually requires expensive equipment and complex fluorescent labeling strategies.Therefore,the development of robust methods with good reproducibility and strong anti-interference ability is significant for the analysis of nucleic acid markers.As a powerful technique,single particle analysis has been widely used in the dynamic monitoring of biomolecules in complex biological systems and the detection of disease-related biomarkers.In this work,two kinds of analytical methods for the quantification of DNA and miRNA based on single particle techniques were proposed.The detailed work is as follows:In Chapter 2,by integrating the plasmonic imaging microscopy with the classical"sandwich"detection strategy,a scattering brightness analysis-based method for quantitative detection of DNA markers was developed.Due to the plasmonic coupling effect,the target DNA induced aggregation of nanoprobes exhibits significantly increased scattering intensity,enabling the discrimination of aggregated nanoprobes under dark-field microscopy.Then we examined the dependence of aggregation-to-monomer ratio of the nanoprobes in the presence of target at different concentrations.A linear range from 1 to 750 pM with a detection limit of 1 pM was obtained,which is comparable to or much better than the previously developed non-amplification detection techniques.Given the advantages of high sensitivity and high compatibility with different amplification methods such as rolling circle amplification reaction（RCA）and hybridization chain reaction（HCR）,this method is promising to achieve the quantitative analysis of extremely low-abundance DNA or miRNA in body fluids.In Chapter 3,a ratiometric strategy for miRNA quantification was developed based on self-calibrated single particle tracking analysis and RCA reaction.Due to the fluctuating local viscosity and temperature of the microenvironment,nanoparticles often exhibit large heterogeneous diffusive behaviors,making it difficult to apply single-particle tracking technique for the quantitative detection of molecules in homogeneous solutions.To address this issue,a self-calibrated single particle tracking technique was introduced based on two-channel imaging of reference probe and detection probe simultaneously.The diffusive analysis indicated that the diffusion coefficient ratio for the two kinds of probes(D_dec/D_ref)was independent on the changes in viscosity as well as temperature of the solutions,indicating the self-calibrated single particle tracking technique might hold the properties such as high reproducibility and strong anti-interference ability.Then we proposed a ratiometric detection method for miRNA detection using the self-calibrated single particle tracking and RCA strategy.This method relied on the target sequence triggering the release of capture DNA,allowing the RCA reaction of primer sequence on the surface of the nanoprobes.Then a lowered diffusivity for the nanoprobe could be detected.A preliminary analysis suggested that the D_dec/D_refgradually decreased with increased of the target concentrations from 50 pM to 50 nM.To achieve satisfying sensitivity,the optimization of RCA reaction and application of lighter detection probes could be performed in the future.We predicted that this ratiometric diffusive analysis based detection strategy,with its high reproducibility and strong anti-interference ability,holds promise for practical detection of micro RNA markers.