Detection Of Biological Molecules Based On HCR And LAMP Isothermal Amplification Techniques

As an important approach to improve the sensitivity of biochemical analysis,nucleic acid amplification methods are widely used in the fields of scientific research,clinical medicine and military forensic identification,which have attracted the attention of researchers.Especially in biochemical research,nucleic acid amplification methods are widely used in the detection and recognition of sensor molecules such as DNA,RNA,cells,proteins and other biomolecules.They can quickly and easily accumulate nucleic acid sequences and realize signal output and amplification so that increase the sensitivity of test substances.In the thesis,some novel biosensing platforms based on the hybridization chain reaction and loop mediated isothermal amplification have been established for a more sensitive and highly selective detection for nucleic acid,enzyme activity and the viral deseases detection.Compared with the traditional detection methods,the analysis platform established in this paper has the advantages of low experimental cost,high sensitivity,fast response and low detection limit.The feasibility and practicability of methods are preliminarily verified through the analysis of the actual test objects.The main contents of the thesis have been summarized as follows:Telomerase,a reverse transcriptase of the RNA-protein complex,promotes the extension of telomeres during chromosomal division and replication and maintains chromosomal integrity.In recent years,the study found that the detection of telomerase activity of tumor markers for the early clinical diagnosis of cancer,biomedical research,even telomerase-related anti-cancer drugs are of great significance.In Chapter 2,we used the Lipofectamine 3000 as the transfection reagent carrier that is less toxic and more efficient to achieve the hybridization chain reaction(HCR)signal on the cell cytoplasm to image telomerase in living cells.A novel biosensing fluorescence assay for detection of Telomerase Activity in Cells was proposed.The 3'end of the H1 hairpin probe is labeled with a Cy3 fluorophore as a signal probe whose fluorophore can be quenched by the BHQ2 quencher.In the absence of the target telomerase,the HCR primer sequence is blocked in the hairpin structure and does not trigger the HCR amplification reaction.With the addition of telomerase,the primer probe can continuously be extended at its 3' end to produce telomeric repeated sequences that are capable of hybridizing to H1 probe 5' end sequence and be utilized as an initiator sequence to trigger HCR in situ.As the HCR reaction progresses,amplification of the fluorescent signal is achieved,thereby further enabling efficient detection and visualization of telomerase activity in single cells.And detection limits down to 158 HeLa cells.The approach established in this chapter is simpler,more efficient and has good selectivity,which is expected to provide a favorable platform for the detection of telomerase-related drug screening,tumor biomarker precise therapy and early-stage cancer diagnosis in the future.Hepatitis C Virus(HCV)is a single-stranded RNA virus that causes chronic hepatitis and progressive liver fibrosis disease.Every year,nearly 3%of the people are infected with the HCV virus in the world,even 30%of patients eventually develop advanced liver cancer.In Chapter 3,we will take full advantage of BEAMing technology to design a more efficient,high-throughput droplet digital PCR approach that combines the superiority of loop-mediated isothermal amplification(LAMP)to monitor and analyze the Hepatitis C virus infection.BEAMing(beads,emulsions,amplification and magnetics)are technique based on PCR amplification with magnetic beads and microemulsions,which can be used to count millions of molecules.The chapter uses the magnetic beads in the droplet compartment to capture the copy sequence of the original target DNA molecule,and then amplificate in each individual emulsion compartment by utilizing the advantages of LAMP amplification technology such as high specificity and high sensitivity,After the step,each microemulsion reaction chamber contains a magnetic beads which bonded with thousands of copies of the target original DNA molecule.Then the millions of magnetic beads coated with target fluorescence products achieved from the LAMP amplification can be counted and analyzed within minutes using flow cytometry.Because each magnetic bead amplifies thousands of target molecules sequence,the signal to noise ratio achieved by hybridization is very high.Besides,the target DNA amplification in each individual droplet compartment increase the number of partitions that facilitates the resolution of low concentration differences between nucleic acid sequences in the sample.The approach established in this chapter will provide a more favorable template for high-throughput sequencing of target molecules in the future.