Bioanalysis Based On Isothermal Amplification Of Nucleic Acid

Isothermal amplification has been developed as a series of fast and efficient nucleic acid amplification methods which can be carried out at a constant temperature.Therefore,comparing to the polymerase chain reaction(PCR),isothermal amplification is more simple and does not need complex instruments to rapidly adjust the reaction temperature.Because of the advantages,isothermal amplification has been widely applied for point of care test and become the international research frontier in analytical chemistry.This dissertation is about developing novel analysis methods with nucleic acid isotherma l amplification to solve the chal enging problems in traditional bioanalysis and provide new ideas for the development of bioanalysis technology.The main contents and results are as follows:1.Quantification of miRNAs in blood can be potentially used for early disease detection,surveillance monitoring and drug response evaluation.However,quantitat ive and robust measurement of miRNAs in blood is still a major chal enge in large part due to their low concentration and complicated sample preparation processes typically required in conventional assays.Here,we present the ‘Integrated Comprehensive Droplet Digital Detection'(IC 3D)system where the plasma sample containing target miRNAs is encapsulated into microdroplets,enzymatically amplified and digitally counted using a novel,high-throughput 3D particle counter.Using Let-7a as a target,we demonstrate that IC 3D can specifically quantify target miRNA directly from blood plasma at extremely low concentrations ranging from 100 to 100,000 copies/mL in ? 3 hours without the need for sample processing such as RNA extraction.Using this new tool,we demonstrate that target miRNA content in colon cancer patient blood is significantly higher than that in healthy donor samples.Our IC 3D system has the potential to introduce a new paradigm for rapid,sensitive and specific detection of low-abundance biomarkers in biologica l samples with minimal sample processing.2.CRISPR/Cas9 is a highly efficient genome engineering tool,yet its off-target effect and the sequence-dependent cleavage activity across different sgRNAs remain major concerns for its application.Here,we propose a nicking triggered exponentia l amplification reaction(NTEXPAR),a fast and sensitive in vitro method,to detect the double strand DNA cleaved by down to 10 pM Cas9 with a linear range of 100 pM to 20 nM.With this newly developed amplification method,Cas9 cleavage activity can be quantified in 40 min and the optimal sgRNA design for specific target sequence can be successfully determined.Using the pre-screened sgRNA,we are able to distinguish single nucleotide mismatch in gene silencing experiment.This fluorescent based isotherma l assay provides a versatile tool for pre-screening of sgRNAs to achieve highly specific and highly efficient CRISPR/Cas9 genome editing.3.Chemically functionalized and nanostructured materials,which mimic the features of natural extracellular matrix,provide a method to organize cell surface receptors into nanoscale clusters and manipulate cell functions.However,the existing materials are mainly based on static structures.Herein,we developed a DNA based structure-switchable and multivalent material acting as ‘nano-spring',enabling reversible control of membrane receptor function at the cell surface.This ‘nano-spring' can be easily synthesized by rolling circle amplification and tuned by changing the circular template design.Using this ‘nano-spring' to interact with cells,we have demonstrated that the movement o f DNA nanostructure is sufficient to direct the cell morphology change from norma l morphology to numerous cell protrusions and affect the mRNA expression leve l of integrin related genes.