Sensing Nucleic Acids Based On DNA Nanotechnology And Catalytic Nucleic Acids With Chemiluminescence

With the gradual reveal of the role of circulating tumor DNA,circulating microRNA and fusion genes in the occurrence and treatment of diseases,cell-free nucleic acid analysis in biological research and medical treatment becomes increasingly important.This paper intends to construct a series of simple,rapid,isothermal,specific,sensitive and label-free chemiluminescence sensor for nucleic acids detection to meet the needs of clinical diagnosis by integrating the latest research results of clinical laboratory diagnosis and bioanalytical chemistry and taking catalytic nucleic acid and DNA self-assembly as nanometer tools.This dissertation mainly contains three parts as follows: 1.Chemiluminescence imaging for microRNA detection based on cascade exponential isothermal amplification machineryA novel G-quadruplex DNAzyme-driven chemiluminescence(CL)imaging method was developed for ultrasensitive and specific detection of circulating miRNA based on the cascade exponential isothermal amplification reaction(EXPAR)machinery.A structurally tailored hairpin probe switch was designed to selectively recognise miRNA and form hybridisation products to trigger polymerase and nicking enzyme machinery,resulting in the generation of product I,which was complementary to a region of the functional linear template.Then,the response of the functional linear template to the generated product I further activated the exponential isothermal amplification machinery,leading to synthesis of numerous horseradish peroxidase mimicking DNAzyme units for CL signal transduction.The amplification paradigm generated a linear response from 10 fM to 100 pM,with a low detection limit of 2.91 fM,and enabled discrimination of target miRNA from a single-base mismatched target.The developed biosensing platform demonstrated the advantages of isothermal,homogeneous,label-free and visual detection for circulating miRNA assays,offering a promising tool for clinical diagnosis.2.Bis-three-way junction nanostructure and DNA machineries for ultrasensitive and specific detection of BCR/ABL fusion gene by chemiluminescence imagingA novel G-quadruplex DNAzyme-driven chemiluminescence(CL)imaging method has been developed for ultrasensitive and specific detection of BCR/ABL fusion gene based on bis-three-way junction(Bis-3WJ)nanostructure and cascade DNA machineries.By NUPACK software,Bis-3WJ probes are designed logically to recognize BCR/ABL fusion gene,which forms the stable Bis-3WJ nanostructure for the activation of polymerase/nicking enzyme machineries in cascade,resulting in synthesis of DNAzyme subunits.These DNAzyme subunits can form integrated DNAzyme by self-assembly to catalyze CL substrate,thus providing an amplified signal for the sensing events or outputs for AND logic operation.The imaging method achieved ultrasensitive detection of BCR/ABL fusion gene with a low detection limit down to 23 fM.And this method exhibited wide linear ranges over seven orders of magnitude and excellent discrimination ability toward target.In addition,an acceptable recovery was obtained in complex matrix.It is notable that this biosensing strategy possesses merits of homogenous,isothermal and label-free assay system.Therefore,these merits endow the developed imaging method with a potential tool for CML diagnosis.3.Triggerred hairpin switch and in situ nonlinear hybridization chain reaction enabling label-free electrochemiluminescent detection of BCR/ABL fusion geneA highly selective and ultrasensitive strategy for lable-fre electrochemiluminescent(ECL)detection of BCR/ABL fusion gene via triggered hairpin switch and in situ nonlinear hybridization chain reaction(HCR)signal amplification is presented herein.The DNA capture probes are firstly self-assembled onto a gold electrode.Then,a droplet of mixture containing nonlinear HCR system is droped on the electrode surface,in which a trigger DNA consisted in the haipin switch initiates self-sustained assembly of double-stranded substrates into dendritic nanostructures.The presence of BCR/ABL fusion gene leads the open of hairpin switch and the growth of dendritic nanostructures onto electrode surface.The in situ,nonlinear HCR-generated nanostructures introduce the intercalation of numerous ECL signal indicators(Ru(phen)32+)into the dsDNA grooves,resulting in exponential amplification of ECL signal output.By introduction of hairpin switch and nonlinear HCR to this sensor design,both the selectivity and sensitivity have been excellently enableded.This developed biosensor achieves a linear range of 10 fM to 1 nM with low detection limit of 5.49 fM towards BCR/ABL fusion gene detection,and the distigush of target fusion gene from normal BCR gene and ABL gene,logically.In addition,this method has the advantages of non-enzyme,low cost and label-free.These advantages make the sensor a promising contender in the determination of fustion gene for medical diagnostics.