Study On New Method Of G-quadruplex/Hemin DNAzyme Based-Enhanced Microchip Electrophoresis Chemiluminescence Detection

Micrototal Analysis System(?-TAS)refers to the lab of chemistry or biology on the construction of a tiny chip.Networks of microchannels on the chip could control the fluid through the system,in order to achieve a variety of functions,result in the miniaturization and automation of experiments.Microchip electrophoresis(MCE)which combines the microfluidic technology with capillary electrophresis is an important part of ?-TAS.Under the control of program,MCE can do a series work automatically,such as sample,enrichment,separation and detection.At present,detection methods used for MCE are various,including electrochemical detection,mass spectrometry detection,fluorescence detection as well as chemiliuminescence(CL)detection,which has developed to be one of the reseach focuses because of its low background,high sensitivity and no external excitation light source.In the past,most MCE-CL platform were based on the targets' CL nature or reagents labeled with CL substance,leading to lots of limits of application and increasement of cost.However,with the development of nucleic acid aptamer and G-quadruplex/Hemin DNAzyme(G4),these bottlenecks of reseach would be broken through.The aim of this work is to establish a MCE-CL platform based on G-quadruplex/Hemin DNAzyme by combining nucleic acid aptamer,G-quadruplex/Hemin DNAzyme and Nucleic acid signal amplification technology.The main research contents as follows:Part 1:A micro analysis platform with chemiluminescence signal amplification based on Nicking endonuclease,G-quadruplex/Hemin DNAzyme and microchip electrophoresis for microRNA detection was developed.G-quadruplex/Hemin DNAzyme is a horseradish peroxidase micking DNAzyme can promote hydrogen peroxide to oxidize luminol for chemiluminescence detection and easy to conbine with Nicking endonuclease signal amplification(NESA).In this work,we established a NESA-G4-MCE-CL platform for microRNA analysis with miR-3 Ob as a model target.The levels of miR-30b in cell lysamples were determined in 90 s,with a limit of detection of 8.9 pmol/L for miR-30b.Part 2:Based on the NESA-G4-MCE-CL platform in part one,we developed a novel MCE-CL assay for two targets,with CEA and thrombin as two model targets.With the design of two different capture hairpin probes with aptamers of two targets,two signal amplification reactions could happen at the same time without interference.In the optimized condition,The levels of CEA and thrombin were determined,with a limit of detection of 0.11 ng/mL for CEA and 2.43 pmol/L for thrombin.Part 3:On the basis of G-quadruplex/Hemin DNAzyme and the specific binding of Biotin-Strepavidin,a novel assay of biotin was developed.The DNA fragment of G-quadruplex/Hemin DNAzyme was tagged with biotin.Biotin to be detected and biotin binding with DNA were mixed together with limited strepavidin and the limited binding sites were bound with two different biotins.At last,there would be two different kinds of G-quadruplex/Hemin DNAzyme,the free one and the one bound with strepavidin.With the increase of biotin to be detected,the free G-quadruplex/Hemin DNAzyme increased as well.According to the chemiluminescence signal of the free G-quadruplex/Hemin DNAzyme,the content of biotin would be determined.The levels of biotin were determined with a limit of detection of 6.41 nmol/L.