New Systems Based On Cobalt-doped Graphene Quantum Dot Catalysis And Microchip Electrophoresis Assisted Chemiluminescence Signal Amplification And Its Application

Chemiluminescence(CL)analysis is an analytical detection technology with simple operation,wide linear range and low background noise.At present,this technology has been widely used in biochemical analysis,drug analysis and other fields.In the course of CL detection techniques,native enzymes are typically used to catalyze substrates to enhance CL strength.The natural enzyme has the advantages of high catalytic activity and strong specificity,and can obviously enhance the CL intensity.However,due to its poor stability and weak resistance to environmental interference,it limits their further application in life analysis.Synthetic nano-enzymes have the advantages of good stability,strong environmental resistance and low cost,and provide new materials for constructing high-efficiency CL analysis methods,and have been applied to optical biosensing analysis.Micro fluidic chip electrophoresis(MCE)technology has the advantages of high integration,low sample and reagent consumption,short analysis time and high resolution.The combination of MCE and CL analysis technology with low background interference(MCE-CL)is an effective strategy to improve the sensitivity of MCE detection.However,due to the small injection volume of MCE and the very small detection window,the sensitivity of this method is still not too high,thus limiting their application in the detection of trace substances in living organisms.In order to further improve the sensitivity of CL analysis and MCE-CL technology,a new type of cobalt-doped graphene quantum dot(Co-GQDs)nano-enzyme was designed and synthesized.A new method for the detection of uric acid by Co-GQDs catalyzed by luminol-hydrogen peroxide CL analysis.Then,based on MCE-CL and signal amplification technology,the DNA strands labeled with Horseradish Peroxidase(HRP)were used as signal molecules to construct two new MCE separation assisted CL signal methods for miR-21 detection.The research content of this thesis includes the following three aspects.First,a novel cobalt-doped graphene quantum dot(Co-GQDs)was designed and synthesized,and a new method for detecting uric acid by luminol-hydrogen peroxide CL was established by using Co-GQDs as nanozyme.The synthesized Co-GQDs have good stability,dispersibility and catalytic activity.Compared with natural enzymes,Co-GQDs have strong environmental resistance and good stability.The method was mediated by hydrogen peroxide for uric acid detection.The CL intensity and uric acid concentration showed a good linear relationship in the range of 0.1-3?mol/L.The detection limit of uric acid was 3.3×10^-8mol/L.The method has good selectivity,and common coexisting substances in the living body,such as lactic acid,ascorbic acid,urea,glucose,amino acids,etc.,do not interfere with the determination of uric acid.It is applied to the detection of uric acid content in human serum with satisfactory results.Then,by using horseradish peroxidase-labeled DNA(HRP-DNA)as a signal probe,the chemiluminescence(CL)reaction between luminol and hydrogen peroxide and the digestion reaction of T7 exonuclease,together with the advantages of microchip electrophoresis(MCE)separation and CL detection,we proposed a new strategy of cascaded chemiluminescence signal amplification based on microchip electrophoresis platform and established a simple ultra-sensitive MCE-CL assay to to detect trace amounts of miRNA-21 in cells.The linear range of miRNA-21 was determined to be 5.0×10^-151.0×10^-8mol/L,and the detection limit was 1.0×10^-15mol/L.It also shows the ability to recognize normal cells and cancer cells.The method has been applied to the determination of miR-21 in human breast cancer cells,bladder cancer cells and epithelial bladder cells,and the results show that the content of miR-21 in cancer cells is much higher than that of normal cells,and there is a significant difference in the content of miR-21 between different types of cancer cells.Therefore,this method has broad application prospects in the early diagnosis of tumors and biomedical research.Based on the MCE separation,the complementary hybridization of single-stranded DNA and the target cycle reaction of strand displacement,a new method for MCE separation-assisted,enzyme-free target loop CL signal amplification to detect miR-21 was established.The method uses HRP-labeled single-stranded DNA as a signal probe,and uses horseradish peroxidase(HRP)-labeled single-stranded DNA(HRP-DNA)to hybridize with P1 and P2 of single-stranded DNA to form HRP-DNA/P1/P2 complex.In the presence of the target miR-21,it hybridizes to the 5'end region of P1 and replaces HRP-DNA;after binding to the single-stranded DNA Fs,miR-21 and P2 can be replaced.The replaced miR-21continues to hybridize with the HRP-DNA/P1/P2 complex to achieve circular amplification.In this reaction,HRP-DNA and P1/P2/HRP-DNA complexes can catalyze the oxidation of Luminol by H₂O₂ to produce CL.In the microchip electrophoresis separation process,based on the difference in mass-to-charge ratio between the two samples,two peaks in tandem will appear in the electropherogram;the peak height of HRP-DNA can be used to quantify miR-21.The linear range of miRNA-21 was determined to be 6 pmol/L to 5 nmol/L,and the detection limit was4.49 pmol/L.The method has good selectivity,and miR-21 single base mismatched miRNAs such as SM-21,miR-141,miR-15 and miR-16 do not interfere with the determination of miR-21.It was applied to the spike recovery experiment of miR-21in human serum samples,and the recovery rate was 99.6-103%,indicating that the established method can be applied to the determination of miR-21 in complex biological samples.