Study On New Systems For Chemiluminescence Signal Amplification Assisted By Microchip Electrophoresis Separation

Microchip electrophoresis（MCE）is a new separation analysis technique developed by capillary electrophoresis.It is one of the earliest applications of micro-analysis system concept（μ-TAS）and it integrates chemical reaction,electrophoretic separation,and signal detection.MCE has the advantages of low cost,simplicity,rapid analysis,short time,and small sample consumption.It can be used in biomedical research,environmental testing,clinical analysis and other fields.However,because the detection window of MCE is very small,the sensitivity of this method is not high,thus limiting their application in the detection of trace substances in biological samples.At present,the detection methods for microfluidic chip electrophoresis systems include electrochemical detection、mass spectrometry detection、fluorescence detection and chemiluminescence detection.Microfluidic chip electrophoresis-chemiluminescent detection（MCE-CL）has been used to detect nucleic acids,proteins and biologically active small molecules.In addition to using the catalytic chemiluminescence property of the analyte itself,the conventional MCE-CL also markers of horseradish peroxidase（HRP）on antigens and antibodies for chemiluminescence detection.With the advent of nucleic acid aptamers and G-quadruplex/Hemin DNAzyme（G4）,chemiluminescent detection can be performed using G-quadruplex/Hemin DNAzyme.In addition,it is also possible to label isoluminol（ABEI）as a molecular beacon on the DNA strand and horseradish peroxidase（HRP）as a signaling probe on the DNA strand.However,the introduction of HRP-DNA and aptamers in the MCE-CL platform is rarely reported.Based on the MCE-CL platform,three different microchip electrophoresis separation-assisted chemiluminescence signal amplification detection systems were constructed using labeled HRP-DNA strand as signal molecular probe combined with nucleic acid signal amplification technology,and applied to the quantitative analysis of p53 gene,HIV-DNA and S1 enzymes.The main contents of this work can be concluded as followings:Part 1:Based on the MCE-CL platform,using DNA hybridization reaction of horseradish peroxidase-labeled DNA（HRP-DNA）,combined with T7Exo-assisted signal amplification,a new method for MCE-assisted chemiluminescence signal amplification was established and applied to the high p53 gene Sensitive detection.The method uses HRP to catalyze the chemiluminescence reaction of luminol and hydrogen peroxide,and utilizes T7Exo cleavage to assist chemiluminescence signal amplification to achieve highly sensitive detection of the target.The injection,separation and detection processes were completed within 1.0 min under optimized electrophoretic separation and chemiluminescence reaction conditions.When the concentration of p53 gene was in the range of 9.0×10^-12mol/L～2.0×10^-8mol/L,the chemiluminescence intensity and the logarithm of concentration showed a good linear relationship and the detection limit of target p53 gene could reach 4.8×10^-12mol/L（S/N=3）.The method was applied to the analysis of spiked recovery of human normal hepatocyte lysates.The recoveries were between 100% and 113%,indicating that this method can be used for the detection of trace p53 gene in complex biological samples.Part 2:A micro analysis platform with chemiluminescence signal amplification based on T7Exo、HRP-DNA and Microchip electrophoresis for HIV-DNA detection was developed.By designing a two-cycle procedure,the target HIV-DNA was double-cycle amplified and ultra-sensitive detection of HIV-DNA was achieved.Under optimized experimental conditions,the concentration of HIV-DNA was in the range of 1.0×10^-14mol/L～5.0×10^-9mol/L,the chemiluminescence intensity and the logarithm of concentration showed a good linear relationship and the detection limit of target HIV-DNA could reach 1.6×10^-15mol/L（S/N=3）.It was applied to the spike recovery analysis of human serum,and the recovery rate was between 93%and 103%,indicating that this method can be used for the detection of trace HIV-DNA in complex biological samples.Part 3:Based on HRP-DNA as a signal probe,we developed a novel MCE-CL platform for S1 enzyme activity.The platform uses HRP to catalyze the chemiluminescence system of luminol and hydrogen peroxide,and uses MCE for rapid separation of samples to achieves a highly sensitive detection of the target within 1 minute.Under optimized experimental conditions,the concentration of S1 nuclease was in the range of 3.0×10^-4U/ml～1.0×10^-1U/ml,the chemiluminescence intensity and the logarithm of concentration showed a good linear relationship and the detection limit of target S1 nuclease could reach 1.56×10^-4U/ml（S/N=3）.The method was applied to the analysis of spiked recovery of human serum and the recovery rate was between 98%and 105%,indicating that this method is also suitable for the detection of complex biological samples.The inhibitory effects of the two inhibitors of ATP,PPi on the S1 enzyme were also investigated.The results show that this method can be used for the screening of S1 enzyme inhibitors.