Ultrasensitive Chemiluminescence Detection In Capillary Electrophoresis With Aptamer And Gold Nanoparticles

Aptamers, first reported by three groups independently in1990, are the artificial single-stranded DNA or RNA sequences. Aptamers have a number of advantages such as high specificity, affinity, easier modification and functionalization. As antibody’s substitute, aptamer has wide perspective on veterinary clinic diagnose, targeted therapy, drugs detection.Capillary electrophoresis (CE), a micro-separation method with advantages of high performance, high speed and low cost, has been widely used in environmental detection, biochemical analysis, pharmaceutical analysis and food analysis. Chemiluminescence (CL) detection has been proven to be one of the most sensitive detection techniques, and the cost of the instrumental setup for CL detection is relatively low. Therefore, CL detection has become an attractive detection scheme for sensitive detection in CE. When coupled with CE, it can meet the demand of sensitivity and selectivity for the analysis of biological samples simultaneously.This thesis exploited a new approach for ultrasensitive detection of protein by capillary electrophoresis with chemiluminescence detection based on aptamer and AuNPs, which are listed as follows:1. A sensitive CE-CL method has been developed for the first time with aptamer as a new recognition reagent. Gold nanoparticles (AuNPs), as a biosensing platform, were conjugated with SH-aptamer to form AuNPs-aptamer. Using thrombin and thrombin binding aptamer as an initial proof-of-concept recognization pair, AuNPs-aptamer was linked to thrombin to produce an AuNPs-aptamer-thrombin complex. The resulted complex and unbound AuNPs-aptamer were separated in CE and detected with luminol-H2O2CL system. The developed strategy produced an ultrasensitive detection of thrombin down to13.5fmol/L (S/N=3) with a linear range from0.033to66.0pmol/L. The application of the present protocol was demonstrated by analyzing thrombin in human plasma samples with the recoveries of87.6-116.8%. This novel strategy has many outstanding merits including high specificity of aptamer, excellent catalysis behavior of AuNPs, high sensitivity of CL detection, and high separation efficiency of CE.2. An aptamer-based CE-CL method was proposed for the detection of IgE for the first time. The CL reaction of luminol-H202system could be strongly enhanced in the presence of AuNPs. SH-aptamer was conjugated with AuNPs to form AuNPs-aptamer. In the presence of human immunoglobulin E (IgE), the AuNPs-aptamer specifically recognized the protein to produce an AuNPs-aptamer-IgE complex. The complex could be effectively separated from the unbounded AuNPs-aptamer by capillary electrophoresis (CE) and sensitively detected with chemiluminescence (CL) system by taking the advantage of the excellent catalytic behaviour of AuNPs. The proposed method produced the ultrasensitive detection of human IgE with a lower detection limit of7.6fmol/L (S/N=3) and a wide linear range from0.025to250pmol/L. The application of the present protocol was demonstrated by analyzing IgE in human serum samples with the recoveries of94.9-103.2%. The excellent assay features of the developed approach were its specificity, sensitivity, adaptability, and very small sample consumption.