Detection Of Proteins By Aptamer-based Capillary Electrophoresis And Fluorescence Switch

Chapter 1:The selection technologies and characteristics of aptamers,capillary electrophoresis assay and fluorescence analysis were briefly introduced.The research background,main research works,and innovation of the thesis were expounded.Chapter 2:Capillary electrophoresis coupled with laser-induced fluorescence（CE-LIF）analysis for detection of thrombin used a tetramethylrhodamine（TMR）-labeled29-nt aptamer as an affinity probe.A further detailed investigation of the effects of metal ions(e.g.Na⁺,K⁺,and Mg²⁺)in sample buffer on the peak area of the aptamer-thrombin complex was performed.Under the optimized conditions,we achieved sensitive detection of thrombin,the detection limit was 0.1 nM.This method showed good specificity and allowed the quantitative detection of thrombin in complex mitrix.Chapter 3:We have established a method for the detection of human neutrophil elastase（HNE）by CE-LIF assay using TMR-labeled aptamers as affinity probes.The HNE-aptamer complexes were well separated from the unbound aptamer probe in CE-LIF analysis.We conjugated a single TMR on different sites of aptamer,including 5’terminal,3’terminal,or the internal T bases of the HNE aptamer.The aptamer with TMR labeling on the 40th base T allowed to form large complex peaks.We investigated the effect of metal cations in sample buffer on the CE-LIF detection of HNE.The high concentration of K⁺or Na⁺caused reduction of peak areas of complex peaks,and the addition of Mg²⁺significantly decreased the complex peak.Under the optimized conditions,we achieved detection of 0.5 nM HNE.This assay enabled the detection of HNE spiked in diluted human serum samples.We also discussed the ratio of HNE to aptamer in the two complex peaks.Chapter 4:We introduced a polyT addition strategy to significantly enhancing affinity of the 15-nt thrombin aptamer（Apt15）.A stable aptamer-thrombin complex can be formed during CE-LIF analysis of thrombin.The addition of polyT having a length of at least 18T nucleotides on the 3’end of Apt15 greatly improved the binding affinity of the aptamer.It was likely that an additional interaction between polyT and humanα-thrombin occurred when the Apt15 section bound to thrombin,causing enhancement of binding affinity of aptamer due to these double interactions.The use of Apt15 with a T25 tail(Apt15-T₂₅)enabled CE-LIF detection of humanα-thrombin at concentrations as low as0.1 nM.The Apt15 part of Apt15-T₂₅ bound to the fibrinogen-binding site of humanα-thrombin and still maintained the capability to distinguish humanα-thrombin from humanβ-andγ-thrombin.In addition,by using this strategy the affinity of human immunoglobulin E（IgE）aptamers could be enhanced..Chapter 5:We developed a sensitive pyrene-based fluorescent aptamer switch for rapid IgE detection.The aptamer having four base pairs at the stem was labeled with a single pyrene molecule at the 3’end and 5’end and used as an affinity probe.The binding of IgE brought the pyrene moiety at each terminal of the aptamer into proximity,generating an excimer and showing a distinct fluorescence peak.The measurement of the fluorescence peak of the excimer allowed the sensitive detection of IgE.We optimized the experimental conditions such as temperature,incubation time,and metal cations.Under the optimized conditions,the detection limit for IgE was about 1.6 nM.This assay displayed good specificity and allowed for the detection of IgE spiked in the diluted human serum sample.