Dissertation > Biological Sciences > Biochemistry > Enzymes

Human thrombin microfluidic chip electrochemical detection

Author YangWeiPei
Tutor YangPiYuan
School Fudan University
Course Chemical Biology
Keywords Microfluidic Chip Aptamer Electrochemistry Aptasensor
CLC Q55-3
Type Master's thesis
Year 2010
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In this work, a microfluidic chip-based aptasensor is presented for the direct detection of human thrombin. Based on PDDA/AuNPs multilayer assembly, the SH-Aptamer/thrombin system was constructed in the microfluidic channels. When H-D-Pro-Phe-Arg-p-aminophenol (PR-3-1-pAP) as an electrochemical probe was injected into the microchannel, with the catalysis of thrombin, the release of the electroactive pAP was monitored using the end-column three-electrode detection system. The intensity of catalytical current peak is linear with the concentration of thrombin. The aptasensor of thrombin shows a broad linear response range of 1pM to 100pM with a detection limit of 1 pM. The on-chip aptasensor is simple, sensitive and selective, which provides a great promise for determining and detecting ultra trace levels of biomarkers in the complex matrices.The detailed points of the thesis are described as follows:1. The introduction of microfluidic system and aptamers, and their applications in bioanalysisA review on Microfluidic systems and aptamers and their applications in bioanalysis is presented. This part includes:(1) Introduction of microfluidic system(2) Introduction of microfluidic fabrication technique and microfluidic detection technique(3) Application of microfluidic in bioanalysis(4) Introduction of Aptamer and its recent research progress(5) SELEX technologies(6) Application of aptamers in bioanalysis(7)Purpose and significance of this thesis2. Aptamer-Based microfluidc biosensor for sensitive thrombin detection in PMMA microchipIn this work, a sensitive and simple thrombin detective method was developed. Human thrombin was determined by using the combination of microfluidic system and aptamer protein specific binding. Thrombin also as known as the pro-thrombin is a coagulation protein in the blood stream and plays an important role in the coagulation cascade. Thrombin is a kind of serine protease which converts soluble fibrinogen into insoluble strands of fibrin, also catalyzing many other coagulation-related reactions. The concentration of thrombin in human blood should always maintain at a balance level:in human adults the normal blood level of thrombin activity has been measured to be around 1.1 units/ml. In this experiment, first, the poly diallyldimenthylmmonium chloride, as known as PDDA, and AuNPs multi-layers were assembled on the PMMA micro-channel to immobilize SH-aptamer. These assembled films have good thermal and mechanical stability. Next, the specific aptamer-protein interaction took place and thrombin was captured meanwhile. Lastly, an end-column three electrode system was employed to record the current response of p-AP. With the method, the detection limit was improved to 1pM, much lower than previous reports.

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