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Studies on Electrochemical Aptasensor for Thrombin Based on HRP and Nanoparticles

Author: ZhaoJie
Tutor: ZhangYouYu
School: Hunan Normal University
Course: Analytical Chemistry
Keywords: Electrochemistry aptasensor Thrombin Gold nanoparticle Apoferritin Chitosan Enzyme amplification DPV
CLC: TP212.3
Type: Master's thesis
Year: 2011
Downloads: 98
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Abstract

Thrombin is the extracellular serine protease involved in the coagulation cascade, and it is thought to regulate many processes in inflammation and tissue repair at the vessel wall. In addition, Thrombin also has hormone-like properties, and it is involved in thrombosis and platelet activation. Therefore, thrombin plays a central role in a number of cardiovascular diseases. It is significant to analyze and examine thrombin in the field of medicine. Among various detecting methods, electrochemical biosensor technology has unique advantages such as high sensitivity, time/cost-effectiveness, and portability of measuring equipments and on line monitoring. With the high selectivity and affinity of aptamers as well as the amplification effect of Nano materials and enzymes, this thesis has developed a series of novel electrochemical aptasensors for highly sensitive detection. The main contents are summarized as follows:1. An ultrasensitive and highly specific electrochemical aptasensor for thrombin based on amplification of aptamer-gold nanoparticles-horseradish peroxidase (aptamer-AuNPs-HRP) conjugates has been developed successful. In this electrochemical protocol, aptamerl (Apt1) was immobilized on core/shell Fe3O4/Au magnetic nanoparticles (AuMNPs) and served as capture probe. Aptamer2 (Apt2) was dual labeled with AuNPs and HRP and used as detection probe. In the presence of thrombin, the sandwich format of AuMNPs-Apt1/thrombin/ Apt2-AuNPs-HRP was fabricated. Remarkable signal amplification was realized by taking the advantage of AuNPs and catalytic reactions of HRP. Other proteins, such as human serum albumin, lysozyme, fibrinogen, and IgG did not show significant interference with the assay for thrombin. Linear response to thrombin concentration in the range of 0.1-60 pM and lower detection limit down to 30 fM (S/N= 3) was obtained with the proposed method. This electrochemical aptasensor is simple, rapid (the whole detection period for a thrombin sample is less than 35 min), sensitive and highly specific, it shows promising potential in protein detection and disease diagnosis.2. Chitosan (CS) -Au nanocomposites have been prepared by chemical method, used to effectively immobilize aptamer and HRP on the surface, resulting in the high performance of CS-Au-HRP/Aptamer as sandwich-type amperometric aptasensor. The prepared CS-Au nanocomposites feature high load and activity of the enzyme label on the electrode surface with ultrasensitive signal readout. Using hydroquinone as the mediator, the proposed aptasensor could detect thrombin down to a concentration of 5.5 fM.3. The use of apoferritin as a novel and biocompatible carrier for stabilizing enzymes and the study of their activities have been completed. An amino-labeled aptamer2 probe and HRP are immobilized on the apoferritin by using glutaraldehyde as the cross-linker. Aptamerl was immobilized on core/shell Fe3O4/Au magnetic nanoparticles (Au MNPs) and served as the capture probe. In the sandwich format performed onto screen printed electrodes, a thrombin bound specifically to the aptamer probe. The experimental results demonstrate that linear response to thrombin was in the range of 0.8-100 pM, and lower detection limit was down to 0.17 pM (S/N=3), opening the possibility of a real application to diagnostics or medical investigation.

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