Studies On Novel Biosensor Based On Aptamer

Protein is the most abundant and important macromolecule in our body. Conventional methods for detecting protein are based on traditional immunoassay. Because the reactions of antigen-antibody are highly specific, immunosensors possess great sensitivity and selectivity. However, recent decades have seen the increasing interest in developing the method of detecting protein based on aptamer. The word, aptamer, comes from Latin aptus, to fit. Aptamers are selected from SELEX (systematic evolution of ligands by exponential enrichment) and have good recognition ability to proteins. The affinity and specificity between aptamers and protein is as well as those between antibodies and antigens. The dissociation constant of these is 10-9~10-12. Comparing to antibodies, aptamers own great advantages, including easy synthesis, good stability, ease modification and so on. It is facile to label the nucleic acid with different kinds of functional and report groups, such as fluorescence and electroactive groups. Therefore, it facilitates the immobilization of nucleic aptamer. In this article, we created a series of biosensors based on aptamers. The details are as following:(1) In the second chapter, we report a label-free electrochemical method to detect thrombin. In this work, we introduced a new kind of sandwich method to detect protein, utilizing antibody as the capturing probe, aptamer as the detecting probe and methylene blue as the electroactive substance intercalating in the nucleic acid, avoiding previous labeling. The sensor shows the linear response for thrombin in the range of 1-60nM with a detection limit of 0.5nM.(2) Based on the principle of antibody-aptamer sandwich in the second part, we reported a new kind of electrochemical sensor, utilizing the enzyme linked aptamer assay to detect immunoglobulin E (IgE). In this method, the biotinlynated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinlynated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then, silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry.(3) A label-free electrochemical sensor based on target-induced displacement is reported with adenosine as the model analyte. Owing to the high affinity of aptamer binding to target, aptamer can displace the DNA which has bound to the capturing probe on the surface of the electrode, leading to the decreasing signal of the electroactive MB.(4) We have designed a novel and simple fluorescence method to detect IgE. We labeled the aptamer with a fluorescence reporter group and its complementary DNA with a quencher (QDNA). After the aptamer reacting with IgE and QDNA, we measured the fluorescence intensity each step.