Aptamer Probe-based Methodologies For High Sensitive Protein Detection

The research on methodologies of high-sensitive protein detection is one of the most important tasks in biomedical diagnostics as well as disease-related mechanism investigation, it also greatly benefits the rapid development of proteomics. Most of current analytical techniques for proteins are employing antibodies as specific capturing probes. However, Several disadvantages, such as the generation and modification precedures for antibodies are laborious, the performance of the same antibody tends to vary from batch to batch, and less stability of antibodies prevent the development of novel protein detection methods to some extent.Aptamers are a short single-strand DNA (ssDNA) or RNA that can form definite tertiary structures and bind to their targets by complementary shape interactions. Aptamers are generated by an in vitro selection process called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) which was firstly introduced in 1990. As a kind of novel functional molecules, the molecular recognition formats of aptamer-target are similar to antibody-antigen. Further, the affinity between aptamers and their targets are sometimes superior to antibodies-antigen. In addition, aptamers with extreme stability can be produced by in vitro chemical synthesis, and various functional groups can be easily attached to aptamers at precise locations. Aptamers can thus serve as the alternative biorecogition molecules for novel analytical techniques for protein researches.The objective of this research is to develop novel aptamer probe-based methodologies for protein analysis. Thrombin-aptamer(s) as a model system is employed in this thesis. Three chapters are included.Chapter 1 summarized recent advances of aptamer probes on protein separation and sensing fields, with special emphasis on aptamer probes in the applications of APCE (Affinity Probe Capillary Electrophoresis) and fluorescent sensors.In Chapter 2, an aptamer-based APCE system was successfully established for high-sensitive thrombin separation and quantitative analysis. The carboxyfluorescein labeled 29-mer anti-thrombin aptamer (F29-mer) was adopted as an affinity probe in APCE with laser-induced fluorescence detection (LIF). The determination of thrombin can be followed by the different CE behaviors of thromin-aptamer complex and free aaptamers. Under optimized conditions, the linear range for thrombin in buffer was from 0 to 400 nmol/L and the limit of detection (LOD) was 2 nmol/L, which is the lowest one than those achieved by previously APCE methods but based on a 15-mer aptamer. Moreover, this established APCE system has high specificity that about 10-fold common proteins, BSA, HSA, IgG and lysozyme did not interfere with the observation of thrombin-aptamer complex. Comparing to peak height of complex, the relative mobility/relative migration time of this conjugate should be considered as a more important feature for binding specificity in such a CE separation method. The APCE method was further employed for thrombin analysis in 5% (v/v) human serum and the LOD were same as that in buffer. In additon to this determination method, this established APCE system has also be used for investigation on binding behaviors of individual/both aptamers to thrombin. The non-fluorescent aptamer competition/binding experiments revealed that a 15-mer-thrombin-29-mer ternary complex with a middle mobility can be formed when both aptamers were coexisted in the free solution with thrombin, and the CE behaviors of complex is directly influenced by the biological functions of thrombin. The technique of aptamer-based APCE offers a promising way for the rapid, selective and sensitive detection of proteins and an efficient approach for the investigation of nucleic acid-protein interactions.The verification of non-covalent labeled fluorescence sensing systems were descirbed in Chapter 3. two kinds of dsDNA intercalating fluorescent dyes, [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanlinthroline; dppz = dipyrido [3,2-a:2',3'-c] phenazine) and BOBO-3 (2,2'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl -1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-benzo[d]thiazoe) were employed in this chapter, and two non-covalent labeled fluorescence sensing systems for thrombin determination were successfully developed and compared based on its 29-mer aptamer. Under the optimized condition, the linear range of [Ru(phen)2(dppz)]2+ and BOBO-3 system in buffer were from 0 to 20 nmol/L and 0 to 40 nmol/L, respectively. The LOD were 100 pmol/L and 280 pmol/L, respectively. Specific studies showed that four widely existed proteins in biological fluids did not interfere thrombin detection. Quantification of thrombin in 1% (v/v) human plasma was also achieved by employing BOBO-3 detection system and the linear range and LOD were 0 - 30 nmol/L and 300 pmol/L.In conclusion, two aptamer-based protein analytical methodologies have been investigated with high sensitivity and specificity for thrombin detection. On the basis of high separation efficiency of CE, APCE makes itself more suitable to protein analysis in biological samples while a super resolution between free aptamer and its target-aptamer complex is achieved. Besides, APCE method can be also considered as an important approach for investigation on binding behaviors among nucleic acid and protein. Free-of-labeling detection can be obtained from a non-covalent labeled fluorescence sensing system that coupled to aptamers. The covalent labeling procedures for aptamers are thus not necessary and the affinity decay between aptamers and its targets are prevented.