| Hemoglobin as a binding protein exists in organism. Hemoglobin has been chosen for blood identification since this protein is specific for blood and present in large amounts. However, hemoglobins of most vertebrates have similar structures. Therefore, the analysis of different hemoglobins remains challenging due to the subtle differences in hemoglobin structures. A lot of hemoglobin-based assays have been used for species identification, including mass spectrometry (MS), immunochemical assays, high-performance liquid chromatography (HPLC), and Raman spectroscopy. However, immunochemical assays may exhibit cross-reaction with animal blood and suffer from reagent instability. MS, HPLC, and Raman spectroscopy are usually costly and time consuming. Sensing arrays have been widely used for discrimination and recognition of different proteins. In these sensing arrays, proteins were chosen according to different sizes and charges. It is urgently need to develop new methods for the dicrimination of hemoglobins with subtle differences.Aptamers, which are often called chemical antibodies. Compared with antibodies, aptamers exhibit a lot of advantages:A wide range of targets, structural diversity, good stability, easy in vitro synthesis, and easy tagging with other functionalities. They have great potential for sensing array. This thesis carried out the selection of aptamers by SELEX, and we investigated the properties of aptamers (Systematic Evolution of Ligands by Exponential Enrichment) for the discrimination of hemoglobins with subtle differences. The research contents including:(1) Selection of aptamers for human hemoglobinIn order to obtain the aptamers bound to human hemoglobin with high affinity, a SELEX strategy was carried out. Firstly, target was captured by hemoglobin antibody immobilized magnetic beads. Then, the ssDNA pool was mixed with magnetic beads. Finally, the DNAs bound to magnetic beads were seperated for PCR amplification, and PCR products were used for next round of selection. After 11 rounds of selection, the pools of the ninth and tenth rounds were cloned and sequenced. After the sequences analysis, repeated sequences were chosen and then classified into eight groups based on their similarity of secondary structure. Finally, eight aptamers were chosen for properties investigation.(2) Discrimination of hemoglobins with subtle differences using aptamers based sensing arrayBefore the construction of sensing array, eight aptamers were selected for their affinity and selectivity determination, and we got four aptamers which have good affinity to hHb. These four aptamers were 9th-1,10th-2,10th-6 and 10th-9, and their Kd values were 73.94 nM,85.34 nM,116.99 nM and 47.64 nM, respectively. Next, the four aptamers were used for the discrimination of hemoglobins with subtle differences, Fluorescence of FAM-labeled aptamers was firstly quenched by GO. When hemoglobins were added, their fluorescence was then recovered. Four aptamers exhibited different responses to five different hemoglobins. Combining with LDA, the discrimination of hemoglobins (human hemoglobin, bovine hemoglobin, porcine hemoglobin, ovine hemoglobin and mouse hemoglobin) with similar characteristics was achieved.(3) Determination of DNAzyme activity of aptamers of human hemoglobinWe investigated the DNAzyme activity of two G-rich sequences (9th-3 and 10th-2) obtained by SELEX. The results showed that these two G-rich sequences could bound hemin, efficiently catalyzing the H2O2-mediated oxidation of 2, 2'-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS). Besides, these two aptamers were further optimized, and the optimized sequences were compared with PW17 (widely used G-quadruplex sequence). Finally, we got two optimized G-quadruplex (9th-3.C and 10th-2.B) which showed better DNAzyme activity than PW17. |
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