Enzyme-linked Aptamer-Based Assays For The Detection Of Platelet-Derived Growth Factor-BB With Absorbance Analysis

Chapter 1:The definition, selection procedures and advantages of aptamer as well as the structure and role of platelet-derived growth factor (PDGF) were introduced briefly in this part. Different aptamer-based methods for the detection of PDGF-BB were reviewed. The research background, main research work and innovation of the thesis were described.Chapter 2:We developed an antibody and aptamer-based method for the detection of platelet-derived growth factor-BB (PDGF-BB) with absorbance analysis. The PDGF-BB was specifically captured by antibody coated on microplate, and then combined with biotinylated aptamer, forming a sandwich complex. The aptamer could bind with streptavidin-horseradish peroxidase (SA-HRP) through the interaction between biotin and streptavidin. Then the HRP catalyzed the conversion of a substrate (TMB) to a colored product, which was measured by absorbance analysis to achieve the quantitative detection of PDGF-BB. The liner relationship was obtained between the concentration of PDGF-BB and absorbance value in the range of 2.5 pM to 40 pM (y=0.056 x-3.14×10-4, R2=0.9992). This method took advantages of simplicity, high sensitivity, good selectivity and high recovery.Chapter 3:We described a double-aptamers based assay for the detection of platelet-derived growth factor-BB (PDGF-BB) with absorbance analysis. The biotinylated aptamer could be modified on the streptavidin coated microplate through the interaction of biotin and strptavidin. When the PDGF-BB was added, it could be captured by the aptamer specifically. The sandwich complex was formed after biotinylated aptamer applied. The aptamer could combine with streptavidin-horseradish peroxidase (SA-HRP) by the interaction between biotin and streptavidin. Then the HRP catalyzed the conversion of a substrate (TMB) to a colored product, which was measured by absorbance analysis to achieve the quantitative detection of PDGF-BB. The good liner relationship between the concentration of PDGF-BB absorbance value was achieved in the range of 12.5 pM to 400 pM. The liner equation was y=0.005x+0.03 (R2=0.998) with the detection limit of 5.8 pM. Besides, it could be achieved the detection of PDGF-BB in diluted bovine serum complex sample.Chapter 4:Taking advantages of aptamer and rolling circle amplification (RCA), we developed a sensitive absorbance method for the detection of platelet-derived growth factor-BB (PDGF-BB). Firstly, PDGF-BB was specifically captured by antibody coated on microplate, then combined with an aptamer connected with a primer sequence, forming a sandwich complex. The primer sequence was extended by RCA reaction to generate a long single stranded DNA with repeated copies. The amplified DNA product hybridized with many biotinylated short DNA probes, and then labeled with streptavidin-horseradish peroxidase conjugate (SA-HRP). The HRP catalyzed the conversion of a substrate to a colored product, which was measured by simple absorbance analysis to achieve the detection of PDGF-BB. By combining RCA and HRP catalysis amplification, sensitive detection of PDGF-BB with a good selectivity was obtained, the detection limit reached 3.1 pM with a linear detection range from 3.1 pM to 200 pM.