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Determination of the platelet-derived growth factor BB by a competitive thrombin-linked aptamer-based Fluorometric assay
| Content Provider | Semantic Scholar |
|---|---|
| Author | Guo, Limin Zhao, Qiang |
| Copyright Year | 2016 |
| Abstract | AbstractThe authors describe a competitive aptamer based assay for detection of the platelet-derived growth factor BB (PDGF-BB; used as a model protein). The assay is making use of thrombin (a serine protease) as an enzyme label for reporting signals. It is taking advantage of a highly selective aptamer and of the fairly specific enzymatic activity of thrombin in terms of cleaving artificial fluorogenic peptide substrates. In a first step, the surface of wells of microplates is coated with PDGF-BB. On addition of a sample containing PDGF-BB, free and bound PDGF-BB compete with each other for binding to a DNA probe that consists of an aptamer sequence for PDGF-BB and a 29-mer aptamer sequence for thrombin. After washing, thrombin is added and will attach to the DNA probe that bound to the PDGF-BB on the microplates. Following addition of a fluorogenic peptide substrate, the bound thrombin will catalyze the cleavage of the substrate to generate a fluorescent product whose fluorescence intensity is measured at excitation/emission wavelengths of 370/440 nm. Fluorescence intensity decreases with increasing PDGF-BB concentration in the sample because less thrombin will bind to the PDGF-BB coated surface of the microplate. Under optimal conditions, PDGF-BB can be quantified in the 0.125 to 3 nM concentration range. This assay was successfully applied to the determination of PDGF-BB in spiked 100-fold diluted human serum. Graphical abstractIn a competitive thrombin-linked aptamer assay, free platelet-derived growth factor BB (PDGF-BB) sample competes with the PDGF-BB coated on microplates for binding to a DNA probe containing PDGF-BB-binding aptamer and thrombin-binding aptamer. The labeled thrombin cleaves substrate into product, achieving signal generation. |
| Starting Page | 3229 |
| Ending Page | 3235 |
| Page Count | 7 |
| File Format | PDF HTM / HTML |
| DOI | 10.1007/s00604-016-1978-1 |
| Alternate Webpage(s) | http://ir.rcees.ac.cn/bitstream/311016/35834/1/Determination%20of%20the%20platelet-derived%20growth%20factor%20BB%20by%20a%20competitive%20thrombin-linked%20aptamer-based%20Fluorometric%20assay.pdf |
| Alternate Webpage(s) | https://doi.org/10.1007/s00604-016-1978-1 |
| Volume Number | 183 |
| Journal | Microchimica Acta |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
