期刊
ANALYTICAL LETTERS
卷 53, 期 1, 页码 140-151出版社
TAYLOR & FRANCIS INC
DOI: 10.1080/00032719.2019.1640718
关键词
Aptamers; chemiluminescence; colorimetry; microplate; thrombin
A microplate-based sandwich assay for the determination of alpha-human thrombin (HTb) was developed. Fluorescein-modified 29-mer thrombin binding aptamer (FAM-TBA29) and biotinylated 15-mer thrombin binding aptamer (Bio-TBA15) reacting with different exosites of HTb were used as the biorecognition components in the assay. FAM-TBA29 (capture aptamer) was immobilized using its interaction with anti-fluorescein antibody adsorbed on the microplate surface. As a sensitive signaling system, a combination of Bio-TBA15 and streptavidin-polyHRP conjugate was used. Under the optimized conditions, the detection limit for HTb was 1.4 nM; this value was the same in both the colorimetric and the chemiluminescent assays. The replacement of colorimetry for HRP measurement with chemiluminescence increased the assay sensitivity from 0.06 to 1.7 x 10(6) nM(-1) that clearly demonstrated advantage of the latter approach.
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