Chemiluminescent and Colorimetric Aptamer-Based Assays of Human [alpha]-Thrombin

• Published in: Analytical letters Vol. 53; no. 1; p. 140
• Main Authors: Kurseev, Sergei A, Solovjev, Anton M, Neumann, Marina M, Medvedko, Alexey V, Sakharov, Ivan Yu
• Format: Journal Article
• Language: English
• Published: Abingdon Taylor & Francis Ltd 01.01.2020
• Subjects: Antibodies; Assaying; Binding; Chemiluminescence; Colorimetry; Fluorescein; Laboratory equipment; Organic chemistry; Signalling systems; Thrombin
• ISSN: 0003-2719; 1532-236X
• DOI: 10.1080/00032719.2019.1640718

A microplate-based sandwich assay for the determination of α-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 × 106 nM-1 that clearly demonstrated advantage of the latter approach.