Chemiluminescent and Colorimetric Aptamer-Based Assays of Human α-Thrombin

Abstract

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.

Keywords:

• Aptamers
• chemiluminescence
• colorimetry
• microplate
• thrombin

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This work was supported by the Russian Science Foundation [grant 17-14-01042].