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

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 compo...

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Published inAnalytical letters Vol. 53; no. 1; pp. 140 - 151
Main Authors Kurseev, Sergei A., Solovjev, Anton M., Neumann, Marina M., Medvedko, Alexey V., Sakharov, Ivan Yu
Format Journal Article
LanguageEnglish
Published Taylor & Francis 02.01.2020
Subjects
Aptamers
chemiluminescence
colorimetry
microplate
thrombin
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ISSN0003-2719
1532-236X
DOI10.1080/00032719.2019.1640718

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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 × 10 6  nM −1 that clearly demonstrated advantage of the latter approach.
AbstractList 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 × 10 6  nM −1 that clearly demonstrated advantage of the latter approach.
Author Sakharov, Ivan Yu
Kurseev, Sergei A.
Medvedko, Alexey V.
Neumann, Marina M.
Solovjev, Anton M.
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Snippet A microplate-based sandwich assay for the determination of α-human thrombin (HTb) was developed. Fluorescein-modified 29-mer thrombin binding aptamer...
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SubjectTerms Aptamers
chemiluminescence
colorimetry
microplate
thrombin
Title Chemiluminescent and Colorimetric Aptamer-Based Assays of Human α-Thrombin
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