Enhanced Fluorescence Anisotropy Assay for Human Cardiac Troponin I and T Detection |
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Authors: | Yanling Qiao Hongmin Tang Gerhard R Munske Prashanta Dutta Cornelius F Ivory Wen-Ji Dong |
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Institution: | (1) Voiland School of Chemical Engineering and Bioengineering, Washington State University, Wegner 205, Pullman, WA 99164, USA;(2) Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA 99164, USA;(3) School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA;(4) School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164, USA;(5) School of Chemistry and Environmental Science, Guizhou University for Nationalities, Guiyang, Guizhou Province, People’s Republic of China; |
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Abstract: | Human cardiac troponin I (hcTnI) and troponin T (hcTnT) are the biomarkers of choice for the diagnosis of cardiac diseases.
In an effort to improve assay sensitivity, in this study we developed a novel approach to simultaneously detect hcTnI and
hcTnT in homogenous solutions by monitoring enhanced-fluorescence-anisotropy changes. Specifically, our design was based on
a competition assay by measuring anisotropy change of fluorophore-labeled peptides bound to primary monoclonal antibodies
in the presence of nano-gold-modified secondary antibody in response to the presence of target proteins. Enhanced-fluorescence-anisotropy
resulted from interaction between the primary antibody and the nano-gold-labeled secondary antibody, which significantly increased
the size and decreased tumbling motion of the complex of peptide-antibodies. The measurements were performed to detect hcTnI
and hcTnT either individually or simultaneously in a homogenous buffer solution and in the solutions containing human plasma.
Our results showed that when fluorescence emission was monitored at a single wavelength selected by a monochromator the assay
at all experimental conditions had excellent linear response to the target proteins within the concentration range of 0.5–40 nM.
The detection limit is 0.5 nM for both hcTnI and hcTnT in the presence of human plasma. However, when fluorescence emission
was monitored using a cutoff filter, the linear response of the assay to the target proteins is within 15–500 pM. The detection
limit is 15 pM which is close to the recommended 99th percentile cutoff point for concentrations of hcTnI and hcTnT tests
to discriminate healthy and diseased conditions. Homogenous nature, rapid response time, and easy implementation of our assay
design make it a useful tool for disease biomarker and protein sensing. |
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