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Enhanced Fluorescence Anisotropy Assay for Human Cardiac Troponin I and T Detection

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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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Abbreviations

hcTnI:

human cardiac troponin I

hcTnT:

human cardiac troponin T

AMI:

acute myocardial infarction

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Authors and Affiliations

  1. Voiland School of Chemical Engineering and Bioengineering, Washington State University, Wegner 205, Pullman, WA, 99164, USA

    Yanling Qiao, Cornelius F. Ivory & Wen-Ji Dong

  2. Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, WA, 99164, USA

    Wen-Ji Dong

  3. School of Molecular Biosciences, Washington State University, Pullman, WA, 99164, USA

    Gerhard R. Munske

  4. School of Mechanical and Materials Engineering, Washington State University, Pullman, WA, 99164, USA

    Prashanta Dutta

  5. School of Chemistry and Environmental Science, Guizhou University for Nationalities, Guiyang, Guizhou Province, People’s Republic of China

    Hongmin Tang

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  1. Yanling Qiao
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  2. Hongmin Tang
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  4. Prashanta Dutta
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Correspondence to Wen-Ji Dong.

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Qiao, Y., Tang, H., Munske, G.R. et al. Enhanced Fluorescence Anisotropy Assay for Human Cardiac Troponin I and T Detection. J Fluoresc 21, 2101–2110 (2011). https://doi.org/10.1007/s10895-011-0909-0

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  • DOI: https://doi.org/10.1007/s10895-011-0909-0

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