Journal of Fluorescence

, 21:2101 | Cite as

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

  • Yanling Qiao
  • Hongmin Tang
  • Gerhard R. Munske
  • Prashanta Dutta
  • Cornelius F. Ivory
  • Wen-Ji Dong
Original Paper

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.

Keywords

Fluorescence anisotropy/polarization Cardiac biomarker Cardiac troponin Peptide-base immunoassay Competition assay 

Abbreviations

hcTnI

human cardiac troponin I

hcTnT

human cardiac troponin T

AMI

acute myocardial infarction

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Yanling Qiao
    • 1
  • Hongmin Tang
    • 5
  • Gerhard R. Munske
    • 3
  • Prashanta Dutta
    • 4
  • Cornelius F. Ivory
    • 1
  • Wen-Ji Dong
    • 1
    • 2
  1. 1.Voiland School of Chemical Engineering and BioengineeringWashington State UniversityPullmanUSA
  2. 2.Department of Veterinary and Comparative Anatomy, Pharmacology, and PhysiologyWashington State UniversityPullmanUSA
  3. 3.School of Molecular BiosciencesWashington State UniversityPullmanUSA
  4. 4.School of Mechanical and Materials EngineeringWashington State UniversityPullmanUSA
  5. 5.School of Chemistry and Environmental ScienceGuizhou University for NationalitiesGuiyang, Guizhou ProvincePeople’s Republic of China

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