ABSTRACT
A novel optical biosensor technique is being developed for the early detection of myocardial infarction by utilizing the distance-dependent chemical transduction method of fluorescence resonance energy transfer (FRET). The FRET process requires two fluorophores termed the donor and the acceptor. When in close proximity, the donor absorbs energy from the excitation source and non-radiatively transfers the energy to the acceptor, which in turn emits fluorescent energy. This distance-dependent property was utilized to detect conformational changes when antibodies combine with their respective antigens. The fluorophores were conjugated to an antibody-Protein A complex and then immobilized via silanization to the distal ends of optical fibers. Three different antibody-Protein A complexes were immobilized: generic IgG, cardiac Troponin T (cTnT), and cardiac Troponin I (cTnI). Results showed that upon the addition of the specific antigens, the antibodies underwent a conformational change, reducing the distance between the FRET fluorophores. The generic IgG responded to 233 nM antigens, whereas the cTnT biosensor had a limit of detection of 75 nM, and the cTnI biosensors had a limit of detection of 94 nM.
Subject(s)
Biosensing Techniques/methods , Fluorescence Resonance Energy Transfer/methods , Immunoassay , Myocardial Infarction/diagnosis , Biosensing Techniques/instrumentation , Fluorescence Resonance Energy Transfer/instrumentation , HumansABSTRACT
We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab')2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody-protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the 'in solution' response. The paper reports the results of three different donor-acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.