Development of a novel FRET immunosensor technique.

Area of Science:

• Biomedical Engineering
• Biophysics
• Analytical Chemistry

Background:

• Development of sensitive and specific diagnostic tools is crucial for disease detection.
• Optical immunosensors offer a promising platform for real-time biological analysis.
• Fluorescence Resonance Energy Transfer (FRET) enables detection of molecular interactions and conformational changes.

Purpose of the Study:

• To develop and validate a novel optical immunosensor utilizing FRET.
• To investigate the efficacy of different FRET pairs and their optimal conjugation ratios for immunosensing.
• To demonstrate the sensor's ability to detect specific antigens through conformational changes.

Main Methods:

• Antibodies (IgG) were labeled with acceptor fluorophores and carrier molecules (Protein A, Protein G, F(ab')2) with donor fluorophores.
• Labeled antibody-carrier complexes were incubated with specific and non-specific antigens in solution.
• Three FRET pairs (FITC/TRITC, Texas Red/Cy5, Alexa Fluor 546/594) were evaluated, examining fluorophore-to-protein (F/P) and acceptor-to-donor (A/D) ratios.

Main Results:

• A conformational change in antibodies upon binding to specific antigens induced FRET, measured as a change in fluorescence.
• The Alexa Fluor 546/594 FRET pair exhibited the largest fluorescence change (35%).
• Optimal F/P and A/D ratios were found to influence FRET efficiency, confirming the feasibility of the FRET immunosensor.

Conclusions:

• The developed FRET-based optical immunosensor is feasible for detecting specific antigens.
• Further development requires immobilizing the sensor complexes on optical substrates for consistent calibration.
• This technique holds potential for sensitive and specific diagnostic applications.