Probes for biomolecules detection based on RET-enhanced fluorescence polarization

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biophysics

Background:

• Fluorescent probes utilizing resonance energy transfer (RET) or fluorescence polarization (FP) are established for biomolecule detection.
• Limited research exists on the synergistic effects of RET on FP.
• Comprehensive design strategies for such dual-principle probes are scarce.

Purpose of the Study:

• To investigate the impact of RET on FP in a novel fluorescent probe design.
• To analyze the influence of substrate peptide structure and composition on probe performance.
• To develop a reliable method for detecting biomolecules using combined RET and FP principles.

Main Methods:

• Construction of a fluorescent probe (SA-488-sub-nanogold) integrating streptavidin labeled Alexa488 (SA-488), nanogold, and a biotinylated substrate peptide.
• Evaluation of substrate peptide structural and compositional effects on probe characteristics.
• Assessment of fluorescence intensity (FI) suppression and rotational relaxation time changes due to RET.
• Detection of trypsin and biotin by monitoring changes in FI and FP.

Main Results:

• RET-induced fluorescence intensity suppression led to increased probe volume/mass and prolonged rotational relaxation time.
• These changes collectively enhanced the fluorescence polarization (FP) performance of the probe.
• The developed probe demonstrated increased reliability, sensitivity, and a lower detection limit for trypsin and biotin.

Conclusions:

• Resonance energy transfer significantly enhances the fluorescence polarization performance of the designed probe.
• The probe offers a reliable and sensitive method for detecting biomolecules by combining FI and FP measurements.
• This work provides insights into designing advanced fluorescent probes with improved detection capabilities.