MASH-FRET: A Simplified Approach for Single-Molecule Multiplexing Using FRET

Area of Science:

• Biomarker analysis
• Single-molecule analysis
• Fluorescence resonance energy transfer (FRET)

Background:

• Multiplexed detection is crucial for cost-effective and reliable biomarker analysis.
• Current FRET-based multiplexing often requires multiple FRET pairs and excitation sources, complicating experiments.
• A single FRET pair approach with tunable interdye distance offers a simplified solution.

Purpose of the Study:

• To evaluate the MASH-FRET program for identifying multiplexed FRET populations using a single FRET pair.
• To demonstrate the capability of MASH-FRET-enabled bootstrap-based analysis of FRET data (BOBA-FRET) for resolving FRET efficiencies.
• To assess the potential for simplified, cost-effective multiplexed sensing.

Main Methods:

• Implementation and evaluation of the MASH-FRET analysis program.
• Utilizing bootstrap-based analysis of FRET data (BOBA-FRET) for population resolution.
• Testing with simulated FRET data to determine detection limits.

Main Results:

• MASH-FRET successfully identified overlapped FRET populations from a single FRET pair.
• BOBA-FRET provided high resolution and statistical confidence for poorly resolved FRET populations.
• The program identified FRET populations with mean differences of ~0.1, enabling up to ~9-fold multiplexing.

Conclusions:

• MASH-FRET offers a simplified approach to multiplexed sensing by analyzing FRET efficiencies from a single FRET pair.
• This method reduces the need for complex labeling and multiple excitation sources.
• The approach broadens the scope of FRET-based single-molecule analyses and improves biomarker detection.