Ensemble multicolour FRET model enables barcoding at extreme FRET levels

Area of Science:

• Biophysics
• Nanotechnology
• Analytical Chemistry

Background:

• Quantitative models of Förster resonance energy transfer (FRET) are foundational but lack mechanistic understanding for multicolour FRET (mFRET).
• Multicolour FRET in fluorescently barcoded microparticles leads to complex responses, hindering encoding and decoding.
• Stochastic mFRET in microparticle systems presents a significant challenge for high-capacity encoding.

Purpose of the Study:

• To develop a mechanistic model for ensemble multicolour FRET (emFRET) in stochastic systems.
• To apply the emFRET model to optimize fluorescent barcoding in microparticles, particularly in extreme FRET regimes.
• To introduce a novel, facile multicolour labelling method for enhanced microparticle barcoding.

Main Methods:

• Development and application of an ensemble mFRET (emFRET) model.
• Design and validation of 580 unique barcodes using four dyes and oligonucleotide linkers.
• Implementation of multiplexed immunoassays with cytometric readout and automated decoding.

Main Results:

• The emFRET model successfully guided barcoding into regimes with extreme FRET efficiencies (up to 76%).
• A facile, proportional multicolour labelling method using oligonucleotide linkers was established.
• Rapid design and validation of 580 barcodes enabled high-capacity multiplexed immunoassays.

Conclusions:

• The emFRET model provides a mechanistic framework for understanding and controlling stochastic mFRET.
• The developed labelling method and emFRET model significantly expand the barcoding capacity of microparticles.
• This approach benefits applications requiring high-throughput multiplexing and automated decoding, particularly those involving common organic dyes.