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FRET Imaging in Three-dimensional Hydrogels

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Published on: August 1, 2016

Three-Dimensional FRET Multiplexing for DNA Quantification with Attomolar Detection Limits.

Xue Qiu¹, Jiajia Guo¹, Jingyue Xu¹

¹NanoBioPhotonics (nanofret.com), Institute for Integrative Biology of the Cell (I2BC) , Université Paris-Saclay, Université Paris-Sud, CNRS, CEA , Orsay 91400 , France.

The Journal of Physical Chemistry Letters

|July 18, 2018

Summary

This study introduces time-gated Förster resonance energy transfer (TG-FRET) for multiplexed DNA detection. The method achieves sensitive, specific quantification of four DNA targets simultaneously using a single excitation source.

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Area of Science:

Biochemistry
Analytical Chemistry
Molecular Biology

Background:

Photoluminescence (PL) multiplexing for biosensing faces challenges in simultaneous spectral, temporal, and intensity-based quantification.
Achieving higher-order multiplexing requires precise control over PL properties like color, lifetime, and intensity for accurate target detection at low concentrations.

Purpose of the Study:

To develop a novel spectrotemporal multiplexing assay for simultaneous detection of multiple DNA targets.
To overcome the limitations of traditional PL multiplexing by integrating time-gated Förster resonance energy transfer (TG-FRET) with rolling circle amplification (RCA).

Main Methods:

Demonstrated TG-FRET using a long-lifetime terbium (Tb) complex as a donor and Cy3.5/Cy5.5 dyes as acceptors.
Employed RCA to generate DNA concatemers with controlled distances between Tb donors and dye acceptors.
Utilized single-color excitation and two-color detection for spectrotemporal multiplexing of four DNA targets.

Main Results:

Achieved higher-order multiplexing of four DNA targets with separation-free quantification.
Demonstrated target-specific photoluminescence decays of FRET pairs due to precise distance tuning.
Obtained high specificity, distinguishing highly homologous DNA sequences, with limits of detection as low as 40 zeptomoles (300 aM).

Conclusions:

The developed RCA-FRET assay enables sensitive and specific multiplexed DNA quantification.
This spectrotemporal multiplexing approach offers a robust platform for advanced biosensing applications.
The method successfully integrates spectral and temporal PL characteristics for enhanced multiplexing capabilities.