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Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning
Xue Qiu1, Jiajia Guo1, Zongwen Jin2
1NanoBioPhotonics (nanofret.com), Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Université Paris-Sud, CNRS, CEA, 91405, Orsay Cedex, France.
Small (Weinheim an Der Bergstrasse, Germany)
|April 4, 2017
Summary
Time-gated (TG) detection of single terbium-donor Förster resonance energy transfer (FRET) pairs enables multiplexed detection of multiple DNAs or microRNAs. This biosensing approach offers high sensitivity and multiplexing capabilities for diverse applications.
Area of Science:
- Biomedical Engineering
- Analytical Chemistry
- Biochemistry
Background:
- Multiplexed photoluminescence (PL) detection is crucial for simultaneous analysis in chemical and biological sensing.
- Förster resonance energy transfer (FRET) is a powerful tool for studying molecular interactions.
- Terbium-donor based FRET systems offer unique luminescent properties for biosensing.
Purpose of the Study:
- To demonstrate the use of single time-gated (TG) FRET pairs for multiplexed quantification of multiple nucleic acids (DNA/microRNA) in a single sample.
- To showcase the applicability of this method for both molecular (terbium-to-dye) and nanoparticle (terbium-to-quantum dot) based biosensing.
- To explore the potential for high-order multiplexing in biosensing.
Main Methods:
- Utilized time-gated (TG) detection to minimize background noise and enhance signal-to-noise ratio.
- Employed single terbium-donor based FRET pairs for molecular recognition.
- Investigated acceptor-sensitization and donor-quenching mechanisms to quantify biomolecular interactions and tune PL decay.
- Demonstrated a one-step assay format for simplified sample analysis.
Main Results:
- Achieved selective quantification of low nanomolar concentrations of multiple DNAs or microRNAs in a single sample.
- Successfully applied single-TG-FRET-pair multiplexing to both molecular and nanoparticle biosensing systems.
- Demonstrated that modifying the donor-acceptor distance tunes the photoluminescence (PL) decays.
- Confirmed that TG intensity detection provides extremely low background noise.
Conclusions:
- Single time-gated FRET-pair multiplexing is a highly selective and sensitive method for quantifying multiple nucleic acids.
- This approach is versatile, applicable to both molecular and nanoparticle-based biosensing.
- The combination with spectral and spatial resolution opens avenues for biosensing with unprecedented multiplexing capabilities.