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Nucleic Acids02:43

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Multiplexed Nucleic Acid Sensing with Single-Molecule FRET.

Anisa Kaur1, Kumar Sapkota1, Soma Dhakal1

  • 1Department of Chemistry , Virginia Commonwealth University , 1001 West Main Street , Richmond , Virginia 23284 , United States.

ACS Sensors
|February 21, 2019
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Summary

Researchers developed interconvertible hairpin-based sensors (iHabSs) for sensitive, quantitative multiplex detection of nucleic acid sequences using a single dye pair. This advance simplifies biosensing and diagnostics for unlabeled biomarkers.

Keywords:
TIRF microscopymultiplexingrecyclable sensorssingle-molecule FRETsingle nucleotide polymorphism (SNP)

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Area of Science:

  • Bionanotechnology
  • Molecular Diagnostics
  • Biomolecular Sensing

Background:

  • Multiplexed biomolecule detection is crucial for diagnostics and bionanotechnology.
  • Existing methods like microarrays and DNA barcodes are complex and often semiquantitative.
  • Single-molecule fluorescence resonance energy transfer (smFRET) offers sensitive, quantitative detection but faces multiplexing challenges.

Purpose of the Study:

  • To develop a simplified, quantitative multiplex detection method for unlabeled nucleic acid sequences.
  • To overcome limitations of current smFRET-based multiplexing strategies.
  • To create novel sensors enabling simultaneous detection with high confidence.

Main Methods:

  • Design of interconvertible hairpin-based sensors (iHabSs).
  • Utilized a single donor/acceptor fluorophore pair for smFRET measurements.
  • Developed sensors with nonoverlapping FRET efficiencies (E_FRET) for distinct signal channels.
  • Validated multiplex detection of unlabeled nucleic acid sequences.

Main Results:

  • Achieved high-confidence multiplex detection of unlabeled nucleic acids.
  • Demonstrated sensor recyclability and sensitivity with a limit of detection around 200 pM.
  • Showcased the ability to discriminate against single base mismatches.
  • Validated reliability through systematic target omission experiments.

Conclusions:

  • The developed iHabSs enable robust, quantitative multiplex detection using a single FRET pair.
  • This approach simplifies labeling schemes and data analysis for smFRET multiplexing.
  • The technology holds significant potential for advancing biosensing and clinical diagnostics.