Tethered Catalytic Hairpin Assembly with Plasmon-Enhanced Fluorescence Readout for Single Molecule Detection

Area of Science:

• Biotechnology
• Analytical Chemistry
• Biosensing

Background:

• Current bioassays often rely on enzymatic amplification or sample compartmenting, limiting their scope and sensitivity.
• There is a need for enzyme-free, ultrasensitive detection methods for trace biomolecules in diagnostics and research.

Purpose of the Study:

• To report a novel digital bioassay readout concept based on counting individual affinity-captured target biomolecules.
• To demonstrate the efficacy of tethered catalytic hairpin assembly (tCHA) with flexible polymer linkers (FPLs) for spatial confinement and signal amplification.
• To evaluate the analytical performance of this enzyme-free approach for detecting DNA and proteins.

Main Methods:

• Utilizing a tethered catalytic hairpin assembly (tCHA) on a solid sensor surface with spatial confinement via flexible polymer linkers (FPLs).
• Employing wide-field plasmon-enhanced fluorescence (PEF) imaging for real-time optical probing of reaction kinetics.
• Testing the dual amplification tCHA-PEF concept with a model single-stranded DNA analyte and in a sandwich immunoassay for protein detection.

Main Results:

• Individual affinity-captured target biomolecules were visualized as distinct fluorescent spots using PEF imaging.
• The effect of FPL length on analytical performance was investigated.
• Sub-femtomolar concentrations of target proteins were detected using a sandwich immunoassay format.
• The tCHA-PEF method demonstrated enzyme-free, ultrasensitive detection capabilities.

Conclusions:

• The tCHA-PEF concept represents a new class of generic, enzyme-free bioanalytical tools.
• This technology enables ultrasensitive analysis of trace amounts of protein and nucleic acid analytes.
• The method holds significant potential for future molecular diagnostics and research applications.