Cu(I)-Catalyzed Click Reaction-Triggered 3D DNA Walker for Constructing an "OFF-ON" Fluorescent Biosensor for Cu²⁺...

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Molecular Biology

Background:

• Reliable detection of intracellular copper ions (Cu²⁺) is crucial for understanding biological processes.
• Existing fluorescent biosensors often face challenges with selectivity and sensitivity for Cu²⁺ detection.
• Spherical nucleic acid (SNA) nanotechnology offers a platform for developing sensitive biosensing systems.

Purpose of the Study:

• To design and develop a highly selective and sensitive "OFF-ON" fluorescent biosensor for intracellular Cu²⁺ detection.
• To address the reliability issues in Cu²⁺ detection by integrating a Cu(I)-catalyzed click reaction with a 3D DNA walker.
• To demonstrate the application of the developed biosensor for Cu²⁺ sensing in biological systems at picomolar levels.

Main Methods:

• Fabrication of a fluorescent biosensor using gold nanoparticles (AuNPs) functionalized with azide-modified DNA (N₃-S1) and a fluorophore-labeled DNA strand (Cy3-HP).
• Utilizing the Cu(I)-catalyzed click reaction, triggered by Cu²⁺ and ascorbic acid, to initiate a 3D DNA walker.
• Employing a 3D DNA walker mechanism driven by N.BstNBI for signal amplification through the generation of multiple Cy3-labeled DNA fragments.

Main Results:

• The biosensor exhibited an "OFF-ON" fluorescence response, with quenched fluorescence in the absence of Cu²⁺ and recovered fluorescence upon its presence.
• The integrated system demonstrated superior selectivity and ultrahigh sensitivity for Cu²⁺ detection, achieving picomolar detection limits.
• Successful application of the biosensor for intracellular Cu²⁺ sensing in biological systems was demonstrated.

Conclusions:

• The developed "OFF-ON" fluorescent biosensor effectively detects intracellular Cu²⁺ with high reliability, selectivity, and sensitivity.
• The innovative integration of a click reaction and a 3D DNA walker provides a robust signal transduction strategy for biosensing.
• This platform offers a valuable tool for evaluating intracellular Cu²⁺ levels at picomolar concentrations in biological research.