Space-Confined Amplification for In Situ Imaging of Single Nucleic Acid and Single Pathogen on Biological Samples

Area of Science:

• Biotechnology
• Molecular Biology
• Materials Science

Background:

• Direct in situ imaging of nucleic acids offers rapid analysis without DNA extraction but faces challenges with molecular diffusion in aqueous solutions.
• Traditional amplification methods can lose spatial information due to high molecular mobility, limiting direct sample analysis.
• Hydrogels with 3D nanoconfined spaces mimic cellular matrices, restricting molecular diffusion and enabling faster in situ reactions.

Purpose of the Study:

• To develop a hydrogel-based method for direct, ultrafast, in situ imaging of single nucleic acids and pathogens on biological samples.
• To overcome the limitations of molecular diffusion in traditional amplification techniques for spatial information preservation.
• To enable rapid, label-free, and spatially resolved detection of targets without requiring formaldehyde fixation.

Main Methods:

• Development of hydrogel-based in situ space-confined interfacial amplification (iSCIA) using polyethylene glycol hydrogel coating.
• Nanoconfinement of nucleic acids within the hydrogel matrix to restrict movement and facilitate in situ enzymatic reactions.
• Integration of a deep learning model for automated multiplex imaging and analysis of multiple targets.

Main Results:

• Achieved direct, in situ imaging of nucleic acids on large-scale surfaces within 20 minutes.
• Demonstrated a low detection limit of 1 copy/10 cm² for nucleic acids.
• Successfully applied iSCIA for pathogen imaging on plant leaves and food, enabling monitoring of plant health and food safety.

Conclusions:

• Hydrogel-based iSCIA provides a rapid, flexible system for direct in situ imaging of nucleic acids and pathogens.
• The technique preserves spatial information and enhances amplification efficiency by nanoconfining molecules.
• iSCIA shows significant potential for diverse applications in food safety, environmental monitoring, and clinical diagnostics.