Spatial Molecular Heterogeneity on Biofunctionalized Particles Quantified by Three-Dimensional Single-Molecule...

Area of Science:

• Biomaterials Science
• Nanotechnology
• Molecular Imaging

Background:

• Controlling spatial molecular distribution on biofunctionalized particles is key for optimizing their performance in bioscience.
• Existing methods lack the precision to quantify molecular heterogeneity on particle surfaces.

Purpose of the Study:

• To develop and validate an analysis framework for quantitatively assessing spatial molecular properties of biofunctionalized particles.
• To enable precise control over molecular arrangements on particle surfaces for improved functionality.

Main Methods:

• Utilized 3D DNA-PAINT (DirectёлPAINT) imaging for high-resolution visualization of conjugated molecules.
• Developed analysis methods to correlate single-molecule data with particle surfaces, count molecules, and quantify spatial distributions.
• Employed simulation-based molecular counting to determine high densities of conjugated molecules.

Main Results:

• The framework successfully quantified spatial molecular distributions and densities on 1 μm particles functionalized with single-stranded DNA.
• Revealed significant interparticle and intraparticle heterogeneities dependent on streptavidin-biotin and click chemistry bioconjugation methods.
• Demonstrated the ability to access high densities of conjugated molecules and quantify their spatial arrangements.

Conclusions:

• The developed analysis framework using 3D DNA-PAINT is a versatile tool for characterizing biofunctionalized particles.
• This technique provides insights into bioconjugation strategies and guides future optimization for diverse bioscience applications.
• Enables precise control over molecular heterogeneity, enhancing the development of advanced biofunctionalized materials.