Programming amphiphilic tetrahedral DNA frameworks for high-throughput single-molecule measurement

This is the authors' abstract. We don't add key points for this paper.

Achieving high-density, uniform distribution, and optical resolvability of immobilized probes is essential for robust single-molecule analysis, yet this remains particularly challenging for amphiphilic DNA nanostructures. Here, we systematically reveal the dominant role of interface charge effects in regulating the adsorption efficiency of amphiphilic tetrahedral DNA frameworks (TDFs) onto solid substrates. We demonstrate that adsorption is primarily governed by interfacial charge effects, which are cooperatively modulated by Mg 2+ concentration, DNA surface charge, and solid-substrate charge. Based on this charge-dominated mechanism, we developed a controllable method that achieves high-density adsorption of optically resolvable amphiphilic TDF probes on imaging substrates (up to 153281 particles/mm 2 ). Leveraging this optimized platform, we performed high-throughput single-molecule fluorescence characterization and found that inserting a single thymine spacer between the TDF and the Cy5 fluorophore enhanced the fluorescence brightness by approximately 3.4-fold. This work provides both mechanistic insights and a practical methodology for the sample preparation of amphiphilic DNA nanostructures, thereby establishing a reliable foundation for their application in quantitative single-molecule biophysics and ultrasensitive biosensing.