1：Experimental Design and Method Selection:

The study utilized glass-made nanofluidic devices for single-molecule detection under equilibrium and non-equilibrium conditions. The devices were designed with parallel nanochannels for equilibrium studies and a T-shaped nanochannel for non-equilibrium studies. Camera-based single-molecule detection was employed to monitor many fluorescent molecules in parallel.

2：Sample Selection and Data Sources:

Fluorescently labelled DNA molecules were used as probes to study DNA-protein interactions, salt-dependent conformational equilibria of DNA hairpins, and enzymatic catalysis of DNA synthesis.

3：List of Experimental Equipment and Materials:

The setup included a home-built TIRF microscope, a fiber-coupled laser engine, a 100× NA 1.49 objective, and an Ixon Ultra 897 emCCD camera. Nano-/microfluidic devices were fabricated by Micronit.

4：49 objective, and an Ixon Ultra 897 emCCD camera. Nano-/microfluidic devices were fabricated by Micronit. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: The experiments involved tracking the movement of fluorescently labelled DNA oligonucleotides through nanochannels, observing conformational changes, and triggering chemical reactions by mixing reactants in a T-shaped nanochannel.

5：Data Analysis Methods:

Data analysis included tracking particles from frame to frame, calculating mean flow speed and diffusion coefficient, and determining FRET efficiency and stoichiometry ratio for each molecule.