Single-molecule detection with high accuracy and specialty plays an important role in biomedical diagnosis and screening. Zero-mode waveguides (ZMWs) enable the possibility of single biological molecule detection in real time. Nevertheless, the absence of a reliable assessment for single effective complex loading has constrained further applications of ZMWs in complex interaction. Both the quantity and activity of the complex loaded into ZMWs have a critical effect on the efficiency of detection. Herein, a fluorescence evaluation at quenching and accumulation checkpoints was established to assess and optimize single effective complex loading into ZMWs. A primer-template-enzyme ternary complex was designed, and then an evaluation for quantity statistics at the quenching checkpoint and functional activity at the accumulation checkpoint was used to validate the effectiveness of complexes loaded into ZMWs. By optimizing the parameters such as loading time, procedures, and enzyme amount, the single-molecule effective occupancy was increased to 25.48%, achieving 68.86% of the theoretical maximum value (37%) according to Poisson statistics. It is of great significance to provide effective complex-loading validation for improving the sample-loading efficiency of single-molecule assays or sequencing in the future.
Keywords: effective loading; fluorescence accumulation; fluorescence quenching; ternary complex; zero-mode waveguides.