Sequential processing from cell lysis to protein assay on a chip enabling the optimization of an Fx-ATPase single molecule assay condition

摘要

We developed an integrated protein assay device, "Single Molecule MicroTAS (SMM)," which enables cell lysis, protein extraction, purification, and activity assay. The assay was achieved at the single-molecule scale for a genetically engineered protein, F1-ATPase, which is the smallest known rotary motor. A cell lysis condition, with a wide range of applied voltages (50-250 V) and other optimized values (pulse width: 50 μs; duty: 0.01%; electrode gap: 25 urn; total flow rate: 5 μL min~(-1)) provided a high enough protein concentration for the assay. Successively, the protein was extracted and purified by specific binding in a microfiuidic channel. During the assay process, the diffusion effect of lysate between a two-phase laminar flow contributes to optimizing the single-molecule assay condition, because the concentration of the original lysate from the E. coli solution is too high to assay. To achieve the most efficient assay condition, the protein diffusion effect on the assay was experimentally and numerically evaluated. The results reveal that, in our experimental conditions, concentrations of F1 and other contaminated effluents are optimized for the F1 rotational assay at a channel position. The adenosine triphosphate (ATP)-driven rotation speed measured in the SMM was compatible with that obtained by conventional purification and assay. Such a sequential process from cell lysis to assay proves that the SMM is an example of a sample-in-answer-out system for F1 protein evaluation.