Diffusivity Measurement by Single-Molecule Recycling in a Capillary Microchannel

Abstract

Microfluidic devices have been extensively investigated in recent years in fields including ligand-binding analysis, chromatographic separation, molecular dynamics, and DNA sequencing. To prolong the observation of a single molecule in aqueous buffer, the solution in a sub-micron scale channel is driven by a electric field and reversed after a fixed delay following each passage, so that the molecule passes back and forth through the laser focus and the time before irreversible photobleaching is extended. However, this practice requires complex chemical treatment to the inner surface of the channel to prevent unexpected sticking to the surface and the confined space renders features, such as a higher viscosity and lower dielectric constant, which slow the Brownian motion of the molecule compared to the bulk solution. Additionally, electron beam lithography used for the fabrication of the nanochannel substantially increases the cost, and the sub-micron dimensions make the molecule difficult to locate. In this paper, we propose a method of single-molecule recycling in a capillary microchannel. A commercial fused-silica capillary with an inner diameter of 2 microns is chopped into a 1-inch piece and is fixed onto a cover slip. Two o-rings on the sides used as reservoirs and an o-ring in the middle used as observation window are glued over the capillary. The inner surface of the capillary is chemically processed to reduce the non-specific sticking and to improve capillary effect. The device does not require high-precision fabrication and thus is less costly and easier to prepare than the nanochannel. 40 nm Fluospheres® in 50% methanol are used as working solution. The capillary is translated by a piezo stage to recycle the molecule, which diffuses freely through the capillary, and a confocal microscope is used for fluorescence collection. The passing times of the molecule through the laser focus are calculated by a real-time control system based on an FPGA, and the commands of translation are given to the piezo stage through a feedback algorithm. The larger dimensions of the capillary overcomes the strong sticking, the reduced diffusivity, and the difficulty of localizing the molecule. We have achieved a maximum number of recycles of more than 200 and developed a maximum-likelihood estimation of the diffusivity of the molecule, which attains results of the same magnitude as the previous report. This technique simplifies the overall procedure of the single-molecule recycling and could be useful for the ligand-binding studies in high-throughput screening.