Working Paper Article Version 1 This version is not peer-reviewed

Diffusivity Measurement by Single Molecule Recycling in a Capillary Microchannel

Version 1 : Received: 25 May 2021 / Approved: 26 May 2021 / Online: 26 May 2021 (10:50:13 CEST)

A peer-reviewed article of this Preprint also exists.

Wang, B.; Davis, L.M. Diffusivity Measurement by Single-Molecule Recycling in a Capillary Microchannel. Micromachines 2021, 12, 800. Wang, B.; Davis, L.M. Diffusivity Measurement by Single-Molecule Recycling in a Capillary Microchannel. Micromachines 2021, 12, 800.


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 the 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 liquid. In this paper, we have fixed a capillary microchannel with an inner diameter of 2 microns on top of a piezo stage to recycle the molecule and collected the fluorescence by a confocal microscope. 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. 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 previous reports. This technique simplifies the overall procedure of the single-molecule recycling and could be useful for the ligand-binding studies of biomolecules.


capillary; microfluidic device; single-molecule recycling; maximum likelihood


Physical Sciences, Acoustics

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