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.
Highlights
Techniques such as fluorescent labeling have enabled the behavioral observation of a single molecule without the description of ensemble thermodynamics, so that enhanced the precision of localizing single beads and conquered the diffraction limit of a conventional microscope [1,2]
The molecule is confined by the one-dimensional microchannel and passes through a Gaussian beam waist ω0 with constant velocity, and the fitting function of autocorrelation function (ACF) is customized to one dimension with four fitting parameters a0, a1, a2, a3, which is given by g ( τ ) = a0 + √
1 + a3 τ where a1 = F2 /( F + B)2, in which F is the rate of fluorescence counts from a molecule at the center of the laser focus and B is the background rate, and a0 = 1, which gives g(∞) = 1, a2 = (v/ω0 )2 is the square of the reciprocal of the time constant for flow, and a3 = 4D/ω02 is the reciprocal of the time constant for diffusion, in which D is the diffusivity of the molecule
Summary
Techniques such as fluorescent labeling have enabled the behavioral observation of a single molecule without the description of ensemble thermodynamics, so that enhanced the precision of localizing single beads and conquered the diffraction limit of a conventional microscope [1,2]. The Brownian motion limits the time the molecule stays inside the focal volume and the observation time is restricted to the scale of milliseconds, which is not enough to witness changes, such as protein folding and molecular interactions. To overcome this disadvantage, surface tethering and optical trapping are developed to use chemical and electromagnetic attractions to immobilize the molecule but alter the behavior in the meantime [4,5]. Feedback-driven tracking and trapping methods measure the displacement of the molecule from the center of the focal volume and apply real-time compensation to the position of the sample or laser focus [6,7,8,9,10,11,12,13].
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