Reusable Microfluidic Chambers for Single-Molecule Microscopy

Abstract

Maintaining a consistent environment in single-molecule microfluidic chambers containing surface-bound molecules requires laborious cleaning and surface passivation procedures. Despite such efforts, variations in nonspecific binding and background signals commonly occur across different chambers. Being able to reuse the chambers without degrading the surface promises significant practical and fundamental advantages; however, this necessitates removing the molecules attached to the surface, such as DNA, proteins, lipids, or nanoparticles. Biotin-streptavidin attachment is widely used for such attachments, as biotin can be readily incorporated into these molecules. In this study, we present single-molecule fluorescence experiments that demonstrate effective resetting and recycling of the chambers at least 10 times by using photocleavable biotin (PC-biotin) and UV-light exposure. This method differs from alternatives as it does not utilize any harsh chemical treatment of the surface. We show that all bound molecules (utilizing various PC-biotin attachment chemistries) can be removed from the surface by a 5 min UV exposure of a specific wavelength. Nonoptimal wavelengths and light sources showed varying degrees of effectiveness. Our approach does not result in any detectable degradation of surface quality as assessed by the nonspecific binding of fluorescently labeled DNA and protein samples and the recovery of the DNA secondary structure and protein activity. The speed and efficiency of the resetting process, the cost-effectiveness of the procedure, and the widespread use of biotin-streptavidin attachment make this approach adaptable for a wide range of single-molecule applications.

Keywords: UV exposure; chamber recycling; fluorescence; photocleavable biotin; polyethylene glycol; single molecule.

Scheme 1. Chemical Structures of Biotin and PC-Biotin Modifications

The photocleavable moiety, photocleavage site, and oligo attachment sites are indicated in the schematics for 5′ PC-biotin. The sequence of DNA constructs given in Supporting Table S1 also lists which of these modifications were incorporated into each strand with cross-reference to relevant figures.

Figure 1

Characteristic time and efficiency of PC-biotin photocleavage. (A) Schematic of the photocleavage assay. Successive UV exposures result in a gradual decrease in the number of surface-bound fluorescent molecules (Cy5-DNA tagged with PC-biotin) due to UV cleavage of the bond between PC-biotin and streptavidin. A small fraction of DNA molecules tagged with biotin (yellow circles) were mixed with those tagged with PC-biotin (purple circles) to serve as an identifiable background when all PC-biotin molecules are photocleaved after a total of 5 min UV exposure (right panel). (B) Representative images showing the decrease in the number of surface-bound molecules after UV exposure. The first image on the left shows the molecules attached via biotin. (C) Left: Schematics of the assay. Right: Number of surface-bound Cy5-DNA molecules as a function of UV-exposure time shows an exponential decay with a characteristic time of 1.6 min. Five minutes is adequate to remove all surface-bound molecules. The flat background level around ∼10 molecules is due to the fluorescent molecules that are attached to the surface via biotin (those shown in the leftmost image in (B)). (D) Left: Schematics of the assay. Right: Similar exponential behavior is observed for partial-duplex DNA molecules attached to the surface via strands that are tagged with PC-biotin. The flat background level (around ∼20 molecules in this case) is due to the fluorescent molecules that are attached to the surface via biotin. (E) Similar experiments performed on molecules attached to the surface via biotin do not show a decrease in the number of surface-bound molecules after multiple rounds of UV exposure (Table S2), demonstrating the absence of detectable UV-induced photobleaching.

Figure 2

Impact of successive UV exposures on the nonspecific binding of Cy5-DNA. (A) Schematic of the assay where the nonspecific binding of Cy5-labeled DNA to a PEG surface is probed over 10 successive rounds of incubations. (B) Data on the nonspecific binding test performed on a UV-exposed or protected (not exposed to UV) chamber. The chambers were either subjected to 10 successive 5 min UV exposures or protected. Each data point represents the average number of molecules based on imaging 35 different regions on the surface. The error bars are standard errors of these 35 measurements. As expected, the number of surface-bound molecules systematically increases with each round of incubation as nonspecific binding is cumulative; however, the UV-exposed surfaces perform as well or better than the protected surfaces in each of these tests. One-way ANOVA analyses within each group show that the change in the number of molecules with each repeat is significant (Supporting Table S3, p = 0.001). t-test analysis comparing the UV-exposed and protected groups does not show significant difference between the two groups for Repeats 1–3 (p > 0.05), while the differences are significant for Repeats 4–10 (Supporting Table S4, p < 0.001). (C) Images showing the surface before UV exposure (just molecules that are specifically attached via biotin–streptavidin) and after 10 exposures (both specifically and nonspecifically bound molecules are present). The increase in the number of molecules in the right image is due to cumulative nonspecific binding over 10 rounds.

Figure 3

Impact of successive UV exposures on the nonspecific binding of labeled protein. (A) Schematic of the assay. Nonspecific binding of Cy3-streptavidin to a PEG surface that is either subjected to 10 successive 5 min UV exposures or protected (not exposed to UV). The biotin-PEG molecules on the surface were saturated with unlabeled streptavidin before introducing Cy3-streptavidin by incubating a high concentration (100-fold excess) of unlabeled streptavidin. Therefore, only molecules due to nonspecific binding attach to the surface. (B) Comparative data showing nonspecific binding in a protected (not exposed to UV) or UV-exposed chamber. Each data point represents the average number of molecules based on imaging 30 different regions on the surface. The error bars are standard deviations of these 30 measurements. The number of surface-bound molecules systematically increases at successive cycles due to the nonspecific binding being cumulative. The channel exposed to UV and the protected channel are essentially indistinguishable in terms of nonspecific protein binding, suggesting no detectable degradation in surface quality due to UV exposure. One-way ANOVA analyses within each group show that the change in the number of molecules with each repeat is significant (Supporting Table S5, p = 0.001). t-test analysis comparing the UV-exposed and protected groups does not show significant difference between the two groups for Repeats 1 and 6 (p > 0.1), while the differences are significant for the other repeats (Supporting Table S6, p < 0.05). (C) Images showing the surface before UV exposure (just molecules that are specifically attached via biotin–streptavidin) and after 10 cycles of Cy3-streptavidin incubation and UV exposures (both specifically and nonspecifically bound molecules are present). The increase in the number of molecules in the right image is due to cumulative nonspecific binding over 10 rounds.

Figure 4

Reproducibility of GQ folding and RPA activity. (A) Schematics of the assay. pdDNA constructs with an overhang that can form a GQ structure are immobilized on the surface using PC-biotin. The compaction of the overhang by GQ results in a high FRET peak. Binding to and unfolding of the GQ by RPA result in an extended overhang and hence a lower FRET state. After GQ folding and RPA-mediated unfolding are recorded, the channel is reset by subjecting it to 5 min of UV exposure. A fresh batch of pdDNA is then introduced and imaged, followed by the introduction of RPA and imaging of the molecules. This cycle is repeated 10 times. A small fraction of biotinylated Cy3-labeled ssDNA molecules is kept in the channel to identify the surface and ensure all molecules attached with PC-biotin are removed. (B) Results of GQ folding (left) and RPA-mediated unfolding (right) at 10 successive rounds of recycling. The similarity of the folding pattern and level of protein activity suggests the recycling process enables consistent and reproducible recovery of the folding pattern and protein activity for at least 10 recycling processes. The solid lines are Gaussian fits to the FRET distributions.