Cloud-point PEG Glass Surfaces for Imaging of Immobilized Single Molecules by Total-internal-reflection Microscopy

[Abstract] This effective, robust protocol generates glass coverslips coated with biotinfunctionalized polyethylene glycol (PEG), making the glass surface resistant to non-specific absorption of biomolecules, and permitting immobilization of biomolecules for subsequent single-molecule tracking of biochemical reactions. The protocol can be completed in one day, and the coverslips can be stored for at least 1 month. We have confirmed that the PEG surfaces prepared according to the protocol are resistant to non-specific adsorption by a wide range of biomolecules (bacterial, mitochondrial, and human transcription factors, DNA, and RNA) and biological buffers.

The current protocol builds upon a technique first introduced by Ha and colleagues (Ha et al., 2002) which, in turn, built upon finding that polyethylene glycol (PEG) is most effective in creating anti-fouling surfaces (Prime and Whitesides, 1993), also see references in (Ostuni et al., 2001). In the original protocol by Ha et al. (2002), glass surfaces were first coated with a silanol-reactive aminopropyltriethoxysilane (APTES) to create amine groups, followed by deposition of a mixture of amine-reactive N-hydroxysuccinimide (NHS)-PEG (to create a passivation layer on the glass) and NHS-PEG-biotin (to create a handle for immobilization of bio-molecules for single-molecule tracking). In our protocol, PEG deposition is performed in cloud-point conditions, which reduces the size of the PEG globule and results in a denser, more adsorption-resistant, PEG layer (Kingshott et al., 2002). In addition, our protocol maximizes the reactivity of NHS-PEG during deposition. Furthermore, the protocol includes an end-capping step intended to eliminate residual amine groups remaining after PEG coupling, which we found to reduce non-specific adsorption of nucleic acids to surfaces in low-ionic-strength buffers required by some enzymes (Zhang et al., 2014). Finally, the protocol provides simple qualitycontrol tips to help trouble-shooting. Despite these key improvements, we found that some proteins are still prone to non-specific adsorption to 'cloud point' PEG surfaces. For instance, we found that the general transcription factor TFIID, a key component of the human transcription machinery, absorbs to 'cloud-point' PEG surfaces, whereas other five components of the basal human transcription machinery (TFIIB, TFIIF, TFIIE, TFIIH and RNA polymerase II) do not (Revyakin et al., 2012). Thus, we recommend testing the 'cloud-point' PEG surfaces using your specific buffers, biomolecules of interest, and biochemical activity assays. 1. Place 12 coverslips into ceramic rack using flat-tip tweezers.

Materials and Reagents
2. In a designated chemical fume hood, place two clean 250 ml PYREX beakers (beaker 1 and 2) in two separate crystallization dishes. The dishes serve as secondary containers to ensure safety.
3. Very carefully add 1 part (50 ml) of 30% hydrogen peroxide (H2O2) to beaker 1, followed by 3 parts (150ml) of concentrated sulfuric acid (H2SO4). Gently stir to mix the solution with a clean glass rod. The solution will immediately form bubbles and heat up to ~100 °C. 4. Using forceps, very carefully transfer the rack with the coverslips into beaker 1 and incubate for 30 min. To ensure safety, leave a note indicating that Piranha is in use. 5. Repeat Piranha treatment one more time with a fresh Piranha solution in beaker 2.
6. Transfer the coverslips from beaker 2 into a Nalgene 125 ml polypropylene jar filled with double-distilled water. Dispose of piranha waste according to your institution's regulations. Rinse the coverslips copiously with double-distilled water until pH stabilizes (verified by pH paper). The cleaned coverslips can be stored in double-distilled water without noticeable changes in reactivity and fluorescent background for at least 1 month.
a. Surface hydrophilicity. Piranha-treated coverslips become uniformly hydrophilic, which can be qualitatively verified by dipping a coverslip in water using flat-end tweezers, taking it out vertically, and observing water slowly receding as a uniform sheet, and forming Young's rings before drying out. In contrast, untreated coverslips form patches of water when dipped into and taken out of water   (Miron, 1982)]. We also highly recommend measuring the percentage of reactive PEG-NHS in new batches of purchased PEG reagents. We have had cases in which completely hydrolyzed, non-reactive PEGs had been shipped by major suppliers.
14. This step is best done during the APTES incubation at step C11. Take out 6 single-use aliquots of dry mPEG-SVA from storage at -80 °C. Each aliquot should be about 5 mg, which is sufficient to treat two coverslips. The mass of dry mPEG-SVA in the 6 aliquots should have been pre-written on each tube with 0.1 mg accuracy prior to storage at -80 °C (e.g., 4.9, 5.0, 5,1, 4.9, 5.0, and 5.1 mg for 6 tubes). In addition, take out 1-2 mg of biotin-PEG-SVA from storage. Let all tubes warm up to room temperature.
15. This step is best done during the APTES incubation at step C11. Calculate the volume of 0.5 M K2SO4 solution to add to each mPEG-SVA tube by multiplying the aliquot mass by 8 (e.g., the tube containing 4.9 mg will require 4. and fluorescent labels that photobleach in >5 seconds in the absence of oxygen scavengers (e.g., Cy3 and Atto633). For a good-quality surface, we typically observe a 'cloud' of 10 nM biomolecules rapidly diffusing in the bulk solution (at 2.5 Hz acquisition rate), and ~10 single-molecule limited spots in any given movie frame (100 x 100 μm field of view). No additional accumulation of spots should be observed within 10 min