We describe a method for passivating a glass surface using polyethylene glycol (PEG). This protocol covers surface cleaning, surface functionalization, and PEG coating. We introduce a new strategy for treating the surface with PEG molecules over two rounds, which yields superior quality of passivation compared to existing methods.
Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.
When performing a single-molecule protein study it is essential to achieve a high quality of surface passivation so that the experiment is free from any surface-induced protein malfunction or denaturation 1,2. While a glass surface treated with surfactants, such as bovine serum albumin, is commonly used for single-molecule nucleic acid studies 3, the degree of its passivation is not high enough for protein studies. A glass surface coated with polymer (polyethylene glycol, PEG) is superior in the passivation performance 4-6. Thereby, it has become universally used for single-molecule protein studies ever since it was introduced to a single-molecule fluorescence study 7-10. The polymer coating procedure requires multiple surface treatments 7,11,12. Therefore, it is difficult to follow the whole procedure without detailed instructions. Often the degree of surface passivation varies depending on which protocol is followed. Here we present a robust protocol with step-by-step instructions, which will remove one of the major bottlenecks of single-molecule protein studies. See Figure 1 for overview.
1. Slide Preparation and Cleaning
A microfluidic chamber is composed of a quartz slide and a coverslip. In prism-type total internal reflection fluorescence (TIRF) microscopy, the surface of the quartz slide is imaged. Therefore, it is important to clean a quartz slide thoroughly using H2O, acetone, KOH, and piranha solutions. This multi-step cleaning eliminates fluorescent organic molecules on a surface which interfere with single molecule fluorescence measurements. Additionally, the piranha etching makes the quartz surface hydrophilic by generating hydroxyl groups. The free hydroxyl groups are essential for the amino-silanization reaction in Step 3.
2. Coverslip Cleaning
A microfluidic chamber is composed of a quartz slide and a coverslip. When a prism-type TIRF microscope is used, the surface of a coverslip is not imaged. Therefore, it is enough to clean a coverslip only with H2O and KOH. In case a coverslip needs to be imaged (e.g. via the objective-type TIRF microscopy), it is recommended to treat the coverslips with piranha solution (Step 1.7). Note that if PEGylation of the coverslip surface is not of high quality, it might act as a sink for proteins and give rise to variations in the protein concentration.
3. Amino-silanization of Slides and Coverslips
Functionalizing the surface of the quartz slides and the coverslips with amine group via the amino-silanization chemistry. Methanol is used as a solvent and acetic acid as a catalyst for the amino-silanization reaction.
4. Surface Passivation Using Polymer (The First Round)
Passivating the amine-coated surface of quartz slides and coverslips by conjugating NHS-ester polyethylene glycol (PEG). This reaction is carried out with the saturating concentration of PEG solution at pH 8.5 overnight.
5. Long-term Storage
Storing the PEGylated slides and coverslips in N2 at -20 ºC.
6. Surface Passivation Using Polymer (The Second Round)
Additional round of PEGylation to make the PEG layer denser and also to quench any remaining amine groups on the surface. The use of short NHS-ester PEG molecules (333 Da) may be effective in penetrating into an existing PEG layer. It is recommended to do this second round of PEGylation right before using a slide.
7. Assembling a Microfluidic Chamber
Assembling a microfluidic chamber using a pair of a PEGylated quartz slide and a coverslip. Double-sided sticky tape is used as a spacer. The chamber is sealed with Epoxy glue and, solutions are introduced through the holes in the quartz slide.
8. Slide Recycling
Recycling quartz slides. Used slides are recycled by taking off the coverslips and the double-sided sticky tapes.
After the microfluidic chamber assembly (Step 7.1 – 7.6) and before carrying out Step 7.7, it is advised to carry out the quality control of the PEGylated surface.
If the surface passivation has been done successfully, there are less than 10 non-specifically adsorbed proteins per imaging area (25 mm x 25 mm) observed when 1-10 nM fluorescently labeled protein is added into the chamber (Figure 3a, left).
When any of the cleaning or reaction steps has not been properly carried out, the number of non-specifically adsorbed proteins significantly increases, and the CCD screen may be saturated by fluorescence signals. For example, if the piranha etching is skipped, there is 100 times a larger amount of non-specific adsorption observed (Figure 3a; compare left with middle). When the second PEGylation step is skipped, there was approximately 3 times a larger amount of non-specific adsorption observed (Figure 3b). A low degree of PEGylation is observed when expired chemicals (e.g. APTES stored at room temperature for several months) are used (data not shown). The quality of the surface also drops down when a significant amount of time has passed by since it was PEGylated (Figure 3a; compare left with right).
To our best knowledge, it is the first time that the two rounds of PEGylation are introduced for single-molecule studies. The two rounds of PEGylation guarantee the highest quality of PEG layer formation (Figure 3b). The superior nature of the double PEGylation is prominently shown in the movies (Compare Movie 1a with Movie 1b). In these movies, the background signals from fluorescent molecules in solution are observed to be much weaker when the double PEGylation was used, which indicates that proteins are repelled more effectively by the doubly PEGylated layer. Though this two-step process is strongly advised, the second PEGylation step might be skipped if your experiment is tolerable to non-optimal passivation.
Figure 1: Schematic of the surface treatments. (a) A microscope slide is cleaned with acetone, KOH, and piranha solutions. It is functionalized with APTES and PEGylated with NHS-ester PEG. (b) A coverslip is cleaned with KOH, as well as with the piranha solution if necessary. It is functionalized with APTES and PEGylated with NHS-ester PEG.
Figure 2: Microfluidic chamber. (a) A single-channel chamber. The microscope slide has two holes drilled. It is assembled with a coverslip using two slices of a double-sided sticky tape. (b) A three-channel chamber. The microscope slide has six holes drilled. It is assembled with a coverslip using four slices of a double-sided sticky tape.
Figure 3: CCD images taken with dye-labeled proteins in solution. (a) CCD images were taken by prism-type total internal reflection fluorescence microscopy using a 60X objective lens with 10 nM Cy3-labeld Rep in solution. (Left) A surface was prepared following the protocol in this article. (Middle) A surface was prepared following the protocol in this article, but piranha etching was skipped. (Right) A surface was prepared following the protocol in this article and stored for 3 months at -20 ºC under nitrogen. (b) CCD images taken with 10 nM Cy3-labeld Rep in solution. (Left) A surface was prepared following the protocol in this article. (Right) A surface was prepared following the protocol in this article, but the second round of PEGylation was skipped. Scale bar = 5 µm.
Movie 1: CCD movies taken with dye-labeled proteins in solution. CCD movies were taken by prism-type total internal reflection fluorescence microscopy using a 60X objective lens with 10 nM Cy3-labeld Rep in solution. The time resolution is 100 msec. (a) A surface was prepared following the protocol in this article. (b) A surface was prepared following the protocol in this article, but the second round of PEGylation was skipped. Click here to view Movie 1a and click here to view Movie 1b.
Critical steps within this protocol
It is essential to make the surface hydrophilic before the amino-silanization reaction. This was achieved through the piranha etching which generates the free hydroxyl groups on a glass/quartz surface. It is recommended not to keep the piranha etched surface exposed to either H2O or air for a long period of time since the hydrophilicity of the surface goes down gradually.
The NHS-ester PEG molecules are reactive. It is advised to make aliquots and store them under nitrogen in -20 ºC. The shelf life of the APTES chemical at the room temperature is short. It is advised to replace it with a new one every month.
Modifications of this protocol
When you cannot use the piranha etching for any practical reason, you may etch using KOH for a long period of time (e.g. overnight), which will also make hydroxyl groups exposed. The pitfall of this alternative approach is that the slide becomes unusable after a few recycles due to severe scratches.
It is often practiced to burn a glass/quartz surface using propane torch, which is effective in eliminating fluorescent organic materials 7. This procedure was not included in this protocol because it is redundant to the piranha etching. Note that this procedure will potentially lead to oxidation of the hydroxyl groups. Therefore, this procedure should not be carried out once a surface was etched using KOH or piranha solution.
When a buffer with pH lower than 7 is used for single-molecule imaging, the conformation of PEG changes from “mushroom” to “brush,” which reduces the degree of passivation. Therefore, when pH lower than 7.0 is used, it is recommended to further passivate the surface using disuccinimidyl tartarate 7.
Perspectives
This work has provided a robust protocol for achieving high quality surface passivation. This protocol will be useful for single-molecule fluorescence studies that are involved with proteins 14 as well as protein complexes within cell extracts and immunoprecipitates 15. It will be widely used for other single-molecule techniques such as force spectroscopy and torque spectroscopy 16. It will be also useful for preventing cells from adsorbing to a surface 17.
The protocol provided in this work is demanding for its multi-step procedures. Surface passivation using lipid-PEG 8 and poly-lysine PEG 9 are available as an alternative approach. Since, they do not require any chemical reactions it is easy to implement. However, the degree of passivation is not as high as that achieved through chemical modification of a surface.
The authors have nothing to disclose.
S.D.C., A.C.H., and C.J. were supported by Starting Grants (ERC-StG-2012-309509) through the European Research Council. J.-M.N. was supported by the National Research Foundation (NRF) (2011-0018198) of Korea; and the Pioneer Research Center Program (2012-009586) through the NRF of Korea funded by the Ministry of Science, ICT, and Future Planning (MSIP). This work was also supported by Center for BioNano Health-Guard funded by MSIP of Korea as the Global Frontier Project (H-GUARD_2013-M3A6B2078947). Y.K.L. and J.-H.H. were supported by the Seoul Science Fellowship Program of Seoul City, Korea. The labeled Rep protein was a generous gift from Dr. Sua Myong.
1. Slide preparation and cleaning | |||
Acetone | Sigma | 32201 | 1 L |
Coverslips | VWR | 631-0136 631-0144 631-0147 | 24x32mm2, 22x40mm2, 24x50mm2 (No.1½, Rectangular) |
Diamond drill bits | MTN-Giethoorn, The Netherlands | LA-0564-00DB | 3/4 mm |
Duran slide holder | LGS, The Netherlands | 213170003 | |
Glass staining dishes | Fisher Scientific | 300101 | |
H2O2 | Boom | 6233905 | Opened bottles should be stored at 4 °C. 1 L, hydrogen peroxide 30% |
H2SO4 | Boom | 80900627.9010 | Sulfuric acid 95-98% |
KOH | Sigma | 30603 | Potassium hydroxide |
Methanol | Sigma | 32213 | 1 L |
Sonicator | Branson | Tabletop ultrasonic cleaner, 3510 | |
Quartz slides | Finkenbeiner | 1” x 3”, 1 mm thick | |
2. Coverslip cleaning | |||
Duran slide holder | LGS, The Netherlands | 213170003 | |
KOH | Sigma | 30603 | Potassium hydroxide |
Sonicator | Branson | Tabletop ultrasonic cleaner, 3510 | |
3. Amino-silanization of slides and coverslips | |||
Acetic acid | Sigma | 320099-500ML | Acetic acid, glacial |
APTES | Sigma-Aldrich | 281778-100ML | Store at the room temperature under N2 . Replace once in a month. 3-aminopropyl trimethoxysilane |
Flask (200mL) | VWR | Flask (200 mL) | Have one flask dedicated for aminosilanization. Pyrex flask |
Methanol | Sigma | 32213 | 1 L |
Sonicator | Branson | Tabletop ultrasonic cleaner, 3510 | |
4. Surface passivation using polymer (the first round) | |||
Biotin-mPEG | Laysan Bio | Biotin-PEG-SVA-5000-100mg | Store aliquots of 1-2 mg (enough for 10 slides) at -20 °C under N2. Biotin-PEG-SVA, MW 5,000 Da |
mPEG | Laysan Bio | MPEG-SVA-5000-1g | Store aliquots of 80 mg (enough for 10 slides) at -20 °C under N2. mPEG-succinimidyl valerate (SVA), MW 5,000 Da |
Sodium bicarbonate | Sigma | S6014-500G | |
6. Surface passivation (the second round) | |||
DMSO | Sigma-Aldrich | D8418-100ML | Dimethyl sulfoxide BioReagent, for molecular biology, ≥99.9% |
DST | Pierce | 20589 | Disuccinimidyl tartarate |
MS4-PEG | Pierce | 22341 | Dissolve 100 mg MS4-PEG in 1.1 mL DMSO. Store 20 mL aliquots at -20 °C. Short NHS-ester PEG, MW 333 Da |
Sodium bicarbonate | Sigma | S6014-500G | |
7. Assembling a microfluidic chamber | |||
Double sticky tape | Scotch | 10mm wide | |
Epoxy | Thorlabs | G14250 | Devcon 5 minute epoxy |
NaCl | Sigma-Aldrich | S9888-1 KG | Sodium chloride |
Neutravidin | Pierce | 31000 | Stock in 5 mg/mL in H2O at 4 °C. ImmunoPure NeutrAvidin Biotin-Binding protein |
Streptavidin | Invitrogen | S-888 | Stock 5 mg/mL in H2O or T50 at 4 °C. |
Trizma base | Sigma-Aldrich | T1503-1 KG | Tris |