Surface Passivation for Single-molecule Protein Studies

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.

The overall goal of this procedure is to yield a ated quartz slide surface of superior quality. This is accomplished by first cleaning drilled quartz slides and cover slips with water and acetone. Then the slide is etched with potassium hydroxide and the piranha solution to enhance the quality of the surface.

Next, the slides and cover slips are functionalized with amine groups followed by two rounds of surface ation using a polymer to make the glass surface inert. The final step is to assemble the microfluidic chamber from the slide and cover slip with the polyethylene glycol coated or pegylated surfaces facing each other. Ultimately, a new strategy of treating the surface with peg molecules over two rounds remarkably improves the quality of passivation as observed by single molecule fluorescence microscopy.

The main advantage of this technique or the existing techniques such as polymer coating after potassium oxide etching is this technique guarantees superior quality. People new to this method will struggle since multiple service treatments have to be performed. The to begin drill, the quartz slides as detailed in the text protocol.

Clean the slides by placing the slides in a glass staining jar. Typically five to 15 slides can be placed in a single jar. Then rinse the slides with UE water after repeating the rinse three times, sonicate the slides with UE water for five minutes to remove dirt, dispose of the water and rinse the slides three additional times with UE water.

Next, replace the UE water with acetone and sonicate the slides with acetone for 20 minutes or longer, discard the acetone and rinse the slides with Milli Q water to remove any acetone residue. Next, replace the water with one molar potassium hydroxide and sonicate the slides for 20 minutes or longer. Excessive etching will enhance the quality of the surface, but will introduce scratches which might interfere with fluorescence imaging.

Rinse the slides with Milli Q water three times to remove traces of potassium hydroxide to perform piranha etching. Transfer the slides to a Duran slide holder or a custom made Teflon holder, and place them in a one liter beaker that is located in a chemical hood. Fill the beaker with 450 milliliters of sulfuric acid.

Then add 150 milliliters of hydrogen peroxide for a three to one ratio between sulfuric acid and hydrogen peroxide. Stir the solution for proper mixing and leave the beaker undisturbed for 20 minutes. Take the slides out of the piranha solution and put them in a slide holder containing millieu water.

Meanwhile, discard the piranha solution into a designated waste bottle Once it reaches room temperature, rinse the slides three times with UE water following cover slip cleaning. Replace the UE water in the staining dishes with methanol. Keep the slides and the cover slips in methanol.

Meanwhile, prepare the amino islandization solution by pouring 100 milliliters of methanol into the flask. Then add five milliliters of acetic acid and three milliliters of three amino propyl, trimethyl, and gently mix by shaking. Replace the methanol in the staining dishes containing the slides and the cover slips with the amino siloization reaction mixture.

Incubate for 20 to 30 minutes during the incubation sonicate once for one minute. Next, replace the amino islandization reaction with methanol. Then discard the methanol and add a fresh methanol solution.

Repeat this procedure three times for the first round of surface ation Using polymer first, dry the slides and the cover slips using nitrogen gas. Then place them in clean pipette boxes partially filled with milli Q water in such a way that the side, which has to be pegylated is facing up. Next, add 64 microliters of the freshly prepared reaction buffer to a prepared peg solution.

Pipette the mixture up and down in order to dissolve completely. Then centrifuge at 16, 100 G for one minute. To remove air bubbles, drop 70 microliters of the pegylation mixture onto a dried quartz slide.

Gently place a dried cover slip over the solution, incubate the slides in a dark and humid environment for two hours to overnight to store the pegylated slides and cover slips. Carefully disassembled the slide and the cover slip by sliding the cover slip to his side and rinsing them with UE water before drying them with nitrogen gas. Place a pair of a slide and cover slip in a 50 milliliter tube such that the pegylated surfaces are facing away from each other.

Partially close the tube and vacuum it before filling it with nitrogen gas and screwing the lid on tightly. Store it at negative 20 degrees Celsius. These steps help preserve the pegylated surface for a long period of time.

Perform a second round of pegylation as described in the text protocol immediately before using the slide to assemble a microfluidic chamber. Place a quartz slide on a flat surface with the pegylated side facing up. Make a channel diagonally on the pegylated surface by putting double-sided sticky tape over the slide.

Make sure that the holes are positioned at the center of the channel. Then gently place a pegylated cover slip on top to complete the chamber. With the pegylated side facing down.

Seal the chamber by pressing the cover slip over the areas where the double sided tape is placed. Do it gently but thoroughly so that the chamber becomes sealed water tight. Finally, close the edges of the chamber with epoxy glue.

Proceed to immobilize streptavidin or neut travain and add biotinylated biological molecules for single molecule imaging as described in the text protocol, if the surface ation has been done successfully, there are less than 10 non-specifically absorbed proteins per imaging area observed. When one to 10 nanomolar fluorescently labeled protein is added into the chamber, when any of the cleaning or reaction steps has not been properly carried out, the number of non-specifically absorbed proteins significantly increases. For example, if the piranha etching is skipped, there is 100 times more non-specific absorption observed.

The superior nature of the double pegylation is prominently shown here. The background signals from fluorescent molecules and solution was observed to be much weaker when double pegylation was used as shown in the left panel. This indicates that proteins are repelled more effectively by the doubly pegylated layer.

Once mastered, this technique can be done in four hours if performed properly. Don't forget that working with AYA can be extremely hazardous precautions such as proper gloves, goggles and apron should always be taken while performing this procedure.