Characterizing Single-Molecule Conformational Changes Under Shear Flow with Fluorescence Microscopy

Quantifying the conformational changes in single bio-molecules under shear flow is useful for understanding the function and biophysics of macromolecules like protein and DNA. Fluorescence microscopy allows real-time in-situ visualization of single molecules in physiological flow environments with high temporal and spatial resolution. This is unmatched by other techniques like AFM.

By characterizing the shear force under which proteins our DNA have global conformational changes, one can better design drugs that mimic these biophysical properties. This protocol can be widely applied to study the behavior or other polymers, especially bio polymers, in varying flow conditions and to investigate the rheology of complex fluids. The timing of steps is critical to success.

We suggest practicing the microfluidic device surface treatment and fluorescence microscopy steps many times to execute them in an efficient fashion. Capturing conformational changes in biomolecules is a visual and dynamic phenomenon best viewed on film. Scientists will better understand this method after seeing the real-time protein and DNA microscopy presented in this video.

To prepare the microfluidic device, add five parts of silicone elastomer base to one part curing agent in a weigh boat. and stir the contents thoroughly for one minute to create a pre-cured PDMS solution. Place the master silicon wafer in a plastic Petri dish and pour the PDMS solution over the wafer to create a five-millimeter layer.

Cover the dish and leave it in a desiccator under vacuum for one hour to remove air bubbles. Then incubate the covered Petri dish at 60 degrees Celsius overnight to cure the PDMS into a flexible solid, which will result in microfluidic channels being molded into the PDMS at the PDMS wafer interface. On the next day, use a razor to cut 20 by 10 millimeter rectangles around each microfluidic channel in the PDMS and remove the rectangular blocks with tweezers.

Use a 25-gauge blunt end needle with sharpened edges to punch a 0.5 millimeter diameter hole at one end of the channel, making sure that the hole goes completely through the PDMS block. Use a thin needle to punch the PDMS out of the hole and repeat the process at the other end of the channel. Place the PDMS block with the channel side up and the coverslip into the chamber of a plasma bonding machine and start the treatment.

When treatment is complete, quickly place the PDMS block on a coverslip so that the channel is in contact with the slip. Apply pressure to the edges of the block, then place the coverslip PDMS assembly on a hot plate at 115 degrees Celsius for 15 minutes to reinforce the permanent bond. Finally, insert a 10-centimeter long.

0.25 millimeter inner diameter tube into the outlet hole at the top of the PDMS block to allow fluid to easily flow out of the channel. For von Willebrand factor or VWF experiments inject up to 10 microliters of 10 micrograms per milliliter BSA-Biotin dissolved in sterile 1X PBS into the inlet of the microfluidic device. Withhold a few microliters of BSA-Biotin in the pipette tip after injection and allow the tip to remain embedded in the inlet.

Allow the BSA-Biotin to incubate in the device for two hours which will result in BSA non-specifically binding to the coverslip surface. Then remove the pipette tip and inject up to 10 microliters of casein blocking solution into the channel. Allow the casein solution to incubate for 30 minutes so it can block any free sites and reduce nonspecific binding of the biomolecules to the surface.

Remove the tip and inject up to 10 microliters of streptavidin in sterile PBS into the channel, which will bind to the biotin groups of the BSA-Biotin. Next, inject up to 10 microliters of detergent solution to wash away excess streptavidin. Remove the tip and inject either VWF in casein solution or lambda DNA.

Incubate VWF for three minutes or lambda DNA for 45 minutes. Then inject five millimolar free biotin to block the excess streptavidin binding sites. Load one milliliter of casein blocking solution into a syringe and secure it in a syringe pump.

Then attach one end of the tubing to the syringe needle and flow in the solution to remove air bubbles. Approximately three minutes after the free biotin injection attach the other end of the tube to the inlet of the microfluidic device. Select the highest magnification objective of the TIRF or confocal fluorescence microscope.

If needed, add a drop of immersion oil on the objective. Place the microfluidic device on the microscope stage so that the coverslip is flush with the objective. Start bright-field microscopy and adjust the focus so that features such as debris and bubbles are visible.

Then adjust the stage in the X and Y directions until the edge of the microfluidic channel is visible and bisects the frame. Switch to the FITC channel and adjust the Z level and TIRF angle as needed until the individual green globular molecules can be distinguished. Adjust laser intensity and exposure time to visualize fluorescent molecules without photobleaching them too quickly.

Then adjust the contrast to better visualize the molecules. Exactly five minutes after free biotin injection start and stop flow from the syringe pump to observe the changes in molecular conformation using flow rates between 5000 and 30, 000 microliters per hour. With VWF it is critical to apply at least a small amount of flow, such as 30 seconds of 10, 000 microliter per hour flow exactly five minutes after free biotin injection.

Repeat this throughout various areas of the microfluidic device and locate molecules that can extend and relax upon multiple cycles of starting and stopping flow. Note how long it takes for molecules to reach maximum extension and complete relaxation and record videos of the continuous behavior of molecules under shear flow. The flexibility of a molecule to change confirmation is often demonstrated by its ability to lengthen as higher shear rates are applied due to increasing flow.

The extension versus shear rate curve of a von Willebrand factor molecule is useful for characterizing the biomechanical properties of the protein. Fluorescence microscopy images of lambda DNA similarly show increased extension upon higher shear rates at 30 seconds and gradual relaxation over two minutes after flow is stopped. When working with VWF, remember to incubate it only for three minutes and free biotin for five minutes before starting flow.

This is critical for forming the optimal number or biotin-streptavidin linkages needed for reversible unraveling. This method can be adapted to visualize real time interactions between multiple biomolecules and characterize their functions. For example, one could better understand platelet plug formation by visualizing VWF unraveling under high shear and its binding with the platelet adhesion molecule GPIb-alpha with this method.