Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

The overall goal of this procedure is to fabricate and operate a microfluidic device for protein crystallization X-ray diffraction data collection at room temperature. The main advantage of this technique is that crystallization inside the chip minimizes the mechanical disturbance of the protein crystals. These X-ray transparent chips are easy to produce, can be directly mounted on goniometers of most synchrotron beamlines, and make efficient use of available crystals when collecting X-ray diffraction data.

First, place a master mold of the X-ray chip design in a 10-centimeter Petri dish lined with aluminum foil. Mix together about 30 grams total of PDMS base and curing agent in a 10-to-one ratio, and pour the PDMS onto the master to a height of four millimeters. Degas the PDMS in a vacuum desiccator for five minutes, and blow air to remove remaining air bubbles on the surface of the PDMS.

Cure the PDMS in an oven at 70 degrees Celsius for one hour. Then, gently peel the cured PDMS mold from the master. Use a scalpel to cut away the excess PDMS.

Prior to X-ray chip fabrication, ensure that the workspace is arranged to allow easy access to all needed equipment and components during epoxy mold casting. To begin the chip fabrication, dilute the precursors of a two-component epoxy resin with ethanol to a final ethanol mass concentration of 40%by weight. Using a vortex mixer, mix epoxy resin and ethanol together.

Degas the PDMS mold in a vacuum desiccator for 30 minutes to ensure that the PDMS can later absorb small air bubbles from the epoxy resin. Cut a 70 millimeter by 70 millimeter piece of 7.5-micron thick polyimide foil, and wrap the foil around a 75 millimeter by 50 millimeter glass slide. Tape the edges of the foil to the back of the slide to form a flat, rigid polyimide surface.

Treat the slide-backed polyimide foil with oxygen plasma for 20 seconds. Then, incubate the foil in a 1%by volume aqueous solution of either APTS or GPTS for five minutes at 20 degrees Celsius. During silanization, thoroughly mix together the epoxy resin precursor solutions with a small spatula.

Transfer the degassed PDMS mold from the vacuum desiccator to a flat surface. Dry the silanized polyimide foil with pressurized air or nitrogen gas. Quickly apply a droplet of the resin mixture to each microstructure on the mold.

Place the slide-backed polyimide foil face down on the epoxy resin, and firmly press down on the slide. Cover the assembly with a plastic foil and a metal sheet. Place weights on the metal sheet to apply a pressure of up to 1.4 newtons per square centimeter to the assembly.

After 15 minutes, gently separate the glass slide from the epoxy-patterned polyimide foil, and wipe the polyimide foil until dry. Place plastic foil, metal sheet, and weights back on top. Allow the epoxy to cure at room temperature for one hour.

Afterwards, remove the weights, the metal sheet, and the plastic foil. Gently peel the patterned polyimide foil from the PDMS mold. Plasma treat the patterned foil for 20 seconds, and silanize the patterned foil with either APTS or GPTS, as previously described.

Silanize a pristine polyimide foil with the other silane solution under the same conditions. Dry both foils with pressurized air. Then, apply a drop of deionized water to a clean, dry, flat surface.

Cut individual chip structures on polyimide foil, and place the patterned polyimide foil epoxy side up on the drop of water, and carefully smooth out the foil until completely flat. Place the second polyimide foil on the patterned foil with the silanized face down. Gently hand smooth the second foil from corner to opposite corner to expel air bubbles and to bond the polyimide sheets together.

To begin fabricating the access ports, cut from a cured, four-millimeter PDMS slab a block of sufficient size to cover all ports in the polyimide X-ray chip without covering the crystallization compartment. Plasma treat, silanize, and dry the polyimide chip and the PDMS block. Press the treated block onto the chip over the ports.

Cover the chip with a plastic foil, a clean glass slide, and a metal sheet. Apply 1.4 newtons per square centimeter of pressure for one hour. Next, use a 0.75-millimeter biopsy punch to punch inlet and outlet holes where marked in the chip design.

Seal the back of the chip with polyimide film electrical insulating tape. Then, dilute a 9%by weight stock solution of transparent fluoropolymer coating material to 0.45%by weight with a fluorinated solvent. Connect a 27-gauge, 5/8-inch needle to the syringe.

Fit polytetrafluoroethylene tubing over the end of the needle. Load the fluoropolymer solution into a one-milliliter Luer lock syringe. Connect the other end of the tubing to the X-ray chip outlet.

Inject the fluoropolymer solution into the chip until all channels are filled, and remove excessive fluoropolymer solution by using a syringe filled with air. Place the filled chip with the flat side down on a hotplate at 190 degrees Celsius. Heat the chip for five minutes to evaporate the solvent, thereby coating the channels with the fluoropolymer.

Prior to the crystallization procedure, filter the chosen protein solution with a 0.2-micron filter. Centrifuge the solution for 15 minutes at 16, 100 times g and 20 degrees Celsius, and recover the supernatant for the crystallization experiments. To begin the crystallization procedure, combine equal volumes of a protein solution and its precipitant.

Draw about 20 microliters of this mixture into a syringe. Use a 27-gauge, 5/8-inch needle and PTFE tubing to connect the syringe to the inlet of the X-ray chip. Connect a syringe of fluorinated oil to the outlet in the same way.

While monitoring the chip under a microscope, fill the crystallization solution through the inlet into the chip with the parallel layout. Inject sufficient fluorinated oil to separate the crystallization compartments in the chip. Then, block both the inlet and outlet ports with paperclips or other narrow plugs.

Store the chip under the appropriate crystallization conditions. The crystal formation can now be observed under a microscope or by dynamic light scattering measurements. When ready for data collection, use double-sided tape to fix the X-ray chip to a 3D-printed adapter for the plate goniometer.

Mount the chip adapter on the plate goniometer at the synchrotron beamline. Collect and analyze the diffraction data. Bright field microscopy was used to quantify the evaporation rate of water from the crystallization chambers of the polyimide foil chip.

Dynamic light scattering could detect the initial nucleation of thaumatin crystals in a PDMS chip on a glass slide with equivalent geometry, allowing crystallization conditions to be optimized. 10 diffraction patterns were collected from each of 83 thaumatin crystals grown in a polyimide chip with a one-degree rotation during each frame. The dataset was split into five sub-datasets by frame, and the intensity decay of the normalized diffraction power over time was then evaluated.

By the fourth dataset, the diffraction power had decreased to below 50%The measurement R factor values of the sub-datasets increased over time, indicating that the crystals sustained radiation damage during data collection. This was attributed to free radicals generated during X-ray exposure degrading neighboring crystals in the same compartment. The bipyramidal thaumatin crystals had a broad distribution of orientations in the X-ray chip.

Orthorhombic glucose isomerase crystals likewise showed a broad distribution of orientations when grown in a polyimide chip. However, thioredoxin crystals predominantly had orientations in the xy, xz, and yz planes, potentially owing to their elongated shape. In general, these chips are very robust and have a low X-ray background.

This makes this fabrication method suitable for other X-ray imaging techniques, such as small-angle X-ray scattering. This procedure can be easily adapted to suit your experimental needs. For instance, the chip design can be modified by adjusting the dimensions of the individual compartments or the total number of compartments per chip.

A particular focus for us was to engineer a workflow that does not require cost-intensive equipment, like specialized tools or microfluidic pumps. After watching this video and reading the manuscript, you should have a good understanding how to crystallize in chip, to monitor the crystallization process, and to record and analyze diffraction data using the chip approach. Once mastered, this technique enables batch fabrication of microfluidic chips from a PDMS master in about three hours without requiring a dedicated cleanroom.