The overall goal of this procedure is to rapidly mix two solutions to start a biochemical reaction on the microsecond timescale. This is accomplished by first fabricating a microfluidic mixer. The second step is to bond a chip to a cover glass to seal the channels.
Next, the chip is mounted to a manifold that contains the solution reservoirs. The final step is to image the mixed solution using confocal fluorescence microscopy. Ultimately, the biochemical reaction is captured on the microsecond timescale by converting distance along the exit channel to time.
The main advantage of this technique over existing methods like stop flow mixing, is that mixing is not limited by turbulence and occurs in a few microseconds. This method can help answer key questions in the protein folding field, such as when does hydrophobic a collapse occur? Demonstrating the procedure will be ling woo, a postdoc for my laboratory To begin fabrication of microfluidic mixing chips.
First clean four inch FU silica wafers with fresh piranha solution as described in the written protocol accompanying this video. Next, use a low pressure chemical vapor deposition furnace to apply a polysilicon coating approximately one micron thick per every intended 10 micron depth of the final microfluidic channels. Following further treatment of the wafers as described in the text spin coat the wafers with a 900 nanometer thick layer of a Z 52 14 photo Resist then soft bake at 90 degrees Celsius directly on a hot plate for one minute or in an oven at 110 degrees Celsius for 30 minutes.
After aligning a photo mask, make a hard and vacuum contact by first exposing the photo resist to UV light for several seconds. Then develop the pattern in the photo resist as listed in the written protocol. Inspect the features on the wafer with a microscope and hard bake at 115 degrees Celsius directly on a hot plate for two minutes.
Next, DCU the wafers with Asher or oxygen plasma for 2.5 minutes at 100 watts RF power. Using a deep reactive ion etcher etch the poly silica layer all the way through to the fused silica below. Strip the remaining photo, resist and treat the wafer as directed in the text.
Measure the etch depth to ensure it is all the way through the poly coating. Using an oxide capable deep reactive ion etcher etch fused silica to a depth of approximately 10 microns per micron thickness of the poly coating. Clean the wafer with the matrix Asher for four minutes, a 100 watts RF power, and strip the polysilicon with a xenon di fluoride etcher prior to drilling holes into the wafer spin coat with a new one micron layer of photo resist to protect the channels during subsequent handling.
Then mount the wafer feature side down on a sacrificial glass plate using Aqua Bond 55. Taking care to keep the bonder free of bubbles cooler computer controlled diamond tip drill with micro 90 cleaning solution. This keeps small bits of glass from sticking to the channels, which then clog the chip during use.
Proceed to drill, entrance and exit holes in each chip. Rinse the wafer with deionized water using a syringe to inject water into each hole. Then soak in soapy water for an hour following removal of the photo.
Resist and bonder as described in the text. Rinse the wafer with deionized water and warm soapy water. Rinse each hole again with a syringe, avoiding the etched features after flowing nitrogen over the wafer.
Continue to dry in a vacuum oven set to 120 degrees Celsius. Inspect the holes to ensure they're free of grit and repeat cleaning steps as necessary. The following steps should be performed in the clean room.
Measure the depth of the channels with either an optical or mechanical surface profiler. Clean the wafer as well as a fuse silica Cover glass as described in the text in a clean room. Dry the wafer thoroughly with nitrogen gas for at least five minutes.
Then place the wafer feature side up on top of a stack of three to four clean room wipes placed on top of a hard flat surface such as a small pane of glass. Repeat with the cover glass. Pick up the cover glass by the edge and invert it such that the polish side is held face down over the wafer.
Align the flat edges carefully drop the cover glass onto the wafer and allow it to settle nudge horizontally only to adjust alignment. Push down with one finger on the center of the wafer. One should see a bonding front begin to radiate outward.
If necessary, apply additional pressure to other positions around the wafer to advance the bonding front. After placing the sealed wafers in a high temperature oven programmed with the temperature profile listed in the text, dice the wafer with a wafer dicing saw into individual chips. The manifold to hold the mixing chip and solutions can be made of plastic such as lexan or plexiglass, or aluminum.
For temperature control, make the chip to the manifold by small o-rings and hold in place with a retaining ring that allows a clear optical view of the center of the chip. The O-ring grooves should be shallower than standard to ensure good mating without applying too much pressure to the chip. Once the chip is mounted, add solutions to the center and side reservoirs, taking care to release any bubbles trapped at the bottom of the wells.
Place the manifold on an inverted microscope and examine the mixing region with the eyepiece or camera output. An incandescent flashlight placed on top of the manifold will provide enough light. Use a dye or high index of refraction solution in the center channel to view the jet.
Because the volumes used in this mixer are so small, the flow can be controlled with air pressure applied above the solution wells. To visualize the jet, start with the side channel pressure at zero pounds per square inch and slowly increase to equal pressure with the center channel. If one side channel is clogged, the jet will be pushed against one of the walls of the exit channel.
If a visible clog appears, it may be dislodged by applying pressure or vacuum to the exit channel with a syringe. If protein or other organic material clogs the chip, it can be cleaned by soaking and piranha solution overnight. Bring a coated laser beam into a research grade microscope using a dichroic mirror either inside or just outside the microscope.
Align the laser so that the focus can be seen in the eyepiece or camera somewhat near the mixing region. Fluorescence from the protein in the mixer is collected by the objective and sent through the dichroic mirror focused by a lens to a pinhole and imaged on a photon counter. Scan the chip using a pizo electric scanner in x and y to image the mixing region.
Choose a position on the jet and scan in X and Y to find the focus at the center of the channel vertically. The depth of field of the microscope is much less than the channel depth, and the flow rate is uniform in the channel except within one micron of the walls. Therefore, the temporal resolution of the observed fluorescence is determined primarily by the size of the confocal spot.
Scan horizontally within the exit channel in 100 micron increments along the jet observing for approximately one millisecond per position. Move the microscope stage by 80 to 90 microns along the jet to capture later times. Alternatively, light can be detected by a spectrograph and CCD after the dichroic mirror using a lens to focus the light onto the entrance slit.
Since data collection is slower than for the photon counter, typically the jet is imaged first with the photon counter and then points along the jet are measured with the spectrograph. Data from the photon counter is analyzed by summing three to five pixels around the jet at each position to create a plot of intensity versus distance. Time is calculated from distance using a calculated velocity based on the applied pressures.
As a final step, analyze the data from the spectrograph using one of the methods described in the text. Shown here is a contour plot of the intensity in the mixing region measured by the photon counter. The background in the exit channel outside of the jet should be close to the noise floor of the detector and is typically not subtracted.
A higher background may indicate poor alignment or that the protein is sticking to the walls of the channel. Conversely, a jet that looks kinked or crooked, especially near the mixing region, indicates a poor etch of the nozzles. A time resolved spectra is shown from the protein ACell coenzyme a binding protein or A CBP labeled with Alexa 4 88 and Alexa 6 47.
The green and red rectangles show the wavelengths designated as donor and accepted channels respectively. This data has been background subtracted to remove the dark charge and laser signal. The background signal was taken with the center channel pressure such as zero pounds per square inch.
The mixing time can be measured by measuring a rapid reaction that is much faster than mixing, such as tryptophan fluorescence quenching by potassium iodide. Because the jet is smaller than the optical resolution of the microscope, there is a large intensity drop in the mixing region. As the jet forms, this instrument response can be removed by making a control measurement in which no solution conditions change.
The ratio of mixing and non mixing experiments of N-Acetyl tryptophan amide fluorescence is displayed in this figure. The line shows the predicted concentration of potassium iodide from console multiphysics modeling and simulations. The mixing time as measured in the time for the concentration to decrease 80%is eight microseconds.
This plot shows the fret changes in the protein A CBP after dilution of denature from six molar to 0.06 molar. This data does not need a control experiment. Since the fret is already a ratio and the instrument response is already removed, the rapid jump near T equals zero represents a rapid change in fret signal within the mixing time.
After this development, this technique paved the way for researchers in the field of protein folding to explore the earliest steps in the formation of protein structure.
