This protocol is important for regulating the thickness of the picture size at the nano scale to successfully observe proteins and nano materials of different sizes using cryo EM.The Mem's fabrication technique allows for the mass production of the microchip. It also enables the selection of depths and designs of the micro pattern wells. Depending on the experimental purposes.
The technique can contribute to enhancing the efficiency of high throughput 3D structure analysis of biomolecules which is utilized for commercial drug discovery, and then thin wafers and microchips with risk tending silicon nitro membranes can be tricky. It is important not to bend the wafer or apply forces perpendicular to the silicon nitrate window. Begin to pattern the photo resist, or PR by covering a silicon nitride deposited silicon wafer with hexamethyldisilazane solution and then spin coating the wafer at 3000 RPM for 30 seconds on a spin coder.
Bake the coated wafer at 95 degrees Celsius for 30 seconds on a hot plate to render the water surface hydrophobic and ensure a good coating performance with the PR.Next, spin coat the wafer with a positive PR and bake it 100 degrees Celsius for 90 seconds. A spin coated PR has a thickness of 500 nanometers. Expose the PR coated wafer to ultraviolet light for five seconds through a chromium mask using an aligner.
Develop the PR for one minute using a developer and rinse twice by immersing the wafer in deionized water. Then dry the PR patterned wafer by blowing nitrogen gas onto the water surface. Following the patterning of the PR.Use a lab built reactive ion etcher at a radio frequency power of 50 watts, and with sulfur hexafluoride gas at three standard cubic centimeters per minute.
To etch the exposed silicon nitride at the rate of six angstroms per second. Eliminate the PR by immersing the silicon nitride patterned wafer in acetone at room temperature for 30 minutes. Followed by rinsing the wafer twice in deionized water and drying the wafer with nitrogen gas.
To etch the exposed si, immerse the silicon nitride patterned wafer in freshly prepared potassium hydroxide solution. With continuous stirring until the free standing silicon nitride windows can be observed at the opposite side of the patterned wafer. Clean the etched wafer by dipping it several times in a deionized water bath.
Then dry the wafer in the air. To eliminate the etching residues, lightly press the boundaries of the chip array with a tweezer and obtain an array of chips to be micro patterned. Then immerse the chip array in freshly prepared potassium hydroxide solution for 30 seconds, followed by rinsing twice, blowing the chips with nitrogen gas and drying them in the air for one hour.
For solid support prepare a blank 525 micrometer silicon wafer with the spin coating as demonstrated earlier. Attached to the chipper ray on the silicon wafer before baking the wafer and follow the procedure as described earlier to obtain the micropattern silicon wafer. Eliminate the PR by immersing the pattern chip set in one methyl two pure littanol solution at 60 degrees Celsius overnight.
The next day, rinse the chip set twice with deionized water. After drying the pattern chip set with nitrogen, eliminate the PR residues with an oxygen plasma process using 100 standard cubic centimeters per minute of oxygen gas at a radio frequency power of 150 watts for one minute with the reactive ion etcher. Later, immerse the micro patterned chips in potassium hydroxide solution for 30 seconds to fully eliminate the PR residues.
Then rinse and completely dry the chip set. Dilute two milligrams per milliliter graphene oxide or solution 10 folds with deionized water and sonicate the diluted solution for 10 minutes to break up aggregates of the sheets. Then centrifuge to the diluted solution at 300 times G for 30 seconds at room temperature.
Use a glow discharger at 15 million amperes to glow discharge the silicon etched side of the micro pattern chip for one minute and render the chip's surface with a positive charge. When done, drop three microliters of the solution onto the glow discharge side of the micropattern chip. After one minute, blot the excess solution on the chip with filter paper.
Wash the transferred chip with deionized water droplets on a paraffin film and blot the excess with filter paper. Repeat the drop casting procedure twice on the transferred side and once on the opposite side. Dry the transferred chip at room temperature overnight.
During the photolithography procedure the designs of the micro pattern chips were manipulated using different designs of the chromium mask. The numbers and the dimensions of the free standing silicon nitride membranes were controlled. It was observed that the fabricated micro patterned chips could have up to 25, 000 suspended holes.
The ramen spectrum at the window displayed the representative peaks of the Additionally, the multiply oriented hexagonal defraction patterns indicated that the windows consisted of the multilayer The structure and depth of the micro hole with windows were studied with scanning electron microscopy and atomic force microscopy. A well type structure of the micro hole with the window was observed in the images confirming the possibility of the design of the micro pattern chip with windows. With the help of the micro patterned chip, a few biological specimens and inorganic nanoparticles were imaged with the cryo electron microscope.
Optimizing the conditions such as pure coating thickness using intensity and developing time for micro patterning depending on the size and design of the patterns is important. By applying nano pattering techniques such as FIB or even litho graphene smaller some micrometer patterns can be produced which may expand applications of this micro device where it's used with other analytical techniques.
