This protocol shows how to pattern oxide free silicon in germanium with reactive organic monolayers using a highly efficient and operationally simple printing protocol. Selective functionalization of the pattern substrates with both small molecules and proteins is also demonstrated. The first step of the protocol is to covalently modify ated silicon or germanium substrates with a highly stable primary organic monolayer.
This protects the underlying inorganic organic interface from reactive degradation and oxidative damage. The covalently linked in hydroxy smide Ester overlayer is formed to provide latent hydrolytic and reactive functionalities As a second step, the uniform bilayered NHS modified substrates are patterned by catalytic micro contact printing using a pattern sul acid modified elastomeric stamp contact with the stamp hydrolyzes NHS groups, thus forming patterns of chemically distinct NHS activated and free carboxylic acids. Next, the pattern substrates are functionalized with small organic molecules and proteins.
This step is completed by first of fixing NI Trello. Tri acetic acid terminated hetero functional linkers to the NHS functionalized regions and second, by selective attachment of hexa histamine tagged green fluorescent protein. The approach produces excellent results with patterns of differential fluorescence intensity that are quite clear in a pattern of integrity that is remarkably stable after multiple surface modifications.
So the main advantage of this technique over existing methods is the accuracy. The pattern is really controlled by the accuracy of the stamp itself as opposed to diffusion. The project was originally initiated from the idea that patterning techniques that rely on catalytic reaction as opposed to simple deposition must have numerous advantages.
For one, the catalysts aren't consumed in the reaction and they can be reused multiple times. Unlike traditional printing or deposition where you continuously need to supply new patterning materials. In our early work, we actually tethered a catalyst molecule to an A FM tip, and then we drug that along the surface in a serial process like a machine tool to pattern.
But the elastomeric stamp was a logical extension for parallel manufacturing applications Ready. One of the most important aspects of the protocol is the use of the Bilayered molecular system. The system allows us to both ate and functionalize oxide free and organic substrates.
Ideally, the initial primary Sam should achieve complete termination of all surfaced exposed atoms and form a close packed molecular system, which can protect the surface from both oxidation and degradation. The secondary overlayer should contain terminal functional groups, which can be further modified with additional chemical transformations. The significant limitations to the resolution of traditional micro contact printing is the diffusion of the pattern and molecules.
Our methods license a chemical reaction between a catalyst currently mobilized on a stem and a substrate attached to silicon or germanium. Because of this properties, our technique experiments replication of very small 100 nanometer size features more. Or because the method creates chemical patterns, it is possible to functionalize them through specific reactions with different biological and organic molecules.
This procedure requires the use of several hazardous chemicals, such as hydrofluoric acid, nano chip solution, and phosphorous penta chloride. When working with these reagents, it's important to wear the proper protective clothing and work in a well-ventilated environment. The most challenging part of this protocol is to quickly move the chlorinated surfaces into the greenard solution.
In order to avoid reformation of the oxide layer, Begin by preparing a silicon one 11 wafer. Cut it into one centimeter square substrates, dust the substrates and rinse them with water and filtered ethanol. Next, remove any organic contamination by submerging the silicon substrates in a glass dish containing nanos.
Strip solution heated to 75 degrees Celsius. Wait 15 minutes, then rinse. Clean each substrate with deionized filtered water.
Give each substrate a five minute bath in a 5%HF solution to remove the native oxide layer and then dry the oxide free silicon with nitrogen. To produce a chlorinated substrate, immediately submerge each oxide free silicon piece in a scintillation vial containing two milliliters of saturated phosphorus Penta chloride in chloro benzene. After reaction is complete.
Let the vials cool at room temperature. Rinse each surface with chloro benzene and dry under filtered nitrogen. Next, place each chlorinated silicon surface in a pressure vial containing four milliliters of propanol Magnesium chloride.
Incubate the pressure vials at 130 degrees Celsius for 24 hours. Once the pressure vials have cooled to room temperature, rinse each surface quickly with di chloro methane and ethanol and dry under filtered nitrogen like the preparation of silicon substrate. Cut a germanium wafer into one centimeter squares and dust and rinse with water and filtered ethanol with germanium.
Remove organic contaminants by submerging the substrates in a dish of acetone for 20 minutes. Then submerge them in a 10%HCL solution for 15 minutes. Dry the substrates with nitrogen and place each chlorinated surface in a pressure vial containing four milliliters of octal magnesium chloride.
Incubate the vials at 130 degrees Celsius for 48 hours. After the incubation, allow the vials to cool to room temperature and quickly rinse each wafer with di chloro methane and ethanol. Dry the wafers under filtered nitrogen.
Begin by pipetting a few drops of NHS Diaz Solution onto the methyl terminated. Allow the solution to spread across the entire surface. Place the surfaces under a UV lamp for 30 minutes.
Then add more drops of NHS Diaz to the surface and let the reaction proceed. For an additional 30 minutes. Rinse the NHS modified surfaces with di chloro methane and ethanol, and dry them under filtered nitrogen.
Then proceed with functionalizing the small molecules. Finally, analyze the surfaces by XPS to determine the elemental composition. Begin the stamp preparation by mixing sodium tumor capto ethane sulfonate into 10 milliliters of four normal HCL solution in dioxane.
Stir the solution at room temperature for two minutes from the solution. Filter off the sodium chloride through a fine glass filter, and then through a 0.2 micron PTFE membrane syringe filter. Now take the clear solution of tumor capto ethane s phonic acid in dioxane and evaporate off the dioxane under reduced pressure.
React the resulting sul acid with two milliliters of polyurethane acrylate, pre polymeric mixture at room temperature, and then under vacuum at 50 degrees Celsius. Be sure to completely free the mixture from trapped arable To ensure the successful polymerization of pre polymeric mixture. It is important not to ever heated when reaction with me.
CAPTA atten phonic acid, and when the oxygenating on the vacuum, While the solution is viscous, pour it onto the pattern silicon master and cover with a flat glass slide wrapped in paraform. Then cure the mold by exposing it to UV light. After polymerization, remove the glass slide and para film and carefully peel the stamp off the master, cut the stamp to the appropriate size and wash it with ethanol and water.
Then dry it with filtered nitrogen. The following is the most important step to the protocol. Place the stamp on top of the NHS modified substrate with no external load to hold them together.
Do not ship the stamp or apply too much pressure. Wait one minute, then rinse the substrate and stamp with ethanol water and then ethanol again, followed by drying under filtered nitrogen. Store the stamps at room temperature to analyze the produced pattern.
Use lateral atomic force microscopy in contact mode and scanning electron microscopy. In this step, we mobilize GFP to the pattern silicon surface. We first start by modifying the activated ester with an NTA derivative, then immobilize the HIIN tag protein to the surface through nickel chelation.
It's important in this step to keep a biomolecule of interest at the appropriate temperature to avoid unwanted degradation. To attach proteins to the NHS pattern, to bifunctional substrate, submerge it in a solution of lysine, nnn dia athetic acid, and triethylamine. After an hour, rinse the substrates with water followed by ethanol.
Now incubate the substrates for five minutes in a chelating solution of nickel sulfate. Next, rinse the substrates with water and binding buffer, followed by submerging them in a bath of ice cold GFP solution. One hour later, rinse the substrates with the same binding buffer, followed by a rinse in PBS.
Then store the substrates in PBS at zero degrees Celsius prior to analysis. Finally analyze the surfaces by fluorescent microscopy to visualize GFP modified areas. Soft B lithographic nano patterning was used to create chemo selective patterns on oxide free silicon, and on germanium, the reaction between the NHS functionalized substrate in the catalytic pattern stamp leads to the hydrolysis of NHS moieties in areas of confirmational contact, yielding a pattern by functional substrate bearing regions of NHS activated and free carboxylic acids.
Due to the diffusion free nature of the method, the resolution is close to that of photolithography as seen in 125 nanometer features. These features were uniformly reproduced across the entire silicon substrate surface. The dimensions of the printed features were identical to those in the corresponding silicon master and the catalytic stamp.
Remarkably, the catalytic stamp can be reused multiple times without losing efficiency. Chemo selective functionalization of pattern semiconductors was accomplished by exploiting the differential reactivities of activated and free carboxylic acids. First niello trice acid terminated hetero bifunctional linkers were affixed to the NHS functionalized regions and then used to the resulting NHS pattern surface as a template for the selective attachment of hexa histamine tag G-F-P-N-H-S pattern.
Silicon was chemo functionalized with protein molecules. Using this approach under fluorescence microscopy, there was a clear intensity difference between GFP modified and hydrolyzed free carboxylic acid regions. The size and shape of the replicated features are consistent between both NHS patterned and GFP modified surfaces, confirming the remarkable stability of carbon passivated surfaces and the selectivity of the stamping approach.
The presented protocol is a form of ink, less micro contact printing that can be universally applied to any substrate capable of supporting simple well-ordered monolayers. Because the process does not rely on ink transfer from stamp to surface. The diffusive resolution limitation of traditional and reactive micro contact printing is eliminated.
Permitting routine manufacturing of nanoscale objects. The incorporation of a primary, highly ordered molecular system provides complete protection of the underlying semiconductor from oxidation damage. The formation of CHE selective patents provides specially resolved attachment points for a variety of biological and organic molecules.
By using different activities of free and activated carc cases, we were able to mobilize histo intact proteins on the created patterns. However, this method is not limited to histo intact proteins, and it can be used to immobilize other biomolecules such as DNAs and antibodies. After watching this video, you should have a good understanding of how to modify passivated silicon or germanium with a molecular system and cataly pattern.
The NHS modified substrates. While attempting this procedure, it's important to remember to work in a clean dust-free environment. It's also important to employ the necessary safety precautions when working with hazardous materials such as HF and nanos.Stripp.
