Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The overall goal of the following experiment is to prepare two types of substrates for DNA NANOSTRUCTURE experiments, MICA and functionalize silicon oxide. This is achieved two different ways for the two substrates. The top layer of MICA is removed using double-sided tape in a quick and easy process.

Conversely, silicon substrates must be cleaned using RCA cleaning, which entails etching of the oxide layer and removal of both metal and organic contaminants. This produces a clean, smooth silicon oxide layer that can be further functionalized. Next, the MICA is flooded with magnesium ions to create a positively charged surface.

An analogous surface is created on the silicon oxide substrate through the formation of a positively charged self-assembled monolayer, such as three amino propyl triethyl. Next, the substrate is exposed to the DNA origami solution in order to promote adhesion of the negatively charged DNA with the positively charged substrates. Coverage is dependent on substrate type solution concentration and deposition time factors that can be varied depending on the experiment performed.

The main advantage of this technique is that the APT is monolayer, is covalently attached to the silicon surface. It can be deposited in a specific pattern through ebeam, lithography, and a technique called molecular liftoff. Generally, individuals new to this method will struggle with unique manipulation techniques needed for the small substrates, as well as perfecting the appropriate protocol for handling deposition and drawing.

Visual demonstration of this method is critical as the substrate manipulation and RCA cleaning and safety protocol steps are difficult to describe in text. Visual portrayal of this necessary safety steps is important to promote safe laboratory practices. First, prepare the substrate by cutting one centimeter by one centimeter squares out of the MICA substrate with scissors.

Cleave the MICA using double-sided tape. Place the MICA rectangle on the double-sided tape still in the tape dispenser. Carefully slide the tweezers between the MICA and the tape and remove the mica.

The top most layer will be removed and remain on the tape. Then pipette four microliters of adequately vortex DNA origami solution onto the mica, ensuring that the pipette tip does not touch the substrate. Leave the DNA origami on the MICA for approximately 10 minutes.

To ensure adequate coverage, rinse the DNA origami solution off of the MICA substrate using 100 microliters of sterile water over sink or other liquid receptacle. To do this, pick up the MICA using tweezers. Pipette the water on the substrate with the flow of the drop towards the tip of the tweezers.

Dry the substrate with a steady stream of nitrogen for one minute. Repeat the RINs with an additional 100 microliters of sterile water before drying the substrate with nitrogen for an additional three minutes. For the Radio Corporation of America, RCA one solution preparation plays 50 milliliters of 18 mega ohm centimeter water into the designated RCA one beaker using a measuring beaker, then add 15 milliliters of concentrated Imodium hydroxide to the beaker.

Rinse the measuring beaker with 25 milliliters of water and add the rinse water to the RCA one beaker. Turn on the heat and stir her on the hot plate and bring the RCA one bath to 70 degrees Celsius. Then add 15 milliliters of 30%hydrogen peroxide to the RCA one beaker.

Be sure to use the RCA one solution within one hour after the hydrogen peroxide has been added. Rinse the measuring beaker thoroughly with water and discard the rinse in an appropriate RCA one waste bottle for the Radio Corporation of America. RCA two solution preparation.

Add 70 milliliters of 18 mega ohm centimeter water to the designated RCA two beaker using the thoroughly written measuring beaker. Next, add 15 milliliters of concentrated hydrochloric acid. Rinse the measuring beaker with 20 milliliters of water and add it to the RCA two beaker.

Increase the heat and stir speed of the hot plate until the solution reaches 70 degrees Celsius. Then add 15 milliliters of 30%hydrogen peroxide. Like the RCA one bath.

Use a solution within one hour from when the hydrogen peroxide is added. To prepare the hydrogen fluoride solution, place 50 milliliters of water in an inert fluorinated polymer beaker. Measure four milliliters of concentrated hydrofluoric acid in the plastic measuring beaker and add it to the inert fluorinated polymer beaker.

Rinse out the plastic measuring beaker with a total of 50 milliliters of water, adding the rinse water to the hydrogen fluoride beaker. Finally, wash out the measuring beaker thoroughly with water and discard the washings in a designated hydrogen fluoride waste container. Cut the silicon wafer into chips by placing the silicon wafer polish side up on a soft surface such as a napkin.

Using the diamond tipped scribe pen, gently nick the bottom of the wafer along the primary flat edge. Place a small wire such as a paperclip below the nick and gently apply pressure to the wafer by placing fingers or tweezers on either side of the nick and pushing down on another napkin with a pencil and ruler. Measure out the desired width of the squares by marking dots on both the top and bottom of the napkin.

Place one of the wafer halves edge first between the measured lines on the napkin, flush against the line and repeat the cutting procedure. The freshly broken perpendicular pieces should now be the width of the cleats squares. Turn the wafer horizontally on the napkin and place it between the perpendicular lines before repeating the cutting procedure again.

When the RCA solution has reached the appropriate temperature submerge eight to 10, one centimeter by one centimeter silicon chips in the solution for 10 to 20 minutes, using an inverted fluorinated polymer basket with a two inch diameter lift the basket containing the silicon chips up and drain. Well then move the basket over to the waste beaker and rinse thoroughly with 18 mega centimeter water. Immerse the wash beaker and jiggle up and down for 20 seconds.

Drain the basket and rinse thoroughly with water over the waste beaker. After RCA one cleaning is complete, place the basket into the beaker containing hydrogen fluoride solution for 10 to 20 seconds. Use a gentle up and down motion to mix the chips and hydrogen fluoride.

Then lift the dunk bucket to allow the hydrogen fluoride to drain completely away. Place the wash beaker and rinse the beaker in a plastic tub. Move the basket over the rinse beaker and rinse with 18 mega ohm centimeter water.

Submerge the basket into the wash, beaker, and agitate for 20 seconds. Next, complete a second drain and rinse cycle with 18 mega ohm centimeter water. Dump the wash water into the rinse beaker and refill the wash beaker with water.

When the RCA two solution has reached the appropriate temperature, submerge the silicon chips in the solutions using the basket. Leave the chips in the solution for 10 to 20 minutes. Remove the chips after the appropriate amount of time and follow the same rinsing procedures.

As for RCA one store clean chips under high purity water in a clean vial for up to three days for self-assembled monolayer formation on silicon, add 1, 980 microliters of 18 mega ohm centimeter water and 20 microliters of apte to a clean simulation vial and swirl. To mix, use the solution immediately. Place a clean silicon chip reflective side up in the scintillation vial, cap it and let it sit for 20 minutes.

Remove the chip using tweezers and rinse with 200 microliters of water before drying it for one minute with a stream of nitrogen gas. To deposit the DNA origami on the APT test functionalized silicon, briefly mix the DNA origami vial and pipette four microliters of solution onto the silicon substrate. Let the solution stand for the amount of time necessary for the concentration used and the coverage desired.

Then rinse a substrate with 100 microliters of sterile 18 mega ohm centimeter water and dry with nitrogen flow. For one minute, repeat the rinsing with an additional 100 microliters of sterile water and dry the substrate with nitrogen for three minutes. Representative results for varying the DNA origami concentration on substrate coverage are shown here.

Increased concentration gives increased coverage on MICA substrates, DNA origami coverage on MICA and silicon Exhibit a high initial absorption with saturation occurring after approximately one hour. The percent coverage on MICA is also consistently higher than for the same concentration and time on functionalized silicon thorough sample. Rinsing and drying protocol is imperative for the successful preparation of DNA origami samples.

Ineffective rinsing and drying can lead to the presence of solvent aggregation islands of DNA origami or bridging of nanostructures on high coverage samples. Once mastered, the Micah substrate preparation techniques can be completed in 20 minutes or less. Silicon samples can be prepared in less than two hours if it is performed properly.

Electron beam lithography and molecular liftoff can be integrated with this process to create novel patterns and devices for further experiments. Don't forget that working with hydrofluoric acid can be extremely hazardous, take appropriate precautions such as wearing personal protective equipment and having emergency supplies of calcium gluconate available while performing this procedure.