This method allows fabrication of three-dimensional chiral plasmonic assemblies with strong chiroptical responses. The main advantage of this approach is that it enables fabrication of complex metal nanostructures using freely available software tools and common biochemistry lab equipment. Demonstrating the procedure will be Yike Huang a graduate student and Minh-Kha Nguyen a postdoc from my laboratory.
Use caDNAno to design a DNA origami template. Route the scaffold in staple strands according to the desired shape of the template. Then generate the staple strand sequences by clicking sequence tool.
Click paint tool and mark the staple strands that require further modification. Click export tool to export the DNA staple sequences to a CSV file. Then import the CSV file into a spreadsheet application.
Add a polyadenine sequence at the end of the staples to be used as handles for gold nanorod assembly. Modify the staple strands on the designed lock sites with lock sequences. Prepare a working stock of staple strands including strands with handles and locks by mixing equal amounts of concentration normalized staple oligonucleotides.
Then prepare the origami mixture as detailed in the text protocol. Anneal the mixture in the thermocycler from 80 degrees to 20 degrees Celsius. For a 1%gel, dissolve one gram of agarose in 100 milliliters of TBE by heating the mixture in the microwave oven.
Add 10 microliters of 10, 000X DNA stain according to the stain specification. To minimize the exposure to UV light at the extraction step use a DNA stain that can be visualized under blue excitation. Cast the gel and incubate for 30 minutes at room temperature.
Then place the gel box in an ice water bath. Add loading buffer to the origami samples and load the samples into the wells with a proper volume according to the cone used. Run the electrophoresis for two hours at 80 volts.
Image the gel with the gel imager. Use a blue light transilluminator to visualize the bands and cut the origami band. Then smash the gel on parafilm and extract the liquid.
The recovery yield is approximately 40%Pipette the liquid into a centrifugal filter unit and spin at 3, 000 times G for five minutes. Measure the absorption of the origami solution at 260 nanometers with a UV visible spectrometer. To prepare the gold nanorods, dissolve 0.55 grams of CTAB and 0.037 grams of 2, 6-Dihydroxybenzoic acid in 15 milliliters of warm water in a round bottom flask.
After cooling down the solution to 30 degrees Celsius, add 600 microliters of four millimolar silver nitrate and stir at 450 RPM for two minutes. Leave the solution undisturbed for 15 minutes at 30 degrees Celsius. Next, add 15 milliliters of one millimolar hydrogen tetrachloroaurate to the solution and stir at 450 RPM for 15 minutes.
Also, add 120 microliters of 64 millimolar L-ascorbic acid and immediately stir at 1, 200 RPM for 30 seconds. Now, add 12 microliters of gold seeds and keeps stirring at 1, 200 RPM for 30 seconds. Incubate the solution in a water bath at 30 degrees Celsius for 18 hours.
Do not disturb the solution and use a cap to close the flask. Transfer the resulted solution to centrifuge tubes and centrifuge at 9, 500 times G for 12 minutes at 20 degrees Celsius. Discard the supernatant and disperse the pellet in 20 milliliters of ultra pure water.
Perform one more centrifugation step and then disperse the final pellet in three milliliters of distilled water. Estimate the concentration of gold nanorods from a UV visible absorption measurement using the extinction coefficient for the longitudinal plasmon resonance. Mix 150 microliters of 10 nanomolar gold nanorods and 40 microliters of 0.5 millimolar TCEP treated thiol DNA.
Add 1%SDS to the gold nanorod solution until a final SDS concentration of 0.05%is reached. Adjust the PH to between 2.5 and three with approximately one microliter of one molar HCL. Incubate for two hours while shaking at 70 RPM.
Add sodium chloride to reach a final sodium chloride concentration of 0.5 molar and incubate for four hours at room temperature while shaking at 70 RPM. Then, adjust the PH to approximately 8.5 with 10X TBE buffer and incubate over night. Wash the DNA gold nanorods four times by mixing the samples with one milliliter of washing buffer and centrifuge at 7, 000 times G for 30 minutes.
Remove the supernatant and resuspend the DNA gold nanorods in the remaining liquid. Estimate the concentration of DNA gold nanorods from a UV visible absorption measurement as before. Add magnesium chloride to the solution of purified DNA gold nanorods to a final concentration of 10 millimolar.
Mix the purified DNA gold nanorods and origami to a 10 to one ratio and yield the mixture in a mixer with a temperature control from 40 degrees Celsius to 20 degrees Celsius while shaking at 400 RPM. Use 0.7%agarose gel electrophoresis to purify the final origami gold nanorod structures. For TEM imaging, mix 200 microliters of 0.75%uranyl formate solution and 1 microliter of five molar sodium hydroxide.
Vortex immediately for two to three minutes. Centrifuge the stain solution for three to four minutes at 14, 000 times G.Then, protect the stain from light exposure by wrapping it in aluminum foil. After increasing the hydrofalicity of the TEM grids as described in the text protocol, pipette five microliter sample drops on the TEM grid.
Following an incubation of five to eight minutes, remove the drop by gently touching a filter paper with the edge of the grid. Now, pipette one big drop and one small drop of the stain solution on a piece of parafilm. Put the grid on the small stain solution drop and dry immediately by touching the filter paper with the edge of the grid.
Then, put it on the big stain solution drop for 30 seconds. Shown here is a TEM image of DNA origami templates. The origami structure consists of two 14 helix bundles linked together by the scaffold strand.
Here, a representative TEM image of gold nanorods is shown. The average dimensions of synthesized gold nanorods are 70 by 30 nanometers. This image shows gold nanorod dimers on origami after annealing.
Due to their binding preference to TEM grids, origami gold nanorod assemblies are usually seen as parallel origami bundles and rods. The protocol enables high yields of the assembly of gold nanorods into chiral meta-molecules with strong plasmonic circular dichroism responses. Shown here are the spectra of the closed structures and the open structures.
After its development, this technique paved the way for researchers to explore plasmonic optical properties of complex self-assembled metal nanostructures. As described in the text protocol, several hazardous chemicals are used in this method. Please carefully check the material safety data sheet and take necessary precautions.
