Our research aims to develop new tools to detect single molecules by Surface-Enhanced Raman Scattering or SERS. This is the only technique that provides a chemical fingerprint of a molecule and is sensitive enough to detect single molecules. In this way, detailed mechanistic information about chemical reactions can be obtained.

DNA origami nano structures have been used to precisely position both plasmonic nanoparticles and target molecules. And this is required because the enhanced Raman scattering originates from a small nano metric volume between the nanoparticles, which we call hotspots. And we have now created new plasmonic DNA origami nano antenna exactly for this purpose.

The main challenge is to place target molecules in such hotspots between two nanoparticles and collect Raman data from exactly one nano antenna structure. To collect large amounts of data and efficient correlation between atomic force microscopy and Raman spectroscopy needs to be done. The plasmonic DNA origami nano antennas allow for a reproducible production of a large number of plasmonic dimers in which the target molecule is precisely positioned in between the nano particles in the hotspot.

And through a correlation of AFM and Raman data, we can now make sure that there's only a single molecule that is detected. Now we can track single molecules such as dye molecules or proteins in real time, and their behavior in the hotspots and their reactions to chemical changes in the environment. For example, the change of the spin state of single henian molecules was recently monitored.

In the future, we aim to monitor chemical reactions at a single molecular level and to study their reaction mechanisms. In addition, we can use this technology to detect medically relevant biomolecules with very high sensitivity. To self-Assemble the DNA origami structure in one pot, begin by preparing a mixture of 2.5 nano molar of the circular scaffold strand M13, MP18 and 100 nano molar of the 201 short polygon nucleotide staples in TAE buffer, supplemented with 15 millimolar magnesium chloride.

Then adjust the total volume of the solution to 100 microliters using ultrapure water. Anele the solution in a thermocycler using a temperature gradient. Start by rapidly heating to 80 degrees Celsius and follow the displayed temperature gradient program step by step.

To purify the mixture from excess staples, add 100 microliters of the DNA origami solution to a 100 kilodalton molecular weight cutoff amanon filter, followed by adding 400 microliters of ultrapure water. Then centrifuge at 6, 000 G for eight minutes at room temperature. After centrifuging, remove the filter and flip the tube to discard the filtrate, add 400 microliters of ultrapure water to the filter and centrifuge as demonstrated previously.

To collect the purified nanostructure solution, flip the filter upside down in a new tube and centrifuge at 1000 G for two minutes at room temperature following the manufacturer's instructions. Proper assembly of the DNA origami fork was insured using atomic force microscopy or AFM imaging. As expected, most forks were solid with no broken arms.

However, the bridge between the arms was difficult to image because of its small diameter and high flexibility. To coat the gold nanoparticles with DNA, take 400 microliters of commercially obtained gold nanoparticle solution and centrifuge it at 3, 500 G and room temperature for five minutes. Using a pipet, remove the supernatant and resolubilize the pellet in 25 microliters of ultrapure water.

Next, add four microliters of commercially obtained 100 micromolar thiol modified DNA. Add one microliter of 100 millimolar TCEP solution. Incubate the mixture for 10 minutes at room temperature.

Following incubation, add five microliters of the mixture to the previously concentrated gold nanoparticle solution. Vortex the resulting mixture for five seconds and freeze for at least two hours at minus 20 degrees Celsius. After thawing the frozen mixture at room temperature, centrifuge the same at 3, 500 G for five minutes at room temperature to remove excess added DNA coating.

Using a pipet, remove the supernatant and resolubilize the pellet in 10 microliters of water. The changing color of the solution at each step indicated proper coating of the gold nanoparticle. The deep red color of the gold nanoparticles changed to dark purplish red as soon as the DNA was added.

Freezing changed the color to purple and thawing returned it to deep red. The absorbance spectra of the 60 nanometer DNA coated gold nanoparticles showed a shift in absorbance peak compared to that of the bare gold nanoparticles. To assemble the DNA origami nano antennas or DONAs, use the DNA coated gold nanoparticles and the already assembled DNA origami forks.

Add the nano fork solution to the coated gold nano particle solution, maintaining a final molar concentration ratio of 1.5 is to one. To the mixture, add magnesium chloride from a 50 Millimolar stock solution to reach a final magnesium chloride concentration of four millimolar. Adjust the final volume to 20 microliters using ultrapure water.

Hybridize the DONAs using a temperature gradient in a thermocycler. Starting with rapid heating to 40 degrees Celsius, follow the displayed temperature gradient program step-by-step. For purification of the DONAs, prepare 1%agaros gel by dissolving 0.8 grams of agaros in 80 milliliters of TAE supplemented with five millimolar magnesium chloride.

Add 2.25 microliters of loading buffer containing 30%glycerol and 13 millimolar magnesium chloride to 18 microliters of DONA solution. Run the gel for 60 minutes at 70 volts in an ice water bath. Cut out the band of interest and place it on a microscopy slide wrapped with paraffin plastic film.

Then squeeze out the solution using a second paraffin film wrapped microscopy slide. Using a pipette, collect the squeezed liquid into a 500 microliter tube. During the agaros gel purification, a dimer band corresponding to the DONAs appeared above the free gold nanoparticles band, the fastest running band in the sample.

To perform co-localized AFM imaging and Raman measurements on the purified DNA origami nano antenna or DONA, first treat a silicone chip with plasma for 10 minutes. Incubate 10 microliters of the purified DONA solution, and 10 microliters of 100 millimolar magnesium chloride on the chip for three hours. Then wash the chip two times with an ethanol water mixture and blow dry with compressed air.

Tape the dried chip on a magnetic disc and insert it into the instrument for atomic force microscopy or AFM imaging. Next, choose the desired laser wavelength, power, and accumulation time for the Raman measurements. For gold nanoparticle fully coated with Tamra, select a 633 nanometer laser, 100 micro watt power.

And one second integration time. For a single Tamra measurement, change to 400 micro watt power. And four second integration time.

After adjusting the parameters, place the cursor on top of the desired DONA in the AFM image. Then start the Raman measurement. For co-localization measurements, AFM imaging of the sample was performed to locate the DONAs.

Following that, Raman spectra from single DONAs were collected and compared to ensure that the spectra obtained were from Tamra molecules, the main Tamra peaks were visible in the fully coated Tamra Gold nanoparticle. Although the signal to noise ratio for the single molecule Tamra Spectra was lower, the main peaks were identifiable.