Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

The overall goal of the following experiment is to fabricate and characterize conjugate nano biological systems interfacing solid nano structured substrates with immobilized biomolecules. This is achieved by utilizing ultra high resolution electron beam lithography or EBL to obtain arrays of tiny metallic nanostructures on fused silica supports. As a second step, the substrates are placed in solution and biomolecules are immobilized on their surfaces, producing the nano biological interfaces.

Next surface enhanced ramen spectroscopy or SIRS is employed in order to probe the biological molecules at the interfaces. The resulting sir spectra exhibit ramen mode's characteristic of the particular molecules with intensities strongly dependent on the substrate design. The main advantage of our approach over existing techniques like infrared spectroscopy is that the sensitivity can be greatly enhanced due to surface plasma and resonance within the metal nanostructures.

Our approach creates highly selective surfaces that can be readily controlled using the combination of molecular functionalization and electron beam lithography. This method can answer key questions across multiple fields from exploration research in structural biology or service mobilized by polymers to the development of various devices involving nano biological interfaces. This methods can provide in science into the structural properties of biomolecules with implications extending towards medical diagnosis or environmental detection.

Generally, individuals new to this method can struggle as it involves aspects of nano fabrication, surface functionalization, biochemistry, and spectroscopy, which have been traditionally unrelated To begin spin coat the polymethylmethacrylate or PMMA, resist and conductive layers on the substrates. To achieve this, use a wafer spinner with a vacuum chuck and place the samples individually in the center on the chuck. Place one drop of PMMA resist on the center of the samples using a glass pipette.

Then spin at 3, 500 RPM for 60 seconds with a two second acceleration time. Next, bake the substrates at 180 degrees Celsius for three to five minutes after baking the substrates, cool the samples to room temperature with the substrates cooled and return to the spinner chuck. Spread a drop of conductive polymer on the substrate.

Spin the substrate for 40 seconds at 3000 RPM with a two second ramp following the spin. Bake the samples at 80 degrees Celsius for one minute after performing electron beam lithography or EBL exposure as described in the text protocol, prepare a beaker with the ionized water for removing the conductive polymer. Then prepare a second beaker with a developer mixture and stir for five minutes, set room temperature.

Finally, prepare isopropanol in a third beaker as a rinse agent using tweezers. Place the samples into the water for three seconds to remove the conductive polymer film. Then place the sample into the developer and move the tweezers slowly up and down for 20 seconds immediately transfer the substrates to the isopropanol and rinse for 10 more seconds before drying the sample with nitrogen, load the samples upside down into the electron beam evaporator system to allow for the evaporated metal to be deposited on the front face of the samples.

Deposit a 10 nanometer thick gold layer onto the samples for designs one and three and a 10 nanometer thick silver layer for design. Two at a rate of approximately 0.1 nanometers per second. Then fill a sonication system to the recommended height with water and fill a separate beaker with acetone.

Place a sample face up in the bottom of the beaker and allow the sample to soak for 10 minutes holding the beaker. Place it into the water bath and allow the height of the acetone to match the height of the water. Then turn on the sonication system.

Allow sonication to occur for up to 60 seconds. To prepare design one samples first, prepare a one millimolar solution of MUA and ethanol at room temperature and sonicate for 10 minutes. Immerse the proper nano structured substrate in the solution of MUA for 48 hours.

Then rinse the sample with ethanol three times and dry for five minutes. Set room temperature. Next, prepare a 75 millimolar solution of EDC and distilled water.

Also prepare a 15 millimolar solution of NHS in distilled water using a micro pipette deposit, 100 microliters of NHS onto the gold on the substrate, and immediately add 100 microliters of EDC on the same area. Incubate for one minute to activate the self-assembled monolayer of MUA. Then place a 100 microliter drop of protein, a solution on the same area of the substrate.

Place the sample into one compartment of a Petri dish with one milliliter of distilled water in another compartment. Seal the Petri dish with a cover and store the sample for 24 hours at five degrees Celsius. Next, rinse the sample in distilled water three times by continuously the samples in separate beakers for 20 seconds in each beaker, do not let the samples dry after rinsing or during the rinse for the design two samples.

Prepare a 0.9 millimolar solution of glucose binding protein or GBP in potassium phosphate buffer. Also prepare a 100 millimolar solution of DG glucose in the buffer. Next mix 30 microliters of the D glucose solution and 30 microliters of the GBP solution.

Using a one milliliter plastic micro tube container and a micro pipette incubate for 30 minutes, then deposit 20 microliters of the ligand free GBP solution and 20 microliters of the ligand bound GBP solution onto the prepared substrates. Using a micro pipette store the samples at five degrees Celsius for 24 hours in a Petri dish with a sealed cover. Rinse the samples three times in the potassium phosphate buffer solution at room temperature To prepare.

Design three samples dilute the dopamine binding apre or DBA solution to a concentration of one micromolar. Using the tris EDTA buffer with a final pH of 7.4, prepare a dopamine solution to a five micromolar concentration by measuring the dopamine powder on an analytical balance and mixing it in the phosphate buffered saline or PBS with a stir bead for five minutes. Then deposit a 20 microliter drop of the DBA solution on the surface of each substrate, and let the sample sit for one hour at room temperature with a cover over the Petri dish.

After rinsing the samples three times in a potassium phosphate buffer, place the samples upright on top of a clean room grade wipe to dry the backside while maintaining a film on the front side of the substrate. Set a sample aside as a control. Next place a five microliter drop of the dopamine solution onto the surface of the existing PBS.

Drop on the remaining samples. Incubate the samples for 10 minutes before rinsing the samples in the potassium phosphate buffer solution three times. To perform ramen spectroscopy.

Place each sample in a waterproof chamber to avoid evaporation by laser exposure. Fill a plastic syringe with chemically inert high vacuum grease. Then place the samples on glass slides and dispense a few millimeters of grease surrounding the samples without touching the samples.

Place a microscope cover slip on top of the substrates. Gently press down to form a seal, creating a thin liquid interface between the substrates and the cover slips without allowing the buffer to come in contact with the vacuum grease. Using an optical Raman microscope system, obtain a focus on the surface of the metal nano pattern region to be sampled without turning on the laser.

Perform Raman sampling as described in the text protocol shown. Here are the nano pattern dot arrays of gold and silver nickel on fused silica. The usage of electron beam lithography or EBL allows for superb position and dimension.

Control dot size and gap width is controlled by EBL exposure. Dose hexagon arrays of gold end of silver nickel are displayed here. Nucleation behavior of such very thin metal layers results in discontinuous structures.

Here is the series spectrum of substrate immobilized protein A in design one signal intensity from array one is much stronger than from array two. Signal from array three was very weak. Gap spacing is clearly a critical parameter to plasma uncoupling and surface enhancement of ramen scattering displayed.

Here are s spectra of glucose binding protein showing the effects of different substrate designs in the ligand free and ligand bound state. Finally, s spectra of dopamine binding aptamer is shown for ligand free and ligand bound molecules. A spectrum of dopamine powder is shown for reference.

While attempting this procedure, it is important to avoid contamination of the substrates. As a spectroscopy technique is highly sensitive and you can have false readings Employing this procedure. Optimal source substrate designs may be identified for a specific analytes.

Then other nphy methods like non-print phy can be performed to improve the scalability of the substrate fabrication After its development. This technique helped researchers in the field of biochemistry and bio sensing to detect biological molecules without certain surfaces in very small quantities while remaining in solution. After watching this video, you should have a good idea how to control metal nanostructure size and film properties using fabrication techniques and how to exploit different biomolecular recognition strategies to target different analytes.