Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.
Sum frequency generation vibrational spectra are fitted to investigate interfaces with interfacial selectivity. However Fresnel coefficient method can help to resolve the interference effect when another interface exists. The main advantage of SFG is the interfacial selectivity and submonolayer sensitivity.
This noninvasive optical technique is suitable for investing various interfaces such as solid-liquid, solid-solid, liquid-liquid, solid-gas, and liquid-gas interfaces. We suggest that beginners get a lot of practice operating the SFG system with the help of experts in optics and the SFG. There are not shortcuts.
SFG has not been generally recognized as powerful technique, so we would like to attract more audience interest and encourage more researchers to apply this technique. To begin, prepare about five millilters of either a two or 4%by weight solution of poly-2-hydroxyethyl methacrylate or PHEMA in anhydrous ethanol. Keep the solution in the lab three days before use.
Next, soak four IR grade calcium fluoride right angled prism in analytical grade anhydrous toluene for at least 12 hours. Then immerse the prisms in 30 millilters of ethanol and wipe the surfaces with degreasing cotton for about 10 minutes. Rinse the prism with ultra pure water for two minutes and dry them with nitrogen gas.
Next, place the prisms in an oxygen plasma cleaner and evacuate the chamber. Treat the prisms with oxygen plasma for four minutes and keep them in the chamber until they are used. Film preparation should occur within one hour of plasma treatment.
In this study, to selectively detect the barrier interface it is of great importance to choose the appropriate film thickness according to the calculated result of the Fresnal coefficient model. When ready to prepare the PHEMA films, fix a clean prism in a prism holder on a spin coater and apply one drop of the solution of PHEMA in ethanol to the prism. Run the spin coater at 1, 500 RPM for one minute to prepare the PHEMA film.
Coat additional plasma treated prisms with PHEMA in the same way. Anneal the films in a vacuum oven set to 80 degrees Celsius for at least 10 hours. To perform the experiment place the PHEMA coated face of the prism in deionized water.
Wait 10 to 20 minutes and then evaluate the interfacial PHEMA structure at the water and prism interfaces with some frequency generation spectroscopy. While operating the FGS system do not let the light beam directly come into your eyes. To begin preparing the silk fibroin solution heat three liters of 0.02 molar aqueous sodium carbonate to boiling.
Boil 7.5 grams of Bombyx mori silk cocoons in this solution while stirring for 30 minutes. Transfer the fibrous matter to a clean container and stir it in two to three liters of deionized water for eight to 10 minutes three times to wash off the unwanted sericin molecules. Dry the fibrous matter in a vacuum oven at 60 degrees Celsius for at least 15 hours.
Next, dissolve one gram of dry, degummed silk fibroin in four millilters of 9.3 molar aqueous lithium bromide. Stir the solution at 60 degrees Celsius for two hours. Then place the silk fibroin solution in a 3, 500 Dalton dialysis bag.
Dialyze the solution against one liter of deionized water for three days, changing the water three times per day. Once dialysis is complete, store the silk fibroin solution at four degrees Celsius. Next, use the previously described methods to clean a calcium fluoride prism and coat it with a thin polystyrene film from a 3.5%by weight polystyrene solution.
Place the polystyrene coated prism in contact with the silk fibroin solution and examine the polystyrene silk fibroin interface with SFG spectroscopy. To begin preparing the oligonucleotide duplex, dissolve 10 nanomoles of the appropriate single-stranded oligonucleotide in 0.5 millilters of ultrapure water. Do the same for the complimentary oligonucleotide.
Combine the solutions to obtain a 10 nanomoles per milliliter duplex oligonucleotide solution. Then combine two milligrams of DPPC, two milligrams of deuterated DPPC, and one milliliter of chloroform to obtain the lipid solution. Next, clean and plasma treat a calcium fluoride prism as previously described.
Attach this prism to the sample hold of a Langmuir-Blodgett trough. Fill the trough with deionized water and lower one face of the prism into the trough at one millimeter per minute. Inject several microliters of the lipid solution onto the surface of the water and wait for the surface pressure to stabilize at about 12 millinewtons per meter.
Then start compressing the lipid monolayer at five millimeters per minute. When the surface pressure reaches 34 millinewtons per meter lift the prism from the trough at one millimeter per minute. Next prepare 500 microliters of a mixture of the duplex oligonucleotide solution and the lipid solution in a one to 100 molar ratio.
Then replace the Langmuir-Blodgett trough with a cylindrical polytetrafluorethylene container filled with deionized water. Inject the oligonucleotide lipid mixture onto the water until the surface pressure reaches 34 millinewtons per meter. Put the lipid monolayer coated face of the prism in contact with the lipid oligonucleotide mixture to form the final sample.
Evaluate the chiral and achiral water vibrational signals with FSG spectroscopy. In this first example, the interface between PHEMA hydrogels and the calcium fluoride prism showed clear sharp peaks in the SFG spectrum. This was attributed to the smooth interface between calcium fluoride and the hydrogel.
The interface between the hydrogel and the surrounding water had broader, less intense features because water molecules could diffuse into the bulk of the hydrogel. Here when the silk fibroin concentration was above the critical overlapping concentration there were do detected chiral secondary structures at the solution polystyrene interface unless methanol was added as an inducing agent. Below the critical overlapping concentration, chiral SFG signals were detected even without adding methanol indicating that ordered secondary structures formed unassisted at the solution polystyrene interface.
In the duplex oligonucleotide anchored lipid bilayer sample varying the calcium ion concentration had no significant effect on the chiral water signal which primarily corresponds to the hydration later of the chiral spine in the minor groove. In contrast, the calcium concentration strong effected the achiral water signals which primarily correspond to the water layer surrounding the duplex chain and the bilayer. Over all this suggested that the chiral spine of the water layer could protect the oligonucleotide from calcium ions.
Calculating the Fresnel coefficient and choosing an appropriate film thickness can resolve interference problems. If multi-reflection and refraction are considered the right electric field distribution at the interfaces can be adjusted. There are alternative methods that can be used to prepare thin films with desired thicknesses such as step-coating and the chemical vapor deposition.
Detecting the surface and interfacial structures related to adhesion, whiting, friction, and other properties can help researchers understand the underlying mechanisms and develop new functional materials. This method can also be applied to other systems where varied interfaces need to be investigated. However, the medium you wish the light beams propagate must be transparent.
