This study was supported by the State Key Development Program for Basic Research of China (2017YFA0700500) and the National Natural Science Foundation of China (21574020). The Fundamental Research Funds for the Central Universities, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the National Demonstration Center for Experimental Biomedical Engineering Education (Southeast University) were also greatly appreciated.

1. SFG experimental
<ol>
	<li>Use a commercial picosecond SFG system (Table of Materials), which provides a fundamental 1064 nm beam with a pulse width of ~20 ps and a frequency of 50 Hz, based on an Nd:YAG laser.</li>
	<li>Convert the fundamental 1064 nm beam into a 532 nm beam and a 355 nm beam by using second and third harmonic modules. Directly guide the 532 nm beam as an input light beam and generate the other input mid-infrared (IR) beam covering the frequency range from 1000 to ...

<ol>
	<li>Shen, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Second+Harmonic+Generation+at+Interfaces.">Optical Second Harmonic Generation at Interfaces.</a> Annual Review of Physical Chemistry. 40, 327-350 (1989).</li><li>Shen, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+properties+probed+by+second-harmonic+and+sum-frequency+generation.">Surface properties probed by second-harmonic and sum-frequency generation.</a> Nature. 337, 519-525 (1989).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+Polymer+Surfaces+and+Interfaces+with+Sum+Frequency+Generation+Vibrational+Spectroscopy.">Studying Polymer Surfaces and Interfaces with Sum Frequency Generation Vibrational Spectroscopy.</a> Analytical Chemistry. 89 (1), 466-489 (2017).</li><li>Chen, X., Clarke, M. L., Wang, J., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum+Frequency+Generation+Vibrational+Spectroscopy+Studies+on+Molecular+Conformation+and+Orientation+of+Biological+Molecules+at+Interfaces.">Sum Frequency Generation Vibrational Spectroscopy Studies on Molecular Conformation and Orientation of Biological Molecules at Interfaces.</a> International Journal of Modern Physics B. 19 (4), 691-713 (2005).</li><li>Eisenthal, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+Interfaces+Probed+by+Second-Harmonic+and+Sum-Frequency+Spectroscopy.">Liquid Interfaces Probed by Second-Harmonic and Sum-Frequency Spectroscopy.</a> Chemical Reviews. 96 (4), 1343-1360 (1996).</li><li>Richmond, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molcular+Bonding+and+Interactions+at+Aqueous+Surfaces+as+Probed+by+Vibrational+Sum+Frequency+Spectroscopy.">Molcular Bonding and Interactions at Aqueous Surfaces as Probed by Vibrational Sum Frequency Spectroscopy.</a> Chemical Reviews. 102 (8), 2693-2724 (2002).</li><li>Wang, H., Gan, W., Lu, R., Rao, Y., Wu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+spectral+and+orientational+analysis+in+surface+sum+frequency+generation+vibrational+spectroscopy(SFG-VS).">Quantitative spectral and orientational analysis in surface sum frequency generation vibrational spectroscopy(SFG-VS).</a> International Reviews in Physical Chemistry. 24 (2), 191-256 (2007).</li><li>Shultz, M. J., Schnitzer, C., Simonelli, D., Baldelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum+frequency+generation+spectroscopy+of+the+aqueous+interface:+Ionic+and+soluble+molecular+solutions.">Sum frequency generation spectroscopy of the aqueous interface: Ionic and soluble molecular solutions.</a> International Reviews in Physical Chemistry. 19 (1), 123-153 (2010).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Surface+Hydration+of+Poly(2-hydroxyethyl+methacrylate)+in+Solution+in+situ.">Detecting Surface Hydration of Poly(2-hydroxyethyl methacrylate) in Solution in situ.</a> Macromolecules. 49, 3116-3125 (2016).</li><li>Li, X., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+Irreversibly+Absorbed+Layer+Promotes+Dewetting+of+Polystyrene+Film+on+Sapphire.">Evolution of Irreversibly Absorbed Layer Promotes Dewetting of Polystyrene Film on Sapphire.</a> Macromolecules. 51, 6653-6660 (2018).</li><li>Lu, X., Spanninga, S. A., Kristalyn, C. B., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Orientation+of+Phenyl+Groups+in+Poly(sodium+4-styrenesulfonate)+and+in+Poly(sodium+4-styrenesulfonate):+Poly(3,4-ethylenedioxythiophene)+Mixture+Examined+by+Sum+Frequency+Generation+Vibrational+Spectroscopy.">Surface Orientation of Phenyl Groups in Poly(sodium 4-styrenesulfonate) and in Poly(sodium 4-styrenesulfonate): Poly(3,4-ethylenedioxythiophene) Mixture Examined by Sum Frequency Generation Vibrational Spectroscopy.</a> Langmuir. 26 (17), 14231-14235 (2010).</li><li>Lu, X., Clarke, M. L., Li, D., Wang, X., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Sum+Frequency+Generation+Vibrational+Study+of+the+Interference+Effect+in+Poly(n-butyl+methacrylate)+Thin+Films+Sandwiched+between+Silica+and+Water.">A Sum Frequency Generation Vibrational Study of the Interference Effect in Poly(n-butyl methacrylate) Thin Films Sandwiched between Silica and Water.</a> Journal of Physical Chemistry C. 115, 13759-13767 (2011).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+Probing+Molecular+Ordering+at+the+Buried+Polymer/Metal+Interface+2:+Using+P-Polarized+Input+Beams.">Directly Probing Molecular Ordering at the Buried Polymer/Metal Interface 2: Using P-Polarized Input Beams.</a> Macromolecules. 45, 6087-6094 (2012).</li><li>Lu, X., Myers, J. N., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Ordering+of+Phenyl+Groups+at+the+Buried+Polystyrene/Metal+Interface.">Molecular Ordering of Phenyl Groups at the Buried Polystyrene/Metal Interface.</a> Langmuir. 30, 9418-9422 (2014).</li><li>Li, B., Lu, X., Ma, Y., Han, X., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+to+Probe+Glass+Transition+Temperatures+of+Polymer+Thin+Films.">Method to Probe Glass Transition Temperatures of Polymer Thin Films.</a> ACS Macro Letters. 4, 548-551 (2015).</li><li>Li, X., Deng, G., Ma, L., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interchain+Overlap+Affects+Formation+of+Silk+Fibroin+Secondary+Structure+on+Hydrophobic+Polystyrene+Surface+Detected+via+Achiral/Chiral+Sum+Frequency+Generation.">Interchain Overlap Affects Formation of Silk Fibroin Secondary Structure on Hydrophobic Polystyrene Surface Detected via Achiral/Chiral Sum Frequency Generation.</a> Langmuir. 34, 9453-9459 (2018).</li><li>Kai, S., Li, X., Li, B., Han, X., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-dependent+hydrolysis+of+supported+planar+lipids+was+triggered+by+honey+bee+venom+phospholipase+A2+with+the+right+orientation+at+the+interface.">Calcium-dependent hydrolysis of supported planar lipids was triggered by honey bee venom phospholipase A2 with the right orientation at the interface.</a> Physical Chemistry Chemical Physics. 20, 63-67 (2018).</li><li>Wang, J., Buck, S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum+Frequency+Generation+Vibrational+Spectroscopy+Studies+on+Protein+Adsorption.">Sum Frequency Generation Vibrational Spectroscopy Studies on Protein Adsorption.</a> Journal of Physical Chemistry B. 106, 11666-11672 (2002).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Amide+I+Signals+of+Interfacial+Proteins+in+Situ+Using+SFG.">Detection of Amide I Signals of Interfacial Proteins in Situ Using SFG.</a> Journal of American Chemical Society. 125, 9914-9915 (2003).</li><li>Nguyen, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Spontaneous+Membrane+Insertion+of+a+Tall-Anchored+Membrane+Protein+by+Sum+Frequency+Generation+Spectroscopy.">Probing the Spontaneous Membrane Insertion of a Tall-Anchored Membrane Protein by Sum Frequency Generation Spectroscopy.</a> Journal of American Chemistry Society. 132, 15112-15115 (2010).</li><li>Li, X., Ma, L., Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Ions+Affect+Water+Molecular+Structures+Surrounding+an+Oligonucleotide+Duplex+as+Revealed+by+Sum+Frequency+Generation+Vibrational+Spectroscopy.">Calcium Ions Affect Water Molecular Structures Surrounding an Oligonucleotide Duplex as Revealed by Sum Frequency Generation Vibrational Spectroscopy.</a> Langmuir. , (2018).</li><li>Sartenaer, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum-frequency+generation+spectroscopy+of+DNA+monolayers.">Sum-frequency generation spectroscopy of DNA monolayers.</a> Biosensors &amp; Bioelectronics. 22, 2179-2183 (2007).</li><li>Asanuma, H., Noguchi, H., Uosaki, K., Yu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+Cation-induced+Deformation+of+DNA+Self-Assembled+Monolayers+on+Silicon:+Vibrational+Sum+Frequency+Generation+Spectroscopy.">Metal Cation-induced Deformation of DNA Self-Assembled Monolayers on Silicon: Vibrational Sum Frequency Generation Spectroscopy.</a> Journal of American Chemistry Society. 130, 8016-8022 (2008).</li><li>Howell, C., Schmidt, R., Kurz, V., Koelsch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sum-frequency-generation+spectroscopy+of+DNA+films+in+air+and+aqueous+environments.">Sum-frequency-generation spectroscopy of DNA films in air and aqueous environments.</a> Biointerphases. 3 (3), FC47 (2008).</li><li>Walter, S. R., Geiger, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+on+Stage:+Showcasing+Oligonucleotides+at+Surfaces+and+Interfaces+with+Second+Harmonic+and+Vibrational+Sum+Frequency+Generation.">DNA on Stage: Showcasing Oligonucleotides at Surfaces and Interfaces with Second Harmonic and Vibrational Sum Frequency Generation.</a> Journal of Physical Chemistry Letters. 1, 9-15 (2010).</li><li>Li, Z., Weeraman, C., Azam, M. S., Osman, E., Gibbs-Davis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thermal+reorganization+of+DNA+immobilized+at+the+silica/buffer+interface:+a+vibrational+sum+frequency+generation+investigation.">The thermal reorganization of DNA immobilized at the silica/buffer interface: a vibrational sum frequency generation investigation.</a> Physical Chemistry Chemical Physics. 17, 12452-12457 (2015).</li><li>Lambert, A. G., Neivandt, D. J., Briggs, A. M., Usadi, E. W., Davies, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Effects+in+Sum+Frequency+Spectra+from+Monolayers+on+Composite+Dielectric/Metal+Substrates.">Interference Effects in Sum Frequency Spectra from Monolayers on Composite Dielectric/Metal Substrates.</a> Journal of Physical Chemistry B. 106, 5461-5469 (2002).</li><li>Tong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+effects+in+the+sum+frequency+generation+spectra+of+thin+organic+films.+I.+Theoretical+modeling+and+simulation.">Interference effects in the sum frequency generation spectra of thin organic films. I. Theoretical modeling and simulation.</a> Journal of Chemical Physics. 133, 034704 (2010).</li><li>McGall, S. J., Davies, P. B., Neivandt, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Effects+in+Sum+Frequency+Vibrational+Spectra+of+Thin+Polymer+Films:+An+Experimental+and+Modeling+Investigation.">Interference Effects in Sum Frequency Vibrational Spectra of Thin Polymer Films: An Experimental and Modeling Investigation.</a> Journal of Physical Chemistry B. 108, 16030-16039 (2004).</li><li>Li, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Fresnel+Coefficients+and+Molecular+Structures+of+Model+Cell+Membranes:+From+a+Lipid+Monolayer+to+a+Lipid+Bilayer.">Interfacial Fresnel Coefficients and Molecular Structures of Model Cell Membranes: From a Lipid Monolayer to a Lipid Bilayer.</a> Journal of Physical Chemistry C. 118, 28631-28639 (2014).</li><li>Zhou, J., Anim-Danso, E., Zhang, Y., Zhou, Y., Dhinojwala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interfacial+Water+at+Polyurethane-Sapphire+Interface.">Interfacial Water at Polyurethane-Sapphire Interface.</a> Langmuir. 31 (45), 12401-12407 (2015).</li><li>Gautam, K. 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We have nothing to disclose.

To investigate the structural information from a molecular level, SFG has its inherent advantages (i.e., monolayer or sub-monolayer sensitivity and interfacial selectivity), which can be applied to study various interfaces, such as the solid/solid, solid/liquid, solid/gas, liquid/gas, liquid/liquid interfaces. Although the equipment maintenance and the optical alignment are still time-consuming, the payoff is significant in that the detailed molecular-level information at the surfaces and interfaces can be obtained.
...

Development of sum frequency generation (SFG) vibrational spectroscopy can be dated back to the work done by Shen et al. thirty years ago1,2. The uniqueness of the interfacial selectivity and sub-monolayer sensitivity makes SFG vibrational spectroscopy appreciated by a large number of researchers in the fields of physics, chemistry, biology, and materials science, etc3,4,5. Currently, a broad range of scientific issues related to surfaces and interfaces are being investigated using SFG, especially for complex interfaces with respect to polymers and biomacromolecules, such as the chain structures and structural relaxation at the buried polymer interfaces, the protein secondary structures, and the interfacial water structures9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26.
For polymer surfaces and interfaces, thin-film samples are generally prepared by spin-coating to obtain the desired surfaces or interfaces. The problem arises due to the signal interference from the two interfaces of the as-prepared films, which leads to inconvenience for analyzing the collected SFG spectra27,28,29. In most cases, the vibrational signal only from one single interface, either film/substrate or film/the other medium, is desirable. Actually, the solution to this problem is quite easy, namely, to experimentally maximize the light fields at the desirable interface and minimize the light fields at the other interface. Hence, the Fresnel coefficients or the local field coefficients need to be calculated via the thin film model and to be validated with respect to the experimental results3,9,10,11,12,13,14,15,30.
With the above background in mind, some polymer and biological interfaces could be investigated in order to understand fundamental science from the molecular level. In the following, taking three interfacial issues as examples: probing poly(2-hydroxyethyl methacrylate) (PHEMA) surface and buried interface with substrate9, formation of silk fibroin (SF) secondary structures on the polystyrene (PS) surface and water structures surrounding model short-chain oligonucleotide duplex16,21, we will show how the SFG vibrational spectroscopy helps to reveal the interfacial molecular-level structures in connection to the underlying science.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)&#160;</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850355P-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethanol</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>100092680</td>
			<td>&#8805;99.7%</td>
		</tr>
		<tr>
			<td>CaF2 prism</td>
			<td>Chengdu YaSi Optoelectronics Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10005817</td>
			<td>&#8805;96.0%</td>
		</tr>
		<tr>
			<td>deuterated DPPC (d-DPPC)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>860345P-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Electromagnetic oven</td>
			<td>Zhejiang Supor Co., Ltd</td>
			<td>C21-SDHCB37</td>
			<td></td>
		</tr>
		<tr>
			<td>Langmuir-Blodgett (LB) trough</td>
			<td>KSV NIMA Co., Ltd.</td>
			<td>KN 2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium bromide anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>20056926</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q synthesis system</td>
			<td>Millipore</td>
			<td></td>
			<td>Ultrapure water</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Chengdu Mingheng Science&#38;Technology Co., Ltd</td>
			<td>PDC-MG</td>
			<td>Oxygen plasma cleaning</td>
		</tr>
		<tr>
			<td>Poly(2-hydroxyethyl methacrylate) (PHEMA)</td>
			<td>Sigma-Aldrich Co., LLC.</td>
			<td>192066 MSDS</td>
			<td>Mw = 300 000</td>
		</tr>
		<tr>
			<td>Polystyrene</td>
			<td>Sigma-Aldrich Co., LLC.</td>
			<td>330345 MSDS</td>
			<td>Mw = 48 kDa and Mn = 47 kDa</td>
		</tr>
		<tr>
			<td>Silk cocoons</td>
			<td></td>
			<td></td>
			<td>From Bombyx mori</td>
		</tr>
		<tr>
			<td>Single complementary strand of oligonucleotide</td>
			<td>Nanjing Genscript Biotechnology Co., Ltd.</td>
			<td>H03596</td>
			<td>5&#39;-CGAAGGCTTCCAGCT-3&#39;</td>
		</tr>
		<tr>
			<td>Single strand of oligonucleotide</td>
			<td>Nanjing Genscript Biotechnology Co., Ltd.</td>
			<td>H04936</td>
			<td>&#160;3&#162;-end modified by cholesterol-triethylene glycol(Chol-TEG) (5&#162;-GCTTCCGAAGGTCGA-3&#162;)</td>
		</tr>
		<tr>
			<td>Sodium carbonate anhydrous</td>
			<td>Sinopharm Chemical Reagent Co., Ltd</td>
			<td>10019260</td>
			<td>&#8805;99.8%</td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>Institute of Microelectronics of the Chinese Academy of Sciences</td>
			<td>KW-4A</td>
			<td>For the prepartion of ploymer films&#160;</td>
		</tr>
		<tr>
			<td>Step profiler</td>
			<td>Veeco</td>
			<td>DEKTAK 150</td>
			<td>For the measurement of film thickness</td>
		</tr>
		<tr>
			<td>Sum frequency generation (SFG) vibrational spectroscopy system</td>
			<td>EKSPLA</td>
			<td></td>
			<td>A commercial picosecond SFG system</td>
		</tr>
	</tbody>
</table>

interfacial molecular-level structures of polymers and biomacromolecules revealed via sum frequency generation vibrational spectroscopy

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various surfaces and interfaces. This non-invasive optical technique can provide the local molecular-level information with monolayer or submonolayer sensitivity. We here are providing experimental methodology on how to selectively detect the buried interface for both macromolecules and biomacromolecules. With this in mind, interfacial secondary structures of silk fibroin and water structures around model short-chain oligonucleotide duplex are discussed. The former shows a chain-chain overlap or spatial confinement effect and the latter shows a protection function against the Ca2+ ions resulting from the chiral spine superstructure of water.

In the Fresnel coefficient part of Protocol Section, we have shown that, theoretically, it is feasible to selectively detect only one single interface at one time. Here, experimentally, we confirmed that this methodology is basically correct, as shown in Figure 5 and Figure 6.&#160;
Figure 5 shows the buried interfacial PHEMA structure after water intrusion with a ~150 nm PHEMA hydrogel film and <strong c...

Watch this Scientific Journal Video about Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy at JoVE.com

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

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.

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various ...

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.

interfacial-molecular-level-structures-polymers-biomacromolecules

Research

JoVE Journal

Biochemistry

9.3K 조회수.  Southeast University. 합계 주파수 생성 진동 스펙트럼은 얼굴 간 선택성을 가진 인터페이스를 조사하기 위해 장착됩니다. 그러나 프레스넬 계수 방법은 다른 인터페이스가 있을 때 간섭 효과를 해결하는 데 도움이 될 수 있습니다. SFG의 주요 장점은 얼굴 간 선택성 및 서브모노레이어 감도입니다.이 비침습적 광학 기술은 고체 액체, 고체 고체, 액체 액체, 고체 가스 및 액체 가스 인터페이스...