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 4000 cm-1 through the optical parametric generation (OPG)/optical parametric amplification (OPA)/difference frequency generation (DFG) process.</li>
	<li>Set the incident angles of two input beams to be 53&#176; (IR) and 64&#176; (visible), respectively, versus the surface normal.</li>
	<li>To detect the polymer interfacial structures (either film/substrate interface or film/the other medium interface), use ssp (s-polarized sum-frequency beam, s-polarized visible beam and p-polarized infrared beam) and ppp polarization combinations.</li>
	<li>To detect the interfacial protein secondary structures and water structures surrounding DNA, besides ssp and ppp, use chiral spp and psp polarization combinations were used.</li>
	<li>To ensure the samples were not damaged, control the infrared and visible pulse energies to be ~70 and ~30 &#956;J, respectively. A schematic of the SFG process with the energy level diagram was shown in Figure 1. Figure 2 shows the SFG system in our clean room.</li>
</ol>
2. Fresnel coefficients
<ol>
	<li>Use right-angle prisms as substrates for all the experiments discussed here. There exist two interfaces for a polymer film on the solid substrate, i.e., polymer surface in air and polymer/substrate interface. Both can generate SFG signals since inversion symmetry is broken at both interfaces. Therefore, a collected SFG spectrum is an interfered one. However, the local field coefficients or the Fresnel coefficients at the two interfaces can be adjustable by varying either the incident angles or the film thickness one at a time or simultaneously31,32. This provides the opportunity for us to probe the SFG vibrational signal from only one interface. Here, the PHEMA film on the CaF2 prism was taken as an example9.</li>
	<li>As shown in Figure 3, employ the right-angle prism geometry to detect the SFG signals generated from the bottom PHEMA film. The SFG output intensity in the reflected mode is expressed as 
	<img alt="Equation 1" src="/files/ftp_upload/59380/59380eq1.jpg" style="opacity: 0.9;" />&#160;(1) 
	where &#160;<img alt="Equation 2" src="/files/ftp_upload/59380/59380eq2.jpg" style="opacity: 0.9;" />&#160;denotes the effective second-order nonlinear susceptibility tensor. 
	<img alt="Equation 2" src="/files/ftp_upload/59380/59380eq2.jpg" style="opacity: 0.9;" />&#160;consists of three parts, namely, the prism/polymer interface, the polymer/bottom medium interface (bottom medium includes gas, liquid or solid.) and the nonresonant background, as shown in the following equation. 
	<img alt="Equation 3" src="/files/ftp_upload/59380/59380eq3.jpg" style="opacity: 0.9;" />&#160;(2) 
	&#8203;Here the bottom medium could be air, water or something else. F represents the corresponding Fresnel coefficient responsible for the local field correction.</li>
	<li>Apply a thin-film model to calculate the Fresnel coefficients in this case. Here only brief calculation procedures are presented.
	<ol>
		<li>For the prism/polymer interface, use 
		<img alt="Equation 4" src="/files/ftp_upload/59380/59380eq4.jpg" style="opacity: 0.9;" />&#160; (3) 
		<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq5.jpg" />&#160; (4) 
		<img alt="Equation 6" src="/files/ftp_upload/59380/59380eq6.jpg" />&#160; (5) 
		The meaning of each parameter shown is presented below.
		<ol>
			<li>&#969;i denotes the beam frequency.</li>
			<li>tp and ts denote the overall transmission coefficients and can be expressed as 
			<img alt="Equation 7" src="/files/ftp_upload/59380/59380eq7.jpg" style="opacity: 0.9;" />&#160; (6) 
			<img alt="Equation 8" src="/files/ftp_upload/59380/59380eq8.jpg" />&#160; (7)</li>
			<li>tp12 and ts12 denote the linear transmission coefficients of the light beam at the prism/polymer interface.</li>
			<li>rp23 and rs23 denote the linear reflection coefficients of the light beam at the polymer/medium interface.</li>
			<li>&#945; represents the phase difference between a reflective beam and its secondary reflective beam after it propagates across the polymer thin film and then reflects back, which can be expressed as 
			<img alt="Equation 9" src="/files/ftp_upload/59380/59380eq9.jpg" style="opacity: 0.9;" />&#160; (8)</li>
			<li>&#955;&#160;represents the wavelength of the light beam and d is the polymer film thickness.</li>
			<li>&#934;1 and &#934;2 represent the incident angles at the prism/polymer interface and the polymer/medium interface respectively.</li>
			<li>n1 and n2 represent the refractive indices of the prism and polymer film respectively.</li>
			<li>n12 represents the refractive indices of the polymer interfacial layers for the prism/polymer.</li>
		</ol>
		</li>
		<li>For the polymer/medium interface, use 
		<img alt="Equation 10" src="/files/ftp_upload/59380/59380eq10.jpg" />&#160; (9) 
		<img alt="Equation 11" src="/files/ftp_upload/59380/59380eq11.jpg" />&#160; (10) 
		<img alt="Equation 12" src="/files/ftp_upload/59380/59380eq12.jpg" />&#160; (11)
		<ol>
			<li>&#916; represents the phase difference of the light electrical fields at two interfaces.</li>
			<li>Because the pulse width for our input beams is ~20 ps, the error from the time delay associated with the dispersion effect can be neglected.</li>
			<li>The expression of such phase difference for the output SFG, the input visible and the input infrared beams can be separately written as 
			<img alt="Equation 13" src="/files/ftp_upload/59380/59380eq13.jpg" />&#160; (12) 
			<img alt="Equation 14" src="/files/ftp_upload/59380/59380eq14.jpg" />&#160; (13) 
			<img alt="Equation 15" src="/files/ftp_upload/59380/59380eq15.jpg" />&#160; (14) 
			&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>From the above discussion, for the prism-polymer film-medium (1-2-3) system, express the total Fresnel coefficients for the prism/polymer and polymer/medium interfaces as the following equations, for ssp and ppp polarization combinations. Of course, both interfaces are considered azimuthally isotropic.
	<ol>
		<li>For the prism/polymer interface, the expressions of the total Fresnel coefficients for both ssp and ppp polarization combinations are presented as follows.
		<ol>
			<li>&#160;For ssp, the equation is 
			<img alt="Equation 16" src="/files/ftp_upload/59380/59380eq16.jpg" />(15)</li>
			<li>And for ppp, the equation is&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq17v3.jpg" style="opacity: 0.9;" /> (16) 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq18v3.jpg" style="opacity: 0.9;" />&#160; (17)&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq19v3.jpg" style="opacity: 0.9;" /> (18) 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq20v3.jpg" style="opacity: 0.9;" />&#160; (19) 
			&#160;</li>
			<li>t10 and t01 denote the linear transmission coefficients at the air/prism and prism/air interfaces respectively.</li>
		</ol>
		</li>
		<li>For the polymer/medium interface, the expressions of the total Fresnel coefficients for both ssp and ppp polarization combinations are described as follows.
		<ol>
			<li>For ssp, the equation is 
			<img alt="Equation 21" src="/files/ftp_upload/59380/59380eq21.jpg" style="opacity: 0.9;" />&#160; (20)</li>
			<li>For ppp, the equations are&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq22v3.jpg" style="opacity: 0.9;" />&#160;(21)&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq23v3.jpg" style="opacity: 0.9;" />&#160;(22)&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq24v3.jpg" style="opacity: 0.9;" /> (23)&#160; 
			<img alt="Equation 5" src="/files/ftp_upload/59380/59380eq25v3.jpg" style="opacity: 0.9;" /> (24) 
			&#160;&#160;&#160; 
			&#160;</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After calculating the Fresnel coefficients using the sandwiched model, plot them as a function of the film thickness, as shown in Figure 4. 
	NOTE: In this case, there exists a thickness range for collecting the SFG signal from the CaF2 prism/PHEMA interface with neglectable contribution from the other interface, which is around 150 nm. Similarly, a suitable thickness can be chosen for detection of the PHEMA/bottom medium interface with neglectable contribution from the CaF2 prism/PHEMA interface.</li>
</ol>
3. Chiral SFG polarization combination
<ol>
	<li>For the normal achiral interface, commonly, use C&#8734;v symmetry in terms of ensemble average33,34. With operation&#160;of inversion symmetry, the nonzero second-order nonlinear susceptibility tensor components can be deduced, which are cxxz, cxzx, czxx, cyyz, cyzy, czyy and czzz (the existing terms can be further reduced if an isotropic interface is assumed, which means x and y are the same). However, for the chiral interface, situation will be different. The chiral interface possesses the C&#8734;&#160;symmetry, only the rotation symmetry operation is allowed. In this case, besides the normal achiral terms, more second-order nonlinear susceptibilities will be nonzero, which can be termed as the chiral terms, namely, czyx, czxy and cyzx under the consideration of non-electronic resonance. Therefore, by using psp, pps and spp polarization combinations, chiral SFG spectra can be collected33,34.</li>
</ol>
4. Sample preparation
<ol>
	<li>Preparation of PHEMA film
	<ol>
		<li>Dissolve PHEMA powder (see Table of Materials) in anhydrous ethanol to prepare the solution with 2 wt% and 4 wt% respectively.</li>
		<li>Before deposition of the PHEMA films, soak the CaF2 right-angle prisms in the toluene solvent firstly and then wash them with ethanol and ultrapure water (18.2 M&#937;&#183;cm).</li>
		<li>Afterwards, expose the substrates (CaF2 right-angle prisms) to oxygen plasma to remove possible organic contaminants by plasma cleaner (see Table of Materials).
		<ol>
			<li>First turn on the plasma cleaner and put the substrates in it.</li>
			<li>Then turn on the vacuum pump to vacuumize the cleaner. Input the oxygen in it.</li>
			<li>Finally set 4 minutes for cleaning. After that, preserve the clean substrates for the sequential PHEMA film preparation.</li>
			<li>Then prepare the PHEMA films on the CaF2 prisms by a spin-coater (see Table of Materials). Adjust the film thicknesses by the solution concentration and spin speed.
			<ol>
				<li>Immobilize the CaF2 prism on the sucking disc of spin-coater.</li>
				<li>Drop one drop of the PHEMA solution prepared before onto the clean substrates at 1,500 rpm for 1 min (film thickness 2 wt% for 100 nm and 4 wt% for 200 nm).</li>
			</ol>
			</li>
			<li>Anneal all the prepared PHEMA films in a vacuum oven at 80 &#176;C overnight.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of silk fibroin (SF) 
	&#8203;NOTE: The protocol suggested by Kaplan et al.35 was adopted.
	<ol>
		<li>Place 7.5 g of silk cocoons of B. mori in the boiling sodium carbonate (Na2CO3, 0.02 M) aqueous solution (3 L) for 30 min. Remove the fibrous SF to a clean container.</li>
		<li>Wash the obtained fibrous SF with deionized water for three times under stirring in order to remove the sericin molecules and leave only the SF molecules in the fibrous sample.</li>
		<li>Dry the fibrous SF sample in a vacuum oven at 60 &#176;C overnight.</li>
		<li>Afterwards, dissolve the degummed fibrous SF sample in a lithium bromide (LiBr, 9.3 M) aqueous solution (1 g of&#160; SF was solved in ~4 mL of LiBr solution.) and incubate it at 60 &#176;C for 2 h under stirring.</li>
		<li>Dialyze the SF solution against deionized water (3,500 Da dialysis bags) for 3 days to remove the dissolved LiBr. Change new deionized water three times every day. Finally store the processed SF solution at 4 &#176;C for later SFG experiments.</li>
	</ol>
	</li>
	<li>Preparation of short-chain oligonucleotide duplex
	<ol>
		<li>Order the single-stranded oligonucleotide sample with its 3&#8217;-end modified by cholesterol-triethylene glycol (Chol-TEG) (5&#8217;-GCTTCCGAAGGTCGA-3&#8217;) from a commercial corporation (see Table of Materials) as well as the complementary one. For each single strand, dissolve 10 nmol of the sample powder in 0.5 ml ultrapure water. Then mix them together to form the duplex oligonucleotide solution (10 nmol/mL).</li>
		<li>Mix 2 mg of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 2 mg of deuterated DPPC (d-DPPC) and dissolve them in 1 mL of chloroform to prepare the lipid solution.</li>
		<li>Preparation of DPPC &#38; d-DPPC monolayer by a Langmuir&#8722;Blodgett (LB) trough
		<ol>
			<li>Attach the right-angle CaF2 prism to a homemade sample holder with one prism face perpendicularly dipped into the aqueous environment of the LB trough.</li>
			<li>Afterwards, inject the mixed lipid solution prepared before onto the water surface until the surface pressure reached a certain value below 34 mN&#183;m&#8722;1.</li>
			<li>After the surface pressure levels off, use two Teflon barriers to compress the lipid monolayer at a ratio of 5 mm/min until a surface pressure of 34 mN&#183;m&#8722;1 was reached.</li>
			<li>Lift the prism with a lipid monolayer out of the water at a rate of 1 mm/min vertically.</li>
		</ol>
		</li>
		<li>Preparation of the other lipid monolayer
		<ol>
			<li>To facilitate the assembly of the duplex oligonucleotide and the lipid molecules via the hydrophobic interaction (cholesterol and a lipid alkyl chain), mix the duplex oligonucleotide solution with the lipid solution in a molar ratio of 1:100 (oligonucleotide to lipid).</li>
			<li>Inject the mixed lipid and duplex oligonucleotide solution onto the water surface in a homemade Teflon container until a surface pressure of 34 mN&#183;m&#8722;1 was reached.</li>
		</ol>
		</li>
		<li>Finally, put the lipid monolayer at the bottom of the prism in contact with the lipid monolayer with inserted duplex oligonucleotides on the water surface to form the final sample for the SFG measurement.</li>
	</ol>
	</li>
	<li>Lorentz equation
	<ol>
		<li>Use the Lorentz equation to fit the SFG spectra to extract the vibrational information for a specific vibrational mode. 
		<img alt="Equation 26" src="/files/ftp_upload/59380/59380eq26.jpg" style="opacity: 0.9;" />&#160; (25) 
		where&#160;<img alt="Equation 27" src="/files/ftp_upload/59380/59380eq27.jpg" />&#160;represents the intensity of the qth vibrational mode, <img alt="Equation 28" src="/files/ftp_upload/59380/59380eq28.jpg" />&#160;represents the resonant frequency,&#160;<img alt="Equation 29" src="/files/ftp_upload/59380/59380eq29.jpg" />&#160;denotes the half width at half maximum (HWHM) and <img alt="Equation 30" src="/files/ftp_upload/59380/59380eq30.jpg" />&#160;represents the scanning frequency of the incident IR beam.</li>
	</ol>
	</li>
</ol>

<ol><li>Shen, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+Second+Harmonic+Generation+at+Interfaces%5BTitle%5D)+AND+%22Annual+Review+of+Physical+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Surface+properties+probed+by+second-harmonic+and+sum-frequency+generation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Studying+Polymer+Surfaces+and+Interfaces+with+Sum+Frequency+Generation+Vibrational+Spectroscopy%5BTitle%5D)+AND+%22Analytical+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Sum+Frequency+Generation+Vibrational+Spectroscopy+Studies+on+Molecular+Conformation+and+Orientation+of+Biological+Molecules+at+Interfaces%5BTitle%5D)+AND+%22International+Journal+of+Modern+Physics+B%22%5BJournal%5D)">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/pubmed?term=((Liquid+Interfaces+Probed+by+Second-Harmonic+and+Sum-Frequency+Spectroscopy%5BTitle%5D)+AND+%22Chemical+Reviews%22%5BJournal%5D)">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/pubmed?term=((Molcular+Bonding+and+Interactions+at+Aqueous+Surfaces+as+Probed+by+Vibrational+Sum+Frequency+Spectroscopy%5BTitle%5D)+AND+%22Chemical+Reviews%22%5BJournal%5D)">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/pubmed?term=((Quantitative+spectral+and+orientational+analysis+in+surface+sum+frequency+generation+vibrational+spectroscopy%28SFG-VS%29%5BTitle%5D)+AND+%22International+Reviews+in+Physical+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Sum+frequency+generation+spectroscopy+of+the+aqueous+interface%3A+Ionic+and+soluble+molecular+solutions%5BTitle%5D)+AND+%22International+Reviews+in+Physical+Chemistry%22%5BJournal%5D)">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/pubmed?term=((Detecting+Surface+Hydration+of+Poly%282-hydroxyethyl+methacrylate%29+in+Solution+in+situ%5BTitle%5D)+AND+%22Macromolecules%22%5BJournal%5D)">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/pubmed?term=((Evolution+of+Irreversibly+Absorbed+Layer+Promotes+Dewetting+of+Polystyrene+Film+on+Sapphire%5BTitle%5D)+AND+%22Macromolecules%22%5BJournal%5D)">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/pubmed?term=((Surface+Orientation+of+Phenyl+Groups+in+Poly%28sodium+4-styrenesulfonate%29+and+in+Poly%28sodium+4-styrenesulfonate%29%3A+Poly%283%2C4-ethylenedioxythiophene%29+Mixture+Examined+by+Sum+Frequency+Generation+Vibrational+Spectroscopy%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">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/pubmed?term=((A+Sum+Frequency+Generation+Vibrational+Study+of+the+Interference+Effect+in+Poly%28n-butyl+methacrylate%29+Thin+Films+Sandwiched+between+Silica+and+Water%5BTitle%5D)+AND+%22Journal+of+Physical+Chemistry+C%22%5BJournal%5D)">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/pubmed?term=((Directly+Probing+Molecular+Ordering+at+the+Buried+Polymer%2FMetal+Interface+2%3A+Using+P-Polarized+Input+Beams%5BTitle%5D)+AND+%22Macromolecules%22%5BJournal%5D)">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/pubmed?term=((Molecular+Ordering+of+Phenyl+Groups+at+the+Buried+Polystyrene%2FMetal+Interface%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">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/pubmed?term=((Method+to+Probe+Glass+Transition+Temperatures+of+Polymer+Thin+Films%5BTitle%5D)+AND+%22ACS+Macro+Letters%22%5BJournal%5D)">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/pubmed?term=((Interchain+Overlap+Affects+Formation+of+Silk+Fibroin+Secondary+Structure+on+Hydrophobic+Polystyrene+Surface+Detected+via+Achiral%2FChiral+Sum+Frequency+Generation%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">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/pubmed?term=((Calcium-dependent+hydrolysis+of+supported+planar+lipids+was+triggered+by+honey+bee+venom+phospholipase+A2+with+the+right+orientation+at+the+interface%5BTitle%5D)+AND+%22Physical+Chemistry+Chemical+Physics%22%5BJournal%5D)">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/pubmed?term=((Sum+Frequency+Generation+Vibrational+Spectroscopy+Studies+on+Protein+Adsorption%5BTitle%5D)+AND+%22Journal+of+Physical+Chemistry+B%22%5BJournal%5D)">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/pubmed?term=((Detection+of+Amide+I+Signals+of+Interfacial+Proteins+in+Situ+Using+SFG%5BTitle%5D)+AND+%22Journal+of+American+Chemical+Society%22%5BJournal%5D)">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/pubmed?term=((Probing+the+Spontaneous+Membrane+Insertion+of+a+Tall-Anchored+Membrane+Protein+by+Sum+Frequency+Generation+Spectroscopy%5BTitle%5D)+AND+%22Journal+of+American+Chemistry+Society%22%5BJournal%5D)">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/pubmed?term=((Calcium+Ions+Affect+Water+Molecular+Structures+Surrounding+an+Oligonucleotide+Duplex+as+Revealed+by+Sum+Frequency+Generation+Vibrational+Spectroscopy%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">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/pubmed?term=((Sum-frequency+generation+spectroscopy+of+DNA+monolayers%5BTitle%5D)+AND+%22Biosensors+%26+Bioelectronics%22%5BJournal%5D)">Sum-frequency generation spectroscopy of DNA monolayers.</a> Biosensors & 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/pubmed?term=((Metal+Cation-induced+Deformation+of+DNA+Self-Assembled+Monolayers+on+Silicon%3A+Vibrational+Sum+Frequency+Generation+Spectroscopy%5BTitle%5D)+AND+%22Journal+of+American+Chemistry+Society%22%5BJournal%5D)">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/pubmed?term=((Sum-frequency-generation+spectroscopy+of+DNA+films+in+air+and+aqueous+environments%5BTitle%5D)+AND+%22Biointerphases%22%5BJournal%5D)">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/pubmed?term=((DNA+on+Stage%3A+Showcasing+Oligonucleotides+at+Surfaces+and+Interfaces+with+Second+Harmonic+and+Vibrational+Sum+Frequency+Generation%5BTitle%5D)+AND+%22Journal+of+Physical+Chemistry+Letters%22%5BJournal%5D)">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/pubmed?term=((The+thermal+reorganization+of+DNA+immobilized+at+the+silica%2Fbuffer+interface%3A+a+vibrational+sum+frequency+generation+investigation%5BTitle%5D)+AND+%22Physical+Chemistry+Chemical+Physics%22%5BJournal%5D)">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/pubmed?term=((Interference+Effects+in+Sum+Frequency+Spectra+from+Monolayers+on+Composite+Dielectric%2FMetal+Substrates%5BTitle%5D)+AND+%22Journal+of+Physical+Chemistry+B%22%5BJournal%5D)">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/pubmed?term=((Interference+effects+in+the+sum+frequency+generation+spectra+of+thin+organic+films.+I.+Theoretical+modeling+and+simulation%5BTitle%5D)+AND+%22Journal+of+Chemical+Physics%22%5BJournal%5D)">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/pubmed?term=((Interference+Effects+in+Sum+Frequency+Vibrational+Spectra+of+Thin+Polymer+Films%3A+An+Experimental+and+Modeling+Investigation%5BTitle%5D)+AND+%22Journal+of+Physical+Chemistry+B%22%5BJournal%5D)">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/pubmed?term=((Interfacial+Fresnel+Coefficients+and+Molecular+Structures+of+Model+Cell+Membranes%3A+From+a+Lipid+Monolayer+to+a+Lipid+Bilayer%5BTitle%5D)+AND+%22Journal+of+Physical+Chemistry+C%22%5BJournal%5D)">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/pubmed?term=((Interfacial+Water+at+Polyurethane-Sapphire+Interface%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Interfacial Water at Polyurethane-Sapphire Interface.</a> Langmuir. 31 (45), 12401-12407 (2015).</li>
<li>Gautam, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+Structure+of+Polystyrene+at+Air%2FPolymer+and+Solid%2FPolymer+Interfaces%5BTitle%5D)+AND+%22Physical+Review+Letters%22%5BJournal%5D)">Molecular Structure of Polystyrene at Air/Polymer and Solid/Polymer Interfaces.</a> Physical Review Letters. 85 (18), 3854-3857 (2000).</li>
<li>Yan, E. Y., Fu, L., Wang, Z., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+Macromolecules+at+Interfaces+Probed+by+Chiral+Vibrational+Sum+Frequency+Generation+Spectroscopy%5BTitle%5D)+AND+%22Chemical+Reviews%22%5BJournal%5D)">Biological Macromolecules at Interfaces Probed by Chiral Vibrational Sum Frequency Generation Spectroscopy.</a> Chemical Reviews. 114, 8471-8498 (2014).</li>
<li>Belkin, M. A., Kulakov, T. A., Ernst, K. H., Yan, L., Shen, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sum-Frequency+Vibrational+Spectroscopy+on+Chiral+Liquids%3A+A+Novel+Technique+to+Probe+Molecular+Chirality%5BTitle%5D)+AND+%22Physical+Review+Letters%22%5BJournal%5D)">Sum-Frequency Vibrational Spectroscopy on Chiral Liquids: A Novel Technique to Probe Molecular Chirality.</a> Physical Review Letters. 85, 4474(2000).</li>
<li>Rockwood, D. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Materials+fabrication+from+Bombyx+mori+silk+fibroin%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Materials fabrication from Bombyx mori silk fibroin.</a> Nature Protocols. 6, 1612-1631 (2011).</li>
</ol>

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.
Probing Poly(2-hydroxyethyl methacrylate) Surface and Buried interface in Solution: As we demonstrated above, the light field coefficients can be adjusted. We can confirm this experimentally. At the buried interface with the substrate, because the CaF2 substrate surface is smooth and cannot be penetrated by PHEMA molecules, this interface is a sharp one. However, at the surface with water, water molecules can interact with PHEMA molecules and diffuse into the bulk. Hence this interface is blurry, and not as sharp as the buried one. Therefore, different SFG spectral profiles would be observed for these two interfaces. Our experimental SFG data did prove this, indicating the capability to selectively probe the buried interface with the substrate or the surface in solution.
Interchain Interaction or Confinement effect on Formation of Silk Fibroin Secondary Structures: A key factor is the critical overlapping concentration (C*). For SF, C* is ~1.8 mg/mL. Experimentally, for the SF solution of ~90 mg/mL (above C*), no chiral (psp) SFG vibrational signals were detected at the SF solution/PS interface unless an inducing agent-methanol was added, as shown in Figure 7. But, for the SF solution of ~1 mg/mL (below C*), chiral (psp) SFG vibrational signals can be directly detected without adding methanol, as shown in Figure 8, which indicates that the ordered secondary structures have already been formed at the SF solution/PS interface. Since C* is a threshold concentration for the chain-chain overlap, the chain-chain interaction or the spatial confinement has to be taken as a regulating factor here for the formation of SF secondary structures at the interface.&#160;
Water Molecular Structures Surrounding Short-chain Oligonucleotide Duplex: For a short-chain oligonucleotide duplex in the water solution, chiral water SFG vibrational signals correspond to the hydration layer of the chiral spine in the minor groove. Achiral water SFG vibrational signals mostly correspond to the water layer surrounding the oligonucleotide duplex chain and the bilayer (the chiral spine of the water layer also contributes)33. In a Ca2+ concentration range from 0.6 to 6 mM, as shown in Figure 9, we found, there was no obvious change for the chiral water vibrational signals in terms of the Ca2+ concentration. However, the achiral water vibrational signals were strongly affected when the Ca2+ concentration was changed. This indicates that the chiral spine of the water layer closely binding to the oligonucleotide duplex may protect the oligonucleotide from the Ca2+ ions, in the normal biological condition.

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><tbody><tr><td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)&amp;nbsp;</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>&amp;ge;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>&amp;ge;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&amp;amp;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&#039;-CGAAGGCTTCCAGCT-3&#039;</td></tr><tr><td>Single strand of oligonucleotide</td><td>Nanjing Genscript Biotechnology Co., Ltd.</td><td>H04936</td><td>&amp;nbsp;3&amp;cent;-end modified by cholesterol-triethylene glycol(Chol-TEG) (5&amp;cent;-GCTTCCGAAGGTCGA-3&amp;cent;)</td></tr><tr><td>Sodium carbonate anhydrous</td><td>Sinopharm Chemical Reagent Co., Ltd</td><td>10019260</td><td>&amp;ge;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&amp;nbsp;</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 Figure 6 shows the surface structure in water with a ~430 nm PHEMA hydrogel film. Panels A and B correspond to the CH and CO ranges respectively for both figures. At the buried interface, all the observed vibrational peaks are sharp and clear. The reason is that the CaF2 substrate is smooth and cannot be penetrated by PHEMA molecules, leading to a sharp CaF2/PHEMA interface. However, at the surface, because water molecules can interact with PHEMA and diffuse into the bulk, the PHEMA/water interface would be not as sharp as the buried one. Therefore, different spectral profiles are observed for these two interfaces.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59380/59380fig1.jpg" /> 
Figure 1. Schematic show of the SFG process (left panel) with the energy transition diagram (right panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59380/59380fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59380/59380fig2.jpg" /> 
Figure 2. The SFG system in the lab. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59380/59380fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59380/59380fig3.jpg" /> 
Figure 3. Schematic shows the light propagation path in prism for SFG experiment. The numbers 0, 1, 2 and 3 represent the air, prism, PHEMA and bottom medium (the bottom medium can be air, solid or liquid.), respectively. Reproduced from Li, X.; Li, B.; Zhang, X.; Li, C.; Guo, Z.; Zhou, D.; Lu, X. Macromolecules 2016, 49, 3116&#8722;3125 (ref 9). Copyright 2016 American Chemical Society. This figure has been modified from [9]. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59380/59380fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59380/59380fig4.jpg" /> 
Figure 4. Calculated Fresnel coefficients as a function of the film thickness for the prism geometry in water for ssp and ppp polarization combinations. Panels A1 to C1 correspond to the CH range and Panels A2 to C2 correspond to the CO range. Reproduced from Li, X.; Li, B.; Zhang, X.; Li, C.; Guo, Z.; Zhou, D.; Lu, X. Macromolecules 2016, 49, 3116&#8722;3125 (ref 9). Copyright 2016 American Chemical Society. This figure has been modified from [9].&#160;
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59380/59380fig5.jpg" /> 
Figure 5. ssp and ppp spectra of the CaF2/PHEMA interface after water exposure. A: CH and OH range; B: CO range. The black curves are the fitted results by using Lorentz equation. The spectra have been offset for clarity. Reproduced from Li, X.; Li, B.; Zhang, X.; Li, C.; Guo, Z.; Zhou, D.; Lu, X. Macromolecules 2016, 49, 3116&#8722;3125 (ref 9). Copyright 2016 American Chemical Society. This figure has been modified from [9].
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59380/59380fig6.jpg" /> 
Figure 6. ssp and ppp spectra of the PHEMA surface on CaF2 prism. A: CH and OH range; B: CO range. The sample was placed into contact with water. The black curves are the fitted results by using Lorentz equation. The spectra have been offset for clarity. Reproduced from Li, X.; Li, B.; Zhang, X.; Li, C.; Guo, Z.; Zhou, D.; Lu, X. Macromolecules 2016, 49, 3116&#8722;3125 (ref 9). Copyright 2016 American Chemical Society. This figure has been modified from [9].
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59380/59380fig7.jpg" /> 
Figure 7. Normalized chiral (psp) SFG spectra in the amide I (Panel A) and N-H (Panel B) ranges for the PS/SF solution (90 mg/mL) interface before and after adding methanol. The dots are experimental data and the solid lines are the fitted curves. Spectra have been offset for clarity. Reproduced from Li, X.; Deng, G.; Ma, L.; Lu, X.; Langmuir 2018, 34, 9453&#8722;9459 (ref 16). Copyright 2018 American Chemical Society. This figure has been modified from [16].&#160;
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59380/59380fig8.jpg" /> 
Figure 8. Normalized chiral (psp) SFG spectra in the amide I (Panel A) and N-H (Panel B) ranges for the PS/SF solution (1 mg/mL) interface before and after adding methanol. The dots are experimental data and the solid lines are the fitted curves (blue). Spectra have been offset for clarity. Reproduced from Li, X.; Deng, G.; Ma, L.; Lu, X.; Langmuir 2018, 34, 9453&#8722;9459 (ref 16). Copyright 2018 American Chemical Society. This figure has been modified from [16].&#160;
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59380/59380fig9.jpg" /> 
Figure 9. Achiral (ssp, A) and chiral (spp, B) SFG spectra for the duplex oligonucleotide-anchored lipid bilayer in contact with the Ca2+ solutions with different concentrations (from 0.6 mM to 6 mM). The data points were approximately fitted by using the Lorentz equation. The change of the integrated area for the water vibrational signals as a function of the Ca2+ concentration was presented (ssp, C; spp, D). All the spectra have been normalized and offset for clarity. Reproduced from Li, X.; Ma, L.; Lu, X.; Langmuir 2018, 34, 14774&#8722;14779 (ref 21). Copyright 2018 American Chemical Society. This figure has been modified from [21].

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 ...

interfacial-molecular-level-structures-polymers-biomacromolecules

Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.4K Views.  Southeast University. Sum frequentie generatie trillingsspectra zijn uitgerust om interfaces met interfaciale selectiviteit te onderzoeken. De Fresnel-coëfficiëntenmethode kan echter helpen om het interferentie-effect op te lossen wanneer er een andere interface bestaat. Het belangrijkste voordeel van SFG is de interfaciale selectiviteit en submonolayer gevoeligheid.Deze niet-invasieve optische techniek is geschikt voor het investeren van verschillende interfaces, zoals vaste-vloeistof, vast-vast, vloeiba...

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

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. At the same time the AFM tip is passivated to prevent unspecific interactions between the tip and the substrate, which is a prerequisite to study single molecules attached to the AFM tip. Analyses to determine the adhesion force, the adhesion length, and the free energy of these molecules on solid surfaces and bio-interfaces are shortly presented and external references for further reading are provided. Example molecules are the poly(amino acid) polytyrosine, the graft polymer PI-g-PS and the phospholipid POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). These molecules are desorbed from different surfaces like CH3-SAMs, hydrogen terminated diamond and supported lipid bilayers under various solvent conditions. Finally, the advantages of force spectroscopic single molecule experiments are discussed including means to decide if truly a single molecule has been studied in the experiment.

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

Atomic force spectroscopy is an ideal tool to study molecules at surfaces and interfaces. An experimental protocol to couple a large variety of single ...

Engineering

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Chemistry

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Bioengineering

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal structure and probing the hydrogen-bond (H-bond) dynamics of the water molecules adsorbed on solid surfaces are fundamental issues of water science, which remains a great challenge owing to the light mass and small size of hydrogen. Scanning tunneling microscopy (STM) is a promising tool for attacking these problems, thanks to its capabilities of sub-&#197;ngstr&#246;m spatial resolution, single-bond vibrational sensitivity, and atomic/molecular manipulation. The designed experimental system consists of a Cl-terminated tip and a sample fabricated by dosing water molecules in situ onto the Au(111)-supported NaCl(001) surfaces. The insulating NaCl films electronically decouple the water from the metal substrates, so the intrinsic frontier orbitals of water molecules are preserved. The Cl-tip facilitates the manipulation of the single water molecules, as well as gating the orbitals of water to the proximity of Fermi level (EF) via tip-water coupling. This paper outlines the detailed methods of submolecular resolution imaging, molecular/atomic manipulation, and single-bond vibrational spectroscopy of interfacial water. These studies open up a new route for investigating the H-bonded systems at the atomic scale.

Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

Visualizing Solution Structure at Solid-Liquid Interfaces using Three-Dimensional Fast Force Mapping

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the solution conditions such as ion concentrations, pH, ligands, and trace additives, as well as the underlying crystallography and chemistry. In this context, three-dimensional fast force mapping (3D FFM) has emerged as a promising tool for investigating solution structure at interfaces. This capability is based on atomic force microscopy (AFM) and allows the direct visualization of interfacial regions in three spatial dimensions with sub-nanometer resolution. Here we provide a detailed description of the experimental protocol for acquiring 3D FFM data. The main considerations for optimizing the operating parameters depending on the sample and application are discussed. Moreover, the basic methods for data processing and analysis are discussed, including the transformation of the measured instrument observables into tip-sample force maps that can be linked to the local solution structure. Finally, we shed light on some of the outstanding questions related to 3D FFM data interpretation and how this technique can become a central tool in the repertoire of surface science.

Here, we present a protocol for using three-dimensional fast force mapping - an atomic force microscopy technique - for visualizing solution structure at solid-liquid interfaces with the subnanometer resolution by mapping the tip-sample interactions within the interfacial region.

Amongst the challenges for a variety of research fields are the visualization of solid-liquid interfaces and understanding how they are affected by the ...

Exploring Multiphase Polymeric Systems by Nanoscale Infrared Spectroscopy

Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition is commonly assessed using infrared spectroscopy, which provides an average fingerprint of the various materials contained in the volume probed. However, this approach does not offer any details on the arrangement of the phases in the material. Interfacial regions between two polymeric phases, often in the nanoscale range, are also challenging to access. Photothermal nanoscale infrared spectroscopy monitors the local response of materials excited by infrared light with the sensitive probe of an atomic force microscope (AFM). While the technique is suitable for interrogating small features, such as individual proteins on pristine gold surfaces, the characterization of three-dimensional multicomponent materials is more elusive. This is due to a relatively large volume of material undergoing photothermal expansion, defined by the laser focalization onto the sample and by the thermal properties of the polymeric constituents, compared to the nanoscale region probed by the AFM tip. Using a polystyrene (PS) bead and a polyvinyl alcohol (PVA) film, we evaluate the spatial footprint of photothermal nanoscale infrared spectroscopy for surface analysis as a function of the position of PS in the PVA film. The effect of the feature position on the nanoscale infrared images is investigated, and spectra are acquired. Some perspectives on the future advances in the field of photothermal nanoscale infrared spectroscopy are provided, considering the characterization of complex systems with embedded polymeric structures.

Author Spotlight: Advances in Nanoscale Infrared Spectroscopy to Explore Multiphase Polymeric Systems  

This protocol describes the application of atomic force microscopy and nanoscale infrared spectroscopy to evaluate the performance of photothermal nanoscale infrared spectroscopy in the characterization of three-dimensional multi-polymeric samples.

Multiphase polymeric systems encompass local domains with dimensions that can vary from a few tens of nanometers to several micrometers. Their composition ...

Using a VSFG Microscope for Multimodal, Rapid, Hyperspectral Chemical Analysis

Multimodal Nonlinear Hyperspectral Chemical Imaging Using Line-Scanning Vibrational Sum-Frequency Generation Microscopy

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 &#181;m. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 &#181;m level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

Author Spotlight: Unveiling the Potential of VSFG Microscopy in Studying Mesoscopically Heterogeneous Self-Assembled Structures 

A multimodal, rapid hyperspectral imaging framework was developed to obtain broadband vibrational sum-frequency generation (VSFG) images, along with brightfield, second harmonic generation (SHG) imaging modalities. Due to the infrared frequency being resonant with molecular vibrations, microscopic structural and mesoscopic morphology knowledge is revealed of symmetry-allowed samples.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a ...

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

Samenvatting

Meer video's verkennen