This work is funded by the National Key R&amp;D Program under Grant No. 2016YFA0300901 2016YFA0300903 and 2017YFA0205003, the National Natural Science Foundation of China under Grant No. 11634001, 11290162/A040106. Y.J. acknowledges support by National Science Fund for Distinguished Young Scholars and Cheung Kong Young Scholar Program. J. G. acknowledges support from the National Postdoctoral Program for Innovative Talents.

NOTE: The experiments are performed on water molecules adsorbed on the Au-supported NaCl(001) film (Figure 1a) at 5 K with an ultrahigh-vacuum (UHV) cryogenic STM equipped with Nanonis electronic controller.
1. Fabrication of Experimental Sample
<ol>
	<li>Clean the Au(111) single crystal
	<ol>
		<li>Pump the gas line to the pressure of ~10-7 mbar and then flush the gas line with Ar gas. Put through the pump/flush cycle for three times. 
		NOTE: Each pump/flush cycle takes about 30 min.</li>
		<li>Fill the gas line with Ar gas to the pressure of 2 bar, thus prohibiting the atmosphere to permeate through the gas line.</li>
		<li>Put the Au(111) crystal on the heater stage, which is mounted on the manipulator in the UHV chamber (base pressure of 1.4&#215;10-10 mbar).</li>
		<li>Clean the Au(111) single crystal by cycles of Ar+ ion sputtering for 15 min (p(Ar) = 5&#215;10&#8722;5 mbar, 1.0 kV, 6 &#181;A) and subsequent annealing at about 900 K for 5 min. 
		NOTE: The annealing temperature should be decreased slowly, otherwise a high density of step edges will form on the Au surface. 3 - 5 sputtering/annealing cycles are usually used.</li>
		<li>Transfer the Au(111) sample to the STM scanning stage, and check the cleanliness with STM (Inset of Figure 1b).</li>
	</ol>
	</li>
	<li>Deposition of NaCl on the Au(111) substrate
	<ol>
		<li>Degas the NaCl source. Slowly increase the current applied on the Knudsen cell until the temperature of the source reaches 670 K. Degas the NaCl source several times until the pressure of the chamber is below 4&#215;10-9 mbar. 
		NOTE: The increasing rate of the current depends on the outgassing rate of the NaCl source to maintain the pressure of the chamber below 1&#215;10-8 mbar.</li>
		<li>Put the Au(111) sample on the manipulator and adjust the position of the Au sample to make the sample face the shutter of the Knudsen cell. 
		NOTE: The temperature of the Au(111) substrate could be decreased below room temperature (77-300 K) by cooling down the manipulator head with a continuous flow of liquid nitrogen</li>
		<li>Increase the current applied on the Knudsen cell until the temperature of the source reaches 640 K, and let the evaporation flux stabilize for 5 min before opening the shutter.</li>
		<li>Open the shutter and deposit the NaCl onto the Au(111) sample held at 290 K for 2 min.</li>
		<li>Transfer the Au-supported NaCl sample to the STM scanning stage. Check the coverage and size of the bilayer NaCl(001) islands on the Au(111) substrate with STM (Figure 1b).</li>
	</ol>
	</li>
	<li>Purify the water under vacuum by freeze-pump-thaw cycles21 to remove remaining impurities.
	<ol>
		<li>Prepare three sealed-off glass-UHV adapters. Place water H2O, D2O, and D2O:HOD:H2O isotopic mixture solutions (2 mL) into three adapters separately, and mount the adapters on the gas line (Figure 2). 
		NOTE: The D2O:HOD:H2O isotopic mixtures can be obtained by mixing the ultrapure H2O and D2O with equal amounts under ultrasonic oscillation for 10 min.</li>
		<li>Freeze the liquid water with liquid nitrogen. Make sure that the gas line is pumped to the pressure of ~10-7 mbar before freezing.</li>
		<li>Open the diaphragm-sealed valve and pump off the atmosphere for 15 min. Then close the diaphragm-sealed valve and thaw the solution. 
		NOTE: Gas bubbles evolve from the solution when it is thawing. 
		CAUTION: Let the frozen solvent thaw by itself. Thawing the solution with water bath may cause the glass vessel to break. To freeze and thaw the solution quickly, replace glass-UHV adapters with metal-UHV adapters, though the solution in the metal vessel is invisible.</li>
		<li>Repeat steps 1.3.2-1.3.3 until no gas bubbles evolve from the solution as the solution thaws. Put through the freeze-pump-thaw cycle at least three times.</li>
		<li>Close the bellows-sealed valve and leave the gas line in vacuum. Then open the diaphragm-sealed valve, and let the water vapor fill in the gas line.</li>
	</ol>
	</li>
	<li>Dose water molecules in situ onto the sample surface
	<ol>
		<li>Decrease the temperature of the sample to 5 K. Open the leak valve slowly to make the pressure of the STM UHV chamber increase to 2&#215;10-10 mbar. 
		NOTE: Water molecules flow into the UHV chamber through the dosing tube, which points to the shutter of the shield. The distance between the shutter and the sample (in the shield) is about 6 cm. The base pressure of STM chamber are better than 7&#215;10-11 mbar. The deposition rate is about 0.01 bilayer min-1.</li>
		<li>Open the shutter. Dose the water molecules onto the Au-supported NaCl surface for 1 min. Then close the shutter and the leak valve.</li>
		<li>Check the coverage of water molecules on the Au-supported NaCl(001) surface with STM. Isolated water monomers form on the sample surface (Figure 1c).</li>
	</ol>
	</li>
</ol>
2. Preparation of the Cl-Terminated Tip
<ol>
	<li>Fabricate an electrochemically etched tungsten (W) tip.
	<ol>
		<li>Place 0.3 mm W wire into a 3 Mol/L NaOH etching solution with an immersion length of about 2 mm.</li>
		<li>Apply a 5 V dc potential to the W wire with respect to a platinum ring electrode inserted into the NaOH solution.</li>
		<li>Stop the etching process when the suspended W wire fell off. Clean the etched W tip with distilled water and ethanol. Then transfer the W tip into the scanner. 
		NOTE: The electrochemically etched W tip can be used for one year before exchange.</li>
	</ol>
	</li>
	<li>Apply voltage pulses (2-10 V) and controlled crashing procedures (0.25-0.4 nm) on the STM tip until the atomic Cl atoms of the NaCl surface are resolved. 
	NOTE: The STM tip is poked on a clean region of the Au(111) surface.</li>
	<li>Position the STM tip over the center of one Cl atom (Figure 3a). Bring the bare STM tip close to the NaCl surface in proximity with the set point of V = 5 mV and I = 5 n A (Figure 3b).</li>
	<li>Retract the tip to original set point (Figure 3c) and scan the same area. Check the obtainment of the Cl-tip by improved resolution and a missing Cl atom in the STM image of NaCl (Figure 3d-e). 
	NOTE: Unsuccessful cases may occasionally occur, for example, when the Cl atom doesn&#39;t transfer to the STM or multiple Cl/Na atoms adsorb on the tip. If this occurs, repeat the steps 2.2-2.5.</li>
</ol>
3. Orbital Imaging of Water Monomer
<ol>
	<li>Shape the STM tip with voltage pulse (2-10 V) and controlled crashing (0.25-0.4 nm) procedures.</li>
	<li>Scan the water molecules adsorbed on the NaCl(001) surface with 10 nm by 10 nm frame at 5 K.</li>
	<li>Focus on one individual water monomer and zoom in. Scan the water monomer in a systematic way as functions of the bias (-400-400 mV) and the tunneling current (50-300 pA). 
	NOTE: With a bare STM tip, the highest occupied (HOMO) and lowest unoccupied (LUMO) molecular orbitals of water appear at positive and negative bias, respectively22. Once the tip is Cl-terminated, only HOMO emerges (Figure 4a), and the LUMO feature is not observed throughout the accessible bias range (from -400 mV to 400 mV). Even under larger bias voltage, the water molecules will be unstable due to vibrational excitation.</li>
</ol>
4. Single-Molecule Vibrational Spectroscopy
<ol>
	<li>Setup of the digital lock-in and bias spectroscopy module (Nanonis electronic controller)
	<ol>
		<li>Setup of bias spectroscopy module: Select the current, LIX1 (dI/dV spectra signal), and LIX2 (d2I/dV2 spectra signal) channels. Set the setting time as 50 ms, and integration time as 300 ms. Increase the integration time and sweep times to obtain smooth spectra. Tune the Z offset to take the bias spectroscopy at different tip heights. Make sure the Z-controller set holds and lock-ins runs during measurement 
		NOTE: Setting time is defined as: the time to wait after changing the bias to the next level and before starting to acquire data to avoid transient effect induced by the bias change. Integration time is defined as: the time during which the data are acquired and averaged.</li>
		<li>Setup of lock-in module 
		NOTE: The scanning tunneling spectroscopy, dI/dV and d2I/dV2 spectra, are acquired simultaneously using a lock-in amplifier by demodulating the first and second harmonics of the tunneling current, respectively.
		<ol>
			<li>Modulate the bias and demodulate the current. Set the modulation frequency as a few hundred Hz and modulation amplitude as 5-7 mV. Make sure there is no mechanical and electronic noise at the set point frequency and the corresponding second harmonic frequency.</li>
			<li>Set the first harmonic phase: Switch to the Z-controller module. Set the tip lift to 10 nm and turn off the feedback. Switch to the lock-in module and turn on the lock-in button (green). Click the first harmonic auto phase and record the phase. Repeat the auto phase at least five times and take the average. Then subtract 90 degrees from the averaged phase to get the phase of the junction.</li>
			<li>Set the second harmonic phase: Position the STM tip on the Au(111) substrate and start the bias spectroscopy sweep from -1.5 V to 1.5 V. Select the channel LIX 1 and function dY/dX, which together show the derivative of dI/dV spectrum. Find a prominent peak feature in the spectrum and set the corresponding energy as the bias. Turn on the lock-in and keep the STM system in tunneling. Click the second harmonic auto phase at least five times and take the average. 
			NOTE: Since the second harmonic signals are usually very weak, the phase can fluctuate wildly. When decreasing the tip height to increase the intensity of the signal, the fluctuation of the phase will be much smaller (a few degrees) and the second harmonic phase will be more accurate.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tip-enhanced IETS of a D2O monomer
	<ol>
		<li>Scan a water monomer with Cl-tip at the set point of V=100 mV and I=50 pA.</li>
		<li>Position the Cl-tip on the NaCl surface and take the bias spectroscopy as the background signal. Then position the Cl-tip on the water monomer and start the bias spectroscopy sweep.</li>
		<li>If the dI/dV and d2I/dV2 spectra of water are featureless, simply follow the background NaCl surface (blue curves of Figure 4c-d). Decrease the tip height by tuning the Z offset until the vibrational features emerge in the spectra (red curves of Figure 4c-d). 
		&#8203;NOTE: For the IETS measurement, a long integration time (~1s) and multiple sweeps are needed. For a D2O water monomer, set the bias range from -360 mV to 360 mV. For H2O/HOD water monomers, sweep the bias from -475 mV to 475 mV. Comparing with D2O, H2O, and HOD, water monomers are more easily disturbed and even swept away during IETS measurement.</li>
	</ol>
	</li>
	<li>H-bond strength
	<ol>
		<li>Repeat steps 4.2.2-4.2.3 and tune the sweep bias range to focus on the stretching mode of water monomers. The IETS of water D2O, H2O, and HOD are presented and discussed in 23.</li>
		<li>Obtain the H-bonding energy by converting from the redshift of the H-bonded OH stretching frequency (relative to the free OH stretching energy) using this empirical formula: 
		&#916;H = 1.3 &#215; &#8730;&#916;v&#160; &#160; (1) 
		NOTE: &#916;H is the H-bonding energy, in kJ/mol; &#916;v is the redshift of the OH stretching mode, in cm-1. Convert the unit of H-bonding strength to meV by: 1kJ/mol=10.4 meV/atom. To apply Eq. 1 to the OD stretching mode, the quantity &#916;v should be multiplied by a factor: v(OH) / v(OD) = 1.3612, where v(OH) and v(OD) are the OH and OD stretching frequencies of the free HOD molecule, respectively.</li>
	</ol>
	</li>
</ol>
5. Molecular Manipulation
<ol>
	<li>Construction of a water tetramer (Figure 5a)
	<ol>
		<li>Scan an area containing four water monomers. Position the Cl-tip on top of a monomer at the set point of V=100 mV and I=50 pA. Decrease the height to the set point of V=10 mV and I=150 pA to enhance the tip-water interaction.</li>
		<li>Move the Cl-tip along the predesigned trajectories. Then retract the tip to the initial set point (V=100 mV, I=50 pA), and rescan the same area to check that the water dimer is formed.</li>
		<li>Repeat the steps 5.1.1-5.1.2 to form the water trimer and tetramer. 
		NOTE: The above manipulation process could be realized by the Nanonis controller (Scan control-Follow me module). Setup of the Scan control-Follow me module: 
		Bias: 10 mV 
		Speed: 500 pm/s 
		Z-ctrl Setpoint: 150 pA 
		Switch on/off Z-Ctrl: green 
		Time to wait: 1s 
		Current Gain: LN 10^9 
		Path: Click RECORD button and draw up the designed trajectories on the image, then click the STOP button. 
		Click the EXECUTE button and the STM tip will move along the predesigned trajectories with the setpoint in the Follow me module. If the water monomer doesn&#39;t move, decrease the tip height (smaller bias and bigger current) during manipulation.</li>
	</ol>
	</li>
	<li>Chirality switching of a water tetramer (Figure 6)
	<ol>
		<li>Scan a water tetramer with the Cl-tip. Change the set point to V=5 mV, I=5 pA, and position the tip slightly off the center of the water tetramer.</li>
		<li>In the Z-controller module, define a distance to lift the tip when the Z-controller is switched off (e.g., Tip Lift: -230 pm). Turn off the Z-controller feedback. Bring the tip close to the water tetramer (~230 pm).</li>
		<li>Record the current trace, which shows two different levels, indicating that tetramer has undergone reversible interconversion between two H-bonding chirality.</li>
		<li>Leave the current at the high level and switch on the Z-controller feedback. Retract the tip to the original set point (V=5 mV, I=5 pA). Then scan the water tetramer with the set point of V=10 mV and I=100 pA to check the chiral state of the water tetramer.</li>
		<li>Repeat steps 5.2.1-5.2.4 at least 10 times to confirm the corresponding chiral state of water tetramer at the high current level.</li>
		<li>Repeat steps 5.2.1- 5.2.4 at least 10 times, but leave the current at the low level to check the corresponding chiral state of water tetramer.</li>
		<li>Record the tunneling trace for 20 min, which contains a few hundred switching events</li>
		<li>Plot the distribution of the times the tetramer spent in the low and high level of the current trace, respectively.</li>
		<li>Fit the distribution to an exponential decay (Figure 7). Then get the fitted time constant. Inverse the time constant to yield the switching rate.</li>
	</ol>
	</li>
</ol>

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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=Nanoscale+nuclear+magnetic+resonance+with+a+nitrogen-vacancy+spin+sensor.">Nanoscale nuclear magnetic resonance with a nitrogen-vacancy spin sensor.</a> Science. 339 (6119), 557-560 (2013).</li><li>Staudacher, 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=Nuclear+magnetic+resonance+spectroscopy+on+a+(5-nanometer)3+sample+volume.">Nuclear magnetic resonance spectroscopy on a (5-nanometer)3 sample volume.</a> Science. 339 (6119), 561-563 (2013).</li><li>Aslam, N., 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=Nanoscale+nuclear+magnetic+resonance+with+chemical+resolution.">Nanoscale nuclear magnetic resonance with chemical resolution.</a> Science. 357 (6346), 67-71 (2017).</li></ol>

Authors have nothing to disclose.

To probe the internal structure, dynamics, and vibrational spectroscopy of water molecules adsorbed on the solid surfaces, paying particular attention to the degrees of freedom of hydrogen, some experimental steps are of crucial importance, which will be discussed in the following paragraphs.
The orbital imaging of water molecules is achieved based on two key steps. First, the insulating NaCl films decouple the water electronically from the Au substrate, second the orbital gating effect of the...

The interactions of water with the surfaces of solid materials are involved in various surface reaction processes, such as heterogeneous catalysis, photoconversion, electrochemistry, corrosion and lubrication et al.1,2,3 In general, to investigate interfacial water, spectroscopic and diffraction techniques are commonly used, such as infrared and Raman spectroscopy, sum-frequency generation (SFG), X-ray diffraction (XRD), nuclear magnetic resonance (NMR), neutron scattering4,5,6,7,8. However, these methods suffer from the limitation of spatial resolution, spectral broadening, and averaging effects.
STM is a promising technique to overcome these limitations, which combines the sub-&#197;ngstr&#246;m spatial resolution, atomic manipulation, and single-bond vibrational sensitivity9,10,11,12,13,14. Since the beginning of this century, STM has been extensively applied to investigate the structure and dynamics of water on solid surfaces3,15,16,17,18,19,20. Additionally, vibrational spectroscopy based on STM could be obtained from the second-derivative differential tunneling conductance (d2I/dV2), also known as inelastic electron tunneling spectroscopy (IETS). However, resolving the internal structure, i.e. the H-bond directionality, and acquiring reliable vibrational spectroscopy of water are still challenging. The main difficulty lies in that water is a close shell molecule, whose frontier orbitals are far away from the EF, thus the electrons from the STM tip can hardly tunnel into the molecular resonance states of water, leading to the poor signal-to-noise ratio of molecular imaging and vibrational spectroscopy.
Water adsorbed on the Au-supported NaCl(001) films provides an ideal system for atomic-scale investigation by STM with a Cl-terminated tip (Figure 1a), which is performed at 5 K in the ultrahigh-vacuum (UHV) environment with a base pressure better than 8&#215;10-11 mbar. On one hand, the insulating NaCl films decouple water molecules electronically from the Au substrate so the native frontier orbitals of water are preserved and the lifetime of the electrons residing in the molecular resonant state is prolonged. On the other hand, the STM tip could effectively tune the frontier orbital of water toward the EF via tip-water coupling, especially when the tip is functionalized with a Cl atom. These key steps enable high-resolution orbital imaging and vibrational spectroscopy of water monomers and clusters. In addition, water molecules could be manipulated in a well-controlled manner, due to the strong electrostatic interaction between the negatively charged Cl-tip and water.
In this report, the preparation procedures of the sample and the Cl-terminated tip for STM investigation are outlined in detail in section 1 and 2, respectively. In section 3, we describe the orbital imaging technique, by which the O-H directionality of water monomer and tetramer are resolved. The tip-enhanced IETS is introduced in section 4, which allows the detection of vibrational modes of water molecules at single-bond limit, and determination of the H-bonding strength with high accuracy from the red shift in the oxygen-hydrogen stretching frequency of water. In section 5, we show how the water tetramer can be constructed and switched by controlled tip manipulation. Based on the orbital imaging, spectroscopy, and manipulation techniques, isotopic substitution experiments can be performed to probe the quantum nature of protons in interfacial water, such as quantum tunneling and zero-point motion.

<table border="0" cellpadding="0" cellspacing="0" style="width:632px;" width="633">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Au(111) single crystal</td>
			<td style="width:131px;">MaTeck</td>
			<td style="width:119px;">NA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td align="right">450006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water, deuterium-depleted&#160;</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td align="right">195294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deuterium oxide&#160;</td>
			<td style="width:131px;">Sigma Aldrich</td>
			<td align="right" style="width:119px;">364312</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sealed-off glass-UHV adapters</td>
			<td style="width:131px;">MDC vacuum products</td>
			<td align="right" style="width:119px;">46300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diaphragm-sealed valve</td>
			<td style="width:131px;">any</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bellows-sealed valve</td>
			<td style="width:131px;">any</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leak valve</td>
			<td style="width:131px;">Kurt J. Lesker&#160;</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scanning tunneling microscopy</td>
			<td style="width:131px;">CreaTec</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electronic controller.</td>
			<td style="width:131px;">Nanonis&#160;</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tungsten wire</td>
			<td style="width:131px;">any</td>
			<td style="width:119px;"></td>
			<td>diameter:0.3 mm; purity: 99.95%</td>
		</tr>
	</tbody>
</table>

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.

Figure 1a illustrates the schematic of the STM experimental setup. First, Au(111) substrate is cleaned by sputtering and annealing cycles in the UHV chamber. The clean Au(111) sample shows 22&#215;&#8730;3 reconstructed surface, where the atoms of the surface layer occupy both the hcp and the fcc sites forming herringbone structures (Inset of Figure 1b). The NaCl is evaporated on the Au(111) subs...

Watch this Scientific Journal Video about Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy at JoVE.com

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

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 ...

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8.7K Views.  Peking University. 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.

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

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

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 ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

Multiscale Sampling of a Heterogeneous Water/Metal Catalyst Interface using Density Functional Theory and Force-Field Molecular Dynamics

A significant number of heterogeneously-catalyzed chemical processes occur under liquid conditions, but simulating catalyst function under such conditions is challenging when it is necessary to include the solvent molecules. The bond breaking and forming processes modeled in these systems necessitate the use of quantum chemical methods. Since molecules in the liquid phase are under constant thermal motion, simulations must also include configurational sampling. This means that multiple configurations of liquid molecules must be simulated for each catalytic species of interest. The goal of the protocol presented here is to generate and sample trajectories of configurations of liquid water molecules around catalytic species on flat transition metal surfaces in a way that balances chemical accuracy with computational expense. Specifically, force field molecular dynamics (FFMD) simulations are used to generate configurations of liquid molecules that can subsequently be used in quantum mechanics-based methods such as density functional theory or ab initio molecular dynamics. To illustrate this, in this manuscript, the protocol is used for catalytic intermediates that could be involved in the pathway for the decomposition of glycerol (C3H8O3). The structures that are generated using FFMD are modeled in DFT in order to estimate the enthalpies of solvation of the catalytic species and to identify how H2O molecules participate in catalytic decompositions.

The goal of the protocol presented here is to generate and sample trajectories of configurations of liquid water molecules around catalytic species on a flat transition metal surface. The sampled configurations can be used as starting structures in quantum mechanics-based methods.

A significant number of heterogeneously-catalyzed chemical processes occur under liquid conditions, but simulating catalyst function under such conditions ...

Performing Spectroscopy on Plasmonic Nanoparticles with Transmission-Based Nomarski-Type Differential Interference Contrast Microscopy

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using visible-range light. The purpose of this protocol is to detail a proven method for preparing plasmonic nanoparticle samples and performing single particle spectroscopy on them with DIC microscopy. Several important steps must be followed carefully in order to perform repeatable spectroscopy experiments. First, landmarks can be etched into the sample substrate, which aids in locating the sample surface and in tracking the region of interest during experiments. Next, the substrate must be properly cleaned of debris and contaminants that can otherwise hinder or obscure examination of the sample. Once a sample is properly prepared, the optical path of the microscope must be aligned, using Kohler Illumination. With a standard Nomarski style DIC microscope, rotation of the sample may be necessary, particularly when the plasmonic nanoparticles exhibit orientation-dependent optical properties. Because DIC microscopy has two inherent orthogonal polarization fields, the wavelength-dependent DIC contrast pattern reveals the orientation of rod-shaped plasmonic nanoparticles. Finally, data acquisition and data analyses must be carefully performed. It is common to represent DIC-based spectroscopy data as a contrast value, but it is also possible to present it as intensity data. In this demonstration of DIC for single particle spectroscopy, the focus is on spherical and rod-shaped gold nanoparticles.

The goal of this protocol is to detail a proven approach for the preparation of plasmonic nanoparticle samples and for performing single particle spectroscopy on them with differential interference contrast (DIC) microscopy.

Differential interference contrast (DIC) microscopy is a powerful imaging tool that is most commonly employed for imaging microscale objects using ...

Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope (AFM-SECM)

Scanning electrochemical microscopy (SECM) is used to measure the local electrochemical behavior of liquid/solid, liquid/gas and liquid/liquid interfaces. Atomic force microscopy (AFM) is a versatile tool to characterize micro- and nanostructure in terms of topography and mechanical properties. However, conventional SECM or AFM provides limited laterally resolved information on electrical or electrochemical properties at nanoscale. For instance, the activity of a nanomaterial surface at crystal facet levels is difficult to resolve by conventional electrochemistry methods. This paper reports the application of a combination of AFM and SECM, namely, AFM-SECM, to probe nanoscale surface electrochemical activity while acquiring high-resolution topographical data. Such measurements are critical to understanding the relationship between nanostructure and reaction activity, which is relevant to a wide range of applications in material science, life science and chemical processes. The versatility of the combined AFM-SECM is demonstrated by mapping topographical and electrochemical properties of faceted nanoparticles (NPs) and nanobubbles (NBs), respectively. Compared to previously reported SECM imaging of nanostructures, this AFM-SECM enables quantitative assessment of local surface activity or reactivity with higher resolution of surface mapping.

Probing Surface Electrochemical Activity of Nanomaterials using a Hybrid Atomic Force Microscope-Scanning Electrochemical Microscope AFM-SECM

Atomic force microscopy (AFM) combined with scanning electrochemical microscopy (SECM), namely, AFM-SECM, can be used to simultaneously acquire high-resolution topographical and electrochemical information on material surfaces at nanoscale. Such information is critical to understanding heterogeneous properties (e.g., reactivity, defects, and reaction sites) on local surfaces of nanomaterials, electrodes and biomaterials.

Scanning electrochemical microscopy (SECM) is used to measure the local electrochemical behavior of liquid/solid, liquid/gas and liquid/liquid interfaces. ...

Analyzing Melts and Fluids from Ab Initio Molecular Dynamics Simulations with the UMD Package

We have developed a Python-based open-source package to analyze the results stemming from ab initio molecular-dynamics simulations of fluids. The package is best suited for applications on natural systems, like silicate and oxide melts, water-based fluids, and various supercritical fluids. The package is a collection of Python scripts that include two major libraries dealing with file formats and with crystallography. All the scripts are run at the command line. We propose a simplified format to store the atomic trajectories and relevant thermodynamic information of the simulations, which is saved in UMD files, standing for Universal Molecular Dynamics. The UMD package allows the computation of a series of structural, transport and thermodynamic properties. Starting with the pair-distribution function it defines bond lengths, builds an interatomic connectivity matrix, and eventually determines the chemical speciation. Determining the lifetime of the chemical species allows running a full statistical analysis. Then dedicated scripts compute the mean-square displacements for the atoms as well as for the chemical species. The implemented self-correlation analysis of the atomic velocities yields the diffusion coefficients and the vibrational spectrum. The same analysis applied on the stresses yields the viscosity. The package is available via the GitHub website and via its own dedicated page of the ERC IMPACT project as open-access package.

Melts and fluids are ubiquitous vectors of mass transport in natural systems. We have developed an open-source package to analyze ab initio molecular-dynamics simulations of such systems. We compute structural (bonding, clusterization, chemical speciation), transport (diffusion, viscosity) and thermodynamic properties (vibrational spectrum).

We have developed a Python-based open-source package to analyze the results stemming from ab initio molecular-dynamics simulations of fluids. The package ...

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20&#8211;100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and ...