We are grateful to the Pacific Northwest National Laboratory (PNNL) Chemical Imaging Initiative-Laboratory Directed Research and Development (CII-LDRD) and Materials Synthesis and Simulation across Scales (MS3) Initiative LDRD fund for support. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) Science Themed Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. The authors thank Mr. Xiao Sui, Mr. Yuanzhao Ding, and Ms. Juan Yao for proof reading the manuscript and providing useful feedback. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

1. Cleaning and Sterilization of the SALVI Microchannel
<ol>
	<li>Sterilization of the Microchannel in SALVI
	<ol>
		<li>Draw 2 ml of 70% ethanol aqueous solution into a syringe, connect the syringe with the inlet end of SALVI, and slowly inject 1 ml of the liquid in 10 min. Remove the syringe at the end of injection. Next, connect the inlet and outlet of SALVI using a polyetheretherketone (PEEK) union. Alternately, use a syringe pump to conduct the same procedure. For example, set the flow speed at 100 &#956;l/min.</li>
		<li>Keep the SALVI microchannel filled with 70% ethanol solution at room temperature for 4 hr.</li>
	</ol>
	</li>
	<li>Introduce Deionized (DI) Water into SALVI
	<ol>
		<li>Draw 2 ml of sterilized DI water into a syringe, connect the syringe with the inlet of SALVI, and slowly inject 1 ml of the liquid in 10 min. Remove the syringe, and connect the inlet and outlet of SALVI using a PEEK union. Alternately, use a syringe pump as mentioned in step 1.1.1.</li>
	</ol>
	</li>
</ol>
2. Immobilization of the Protein Film in SALVI
<ol>
	<li>Immobilize the Protein Film in SALVI
	<ol>
		<li>Draw 2 ml of fibronectin (10 &#956;g/ml in phosphate buffered saline, PBS) solution into a syringe, connect the syringe with the inlet of SALVI, slowly inject 1 ml of the liquid in 10 min, remove the syringe, and connect the inlet and outlet of SALVI using a PEEK union.</li>
		<li>Incubate the SALVI filled with the fibronectin solution in a clean culture dish, keep the dish in the hood at room temperature for 12 hr.</li>
		<li>Draw 2 ml of sterilized DI water into a syringe and connect the syringe with the inlet of SALVI. Slowly inject 1 ml of water in 10 min, remove the syringe, and connect the inlet and outlet of SALVI. 
		Note: If equipped with a light microscope, check the SALVI under the microscope to ensure that the SiN membrane is intact before ToF-SIMS analysis. Now the SALVI device is ready for ToF-SIMS analysis.</li>
	</ol>
	</li>
</ol>
3. Install SALVI into the ToF-SIMS Loadlock Chamber
Note: Gloves should be worn at all times when handling the SALVI device and installing it onto the ToF-SIMS stage to avoid potential contaminations during surface analysis.
<ol>
	<li>Mount SALVI onto the Stage
	<ol>
		<li>Lay the ToF-SIMS stage on a flat and clean surface. Put the SALVI device onto the stage with the silicon wafer and SiN membrane facing upward. Fix the SALVI using four metal clamping pieces and screw the metal pieces holding the corner of the device into the metal plate by four screws.</li>
		<li>Gently roll up the PFTE tubing connected to the microfluidic channel. Use four metal pieces and four screws to hold the stiff part down. Make sure that the device is positioned in the open space on the SIMS stage so that it does not interfere with the primary ion beam during analysis. Additional image and illustration of the SALVI fabrication and installation are available in previous publications.8,9,11</li>
	</ol>
	</li>
	<li>Mount the Faraday Cup onto the Stage
	<ol>
		<li>Fix the Faraday cup on the stage by a screw.</li>
	</ol>
	</li>
	<li>Load the Stage into the ToF-SIMS Loadlock Chamber
	<ol>
		<li>Open the ToF-SIMS loadlock, hold and set the stage horizontally onto the loading platform, and close the loadlock door.</li>
	</ol>
	</li>
</ol>
4. ToF-SIMS Data Acquisition
<ol>
	<li>Turn on the vacuum pump and ensure suitable vacuum (e.g., on the order of 10-6 mbar or Torr) is reached in the loadlock chamber.</li>
	<li>Move the SALVI into the main chamber once the vacuum is stabilized after about 30 min. 
	Note: The vacuum should be lower than 5 x 10-6 mbar vacuum in the main chamber during data acquisition. Otherwise, higher vacuum is indicative of a leak in the SALVI system.</li>
	<li>Adjust the primary ion beam and analyzer of ToF-SIMS with a spatial resolution of approximately 400 nm according to manufacturer recommendations. The current of primary beam is 1,200 pA while the cycle time is 30 &#956;sec under DC mode. 
	Note: This process largely follows manufacturer&#39;s recommendations.</li>
	<li>Find the microfluidic channel using the optical microscope. Use the optical image center as a reference and align it to be consistent with the secondary ion image center.</li>
	<li>Before each measurement, use a 1 KeV O2+ beam (~40 nA) to scan on the SiN window with a 500 x 500 &#181;m2 area for ~15 sec to remove surface contamination. Also, use an electron flood gun to compensate surface charging during all measurements. Use the automatic function of the flood gun in the default mode for this step.</li>
	<li>Select positive or negative mode before data acquisition. Use the 25 keV Bi3+ beam as the primary ion beam in all measurements. 
	Note: The steps are similar for positive and negative data acquisitions. For the interest of space, the positive mode is described in details here.
	<ol>
		<li>Depth Profiling
		<ol>
			<li>Scan the Bi3+ beam on a round area with a diameter of ~2 &#181;m with 32 pixels by 32 pixels resolution.</li>
			<li>To minimize the time required to punch-through the SiN membrane, use a long pulse width (i.e., 180 nsec, beam current ~1.0 pA at a cycle time of 100 &#956;sec) Bi3+ beam. The intensities of secondary ions representative from the liquid surface increase when punch-through is reached. The punch-through time varies from 300 sec to 500 sec, depending on the batch of the SiN membrane. 
			Note: This difference seems to be related to the batch of SiN membranes acquired according to our experience. However, the punch-through time does not really affect the data analysis.</li>
			<li>After the SiN membrane punch-through, keep the same current for about 200 sec to obtain sufficient data for 2D image reconstructions.</li>
			<li>Reduce the pulse width to 80 nsec for data collection to acquire spectra with better mass resolution. Continue this acquisition for about another 200 sec. The spectrum data shown in Figure 1 are obtained from this region.</li>
		</ol>
		</li>
		<li>2D Imaging and Mass Spectra
		<ol>
			<li>Collect the 2D imaging and mass spectra data at the same time when measuring the depth profiling data. The ToF-SIMS instrument saves all the information of each spot during the experiment. The data are shown in different windows (FPanel - Spectra, FPanel - Profiles and FPanel - Images separately) of the digital control software. Check data in each panel judiciously during data acquisition.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. ToF-SIMS Data Analysis
Note: Data analysis is initially performed using the IonToF ToF-SIMS instrumental software.
<ol>
	<li>Open the analysis software of SIMS (Measurement Explorer) and then click the &#34;profiles&#39;, &#39;spectra&#39; and &#39;image&#39; buttons, respectively, to process depth profile, m/z spectrum, and image data.</li>
	<li>Open the data files that have the extension &#34;.ita&#34;.</li>
	<li>Select peaks of interest, type the species name in &#34;ion&#34; box, and label them with different colors in the spectra. Use the mouse scroll wheel to drag along the x-axis. Use Ctrl and scroll wheel to adjust peak heights and widths. Letter &#34;L&#34; switches between Linear and Log modes along the y-axis.</li>
	<li>Conduct data reconstruction before mass calibration. Otherwise, it is difficult to choose the spacing between peaks in the mass calibration step.
	<ol>
		<li>Select the depth profiling data of interest and reconstruct the spectra according to the depth profile temporal series.</li>
		<li>Click the reconstruction button. In the &#34;Start Reconstruction&#34; window, type in a scan range of interest. Click &#34;OK&#34; to reconstruct the depth profile data.</li>
	</ol>
	</li>
	<li>Perform a mass calibration. Press &#34;F3&#34; to open the &#34;mass calibration&#34; window. Choose peaks for calibration based on the specific sample.21 In this work, use the following peaks CH3+ (m/z 15), H3O+ (m/z 19), C2H5+ (m/z 29), H5O2+ (m/z 37), C3H7+ (m/z 43), H7O3+ (m/z 55), H9O4+ (m/z 71), H11O5+ (m/z 99) and H13O6+ (m/z 109) for the positive mode. Use the following peaks CH- (m/z 13), O- (m/z 16), OH- (m/z 17), C2H- (m/z 25), C3H- (m/z 37), and C4H- (m/z 49) for the negative mode.
	<ol>
		<li>Select a peak, make sure that the downward arrow is pointed at the peak in the left bottom corner of the window. Click that peak and type the peak name in the &#34;mass calibration&#34; window, click &#34;Add&#34;, and the ion of interest is included in the mass calibration.</li>
		<li>Finally, click the &#34;Recalibrate&#34; button. Check if the areas of peaks are in the right places according to their mass to charge ratios. In the &#34;Spectrum&#34; menu, select and click &#34;Apply Mass Calibration&#34; to &#34;All Spectra&#34; or &#34;Selected spectra&#34;, depending on the specific analysis being conducted.</li>
	</ol>
	</li>
	<li>As peak selection is necessary for sample analysis, determine characteristic peaks of the sample according to literature or other previous findings. Compare the intensity of peaks of interest in the m/z spectra. If necessary, add new peaks or delete useless peaks in the peak list.
	<ol>
		<li>Add peaks. Click on a peak, find the red lines at the left bottom window. Hold the &#34;Ctrl&#34; key, scroll mouse horizontally to define peak width, which adds a peak to the peak list automatically. Another way to add a peak is to select a peak in the main spectrum window, and then to click the &#34;add peak&#34; button above the window. If there are many peaks to add, in the &#34;Spectrum&#34; menu, select &#34;search peaks&#34;, check related specifications in the newly opened window, then add peaks as needed.</li>
		<li>To export a peak list, in the &#34;Peak List&#34; menu, select &#34;Save&#8230;&#34; the peak list in the &#34;itmil&#34; format. Use the Ctrl and left button to drag along the spectrum. In the spectra window, select a peak of interest and drag the two dashed lines to desirable positions.</li>
		<li>To import a peak list, in the &#34;Mass interval List&#34; menu, choose &#34;Import&#8230;&#34;, locate an existing peak list file with the file extension &#34;.ITPL&#34;. Click &#34;Open&#34;.</li>
		<li>To use an &#34;itmil&#34; peak list, in the &#34;Mass Interval List&#34; menu, click &#34;Open&#8230;&#34;, find the file, then click &#34;Open&#34;.</li>
	</ol>
	</li>
	<li>Apply a peak list to further analyze data. At the left-bottom of &#34;Spectra&#34; window, there is a window called &#34;Mass Interval List&#34;. The imported peak list is shown up here.
	<ol>
		<li>Right-click the peak list file to be used, apply it to the &#34;active spectrum&#34; or &#34;all spectra&#34;.</li>
	</ol>
	</li>
	<li>View the data profiles. In the &#34;Profile&#34; window, Use letter &#34;M&#34; to fit to window (full range) and letter &#34;N&#34; to fit to the y-axis. Use Ctrl and scroll wheel to adjust the profile width. Or use &#34;/&#34; and &#34;*&#34; on the key board to change the y-axis range and height.</li>
	<li>Export data for further plotting using other graphic software after the initial analysis.
	<ol>
		<li>To export a depth profile, in the &#34;Profile&#34; window, click on the &#34;File&#34; menu, then select &#34;Export&#34;, and then select &#34;Save as&#34; a &#34;.txt&#34; file.</li>
		<li>To export an m/z spectrum file, in the &#34;Spectra&#34; window, click on the &#34;File&#34; menu, then select &#34;Export&#34;, and then select &#34;Save as&#34; a &#34;.txt&#34; file.</li>
		<li>To export an image file, in the &#34;Image&#34; window, print screen and save as the image file.</li>
	</ol>
	</li>
</ol>
6. ToF-SIMS Data Plotting and Presentation
Note: Use a graphic tool to plot the data after the SIMS data are processed using the instrumental software.
<ol>
	<li>Use a graphic tool to import the data.</li>
	<li>Make a plot using the m/z as the x-axis and peak intensity as the y-axis to show the reconstructed spectrum. An example is given in Figure 1.</li>
	<li>Make a plot using the sputter time as the x-axis and peak intensity as the y-axis to show the reconstructed depth profile temporal series. An example is given in Figure 2.</li>
	<li>Combine reconstructed 2D images of different m/z and form a matrix to show the ion mapping. An example is given in Figure 2.</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+of+plasma+proteins+and+fibronectin+on+poly(hydroxylethyl+methacrylate)+brushes+of+different+thickness+and+their+relationship+with+adhesion+and+migration+of+vascular+smooth+muscle+cells.">Adsorption of plasma proteins and fibronectin on poly(hydroxylethyl methacrylate) brushes of different thickness and their relationship with adhesion and migration of vascular smooth muscle cells.</a> Regen Biomater. , 17-25 (2014).</li></ol>

The authors have nothing to disclose.

SALVI is a microfluidic interface that allows dynamic liquid surface and liquid-solid interface analysis by vacuum based instruments, such as ToF-SIMS and scanning electron microscopy (SEM). Due to the usage of small apertures to expose liquid directly in vacuum, SALVI is suitable for many finely focused spectroscopy and imaging techniques without any modifications;22 the portability and versatility of microfluidics make it a true multimodal imaging platform. Distinct features and significance of SALVI compare...

Hydration is crucial to the structure,1 conformation,2 and biological activity3 of proteins. Proteins without water molecules surrounding them would not have viable biological activities. Specifically, water molecules interact with the surface and internal structure of proteins, and different hydration states of proteins make such interactions distinct.4 The interaction of proteins with solid surfaces is a fundamental phenomenon with implications in nanotechnology, biomaterials and tissue engineering processes. Studies have long indicated that conformational changes may occur as a protein encounters a surface. ToF-SIMS has been envisioned as the technique that has the potential to study the protein-solid interface.5-7 It is important to understand the hydration of proteins on solid surfaces, which potentially provides a fundamental understanding of the mechanism of their structure, conformation, and biological activity.
However, major surface analytical techniques are mostly vacuum-based and direct applications for volatile liquid studies are difficult due to the rapid evaporation of volatile liquid under the vacuum environment. We developed a vacuum compatible microfluidic interface, System for Analysis at the Liquid Vacuum Interface (SALVI), to enable direct observations of liquid surfaces and liquid-solid interactions using time-of-flight secondary ion mass spectrometry (ToF-SIMS).8-11 The unique aspects include the following: 1) the detection window is an aperture of 2-3 &#956;m in diameter allowing direct imaging of the liquid surface, 2) surface tension is used to hold the liquid within the aperture, and 3) SALVI is portable among multiple analytical platforms.11,12
SALVI is composed of a silicon nitride (SiN) membrane as the detection area and a microchannel made of polydimethylsiloxane (PDMS). It is fabricated in the clean room, and the fabrication and key design factors have been detailed in previous papers and patents.8-12 The applications of ToF-SIMS as an analytical tool were demonstrated using a variety of aqueous solutions and complex liquid mixtures, some of which contained nanoparticles.13-17 Specifically, SALVI liquid ToF-SIMS allows dynamic probing of the liquid-solid interface of live biological systems (i.e., biofilms), single cells, and solid-electrolyte interface, opening new opportunities for in situ condensed phase studies including liquids using ToF-SIMS. However, the current design does not permit gas-liquid interactions yet. This is a direction for future development. SALVI has been used to study the hydrated protein film in this work for the first time.
Fibronectin is a commonly used protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds,18 which is well-known for its ability to bind cells.19,20 It was chosen as a model system to illustrate that the hydrated protein film could be dynamically probed using the SALVI liquid ToF-SIMS approach. The protein solution was introduced into the microchannel. After incubating for 12 hr, a hydrated protein film formed on the back side of the SiN membrane. Deionized (DI) water was used to rinse off the channel after protein introduction. Information was collected from hydrated fibronectin protein molecules in the SALVI microchannel using dynamic ToF-SIMS. DI water was also studied as a control to compare with results obtained from hydrated fibronectin thin film. Distinct differences were observed between the hydrated protein film and DI water. This work demonstrates that protein adsorption on surface in the liquid environment can be studied using the novel SALVI and liquid ToF-SIMS approach. The video protocol is intended to provide technical guidance for people who are interested in utilizing this new analytical tool for diverse applications of SALVI with ToF-SIMS and reduce unnecessary mistakes in liquid handling as well as ToF-SIMS data acquisition and analysis.

<table border="0" cellpadding="0" cellspacing="0" width="1170">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">ToF-SIMS</td>
			<td style="width:260px;">IONTOF</td>
			<td style="width:121px;">TOF.SIMS 5</td>
			<td style="width:400px;">Resolution: &#62; 10,000 m/&#916;m for mass resolution; &#62; 4,000 m/&#916;m for high spatial resolution&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">System for Analysis at the Liquid Vacuum Interface (SALVI)</td>
			<td style="width:260px;">Pacific Northwest National Laboratory</td>
			<td style="width:121px;">N/A</td>
			<td style="width:400px;">SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum-based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">PEEK Union</td>
			<td style="width:260px;">Valco</td>
			<td style="width:121px;">ZU1TPK</td>
			<td>for connecting the inlet and outlet of SALVI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">5 Axes Sample Stage</td>
			<td style="width:260px;">IONTOF</td>
			<td style="width:121px;">N/A</td>
			<td>Stage is self-made for mounting SALVI in ToF-SIMS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Barnstead Nanopure Water Purification System</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">D11921</td>
			<td>ROpure LP Reverse Osmosis filtration module (D2716)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Syringe</td>
			<td style="width:260px;">BD</td>
			<td style="width:121px;">309659</td>
			<td style="width:400px;">1 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Pipette</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">21-377-821</td>
			<td>Range: 100 to 1000 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Pipette Tip</td>
			<td style="width:260px;">Neptune</td>
			<td style="width:121px;">2112.96.BS</td>
			<td>1000 &#181;L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Centrifuge Tube</td>
			<td style="width:260px;">Corning</td>
			<td style="width:121px;">430791</td>
			<td>15 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Fibronectin</td>
			<td style="width:260px;">Sigma-Aldrich</td>
			<td style="width:121px;">F1141</td>
			<td>1 mg/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Ethanol</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">S25310A</td>
			<td>95% Denatured</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:389px;">Gibco PBS</td>
			<td style="width:260px;">Thermo Fisher Scientific</td>
			<td style="width:121px;">10010-023</td>
			<td>pH 7.4</td>
		</tr>
	</tbody>
</table>

in situ characterization of hydrated proteins in water by salvi and tof-sims

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

A couple of representative results are presented to demonstrate the advantages of the proposed protocol. By using the SALVI microfluidic interface, the primary ion beam (Bi3+) can directly bombard on the hydrated fibronectin film in DI water. Thus the molecular chemical mapping of the liquid surface can be successfully acquired.
Figure 1a and 1b show the positive ToF-SIMS m...

Watch this Scientific Journal Video about In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS at JoVE.com

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

in-situ-characterization-hydrated-proteins-water-salvi-tof

Universidad San Sebastian

Research

JoVE Journal

Chemistry

8.2K Views.  Pacific Northwest National Laboratory. This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

Video: In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the time-profile volume and enthalpy changes of light induced conformational changes in proteins on microsecond to millisecond time-scales that are not accessible using traditional stop-flow instruments. In addition, since overall changes in volume and/or enthalpy are probed, these techniques can be applied to proteins and other biomacromolecules that lack a fluorophore and or a chromophore label. To monitor dynamics and energetics of structural changes associated with Ca2+ binding to calcium transducers, such neuronal calcium sensors, a caged calcium compound, DM-nitrophen, is employed to photo-trigger a fast (&#964; &#60; 20 &#956;sec) increase in free calcium concentration and the associated volume and enthalpy changes are probed using photothermal beam deflection technique.

Here we report an application of the photothermal beam deflection technique in combination with a caged calcium compound, DM-nitrophen, to monitor microsecond and millisecond dynamics and energetics of structural changes associated with the calcium association to a neuronal calcium sensor, Downstream Regulatory Element Antagonist Modulator.

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface (SALVI) and Scanning Electron Microscopy (SEM). This paper describes the method and key steps in integrating the vacuum compatible SAVLI to SEM and obtaining secondary electron (SE) images of particles in liquid in high vacuum. Energy dispersive x-ray spectroscopy (EDX) is used to obtain elemental analysis of particles in liquid and control samples including deionized (DI) water only and an empty channel as well. Synthesized boehmite (AlOOH) particles suspended in liquid are used as a model in the liquid SEM illustration. The results demonstrate that the particles can be imaged in the SE mode with good resolution (i.e., 400 nm). The AlOOH EDX spectrum shows significant signal from the aluminum (Al) when compared with the DI water and the empty channel control. In situ liquid SEM is a powerful technique to study particles in liquid with many exciting applications. This procedure aims to provide technical know-how in order to conduct liquid SEM imaging and EDX analysis using SALVI and to reduce potential pitfalls when using this approach.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...