Name: in situ characterization of hydrated proteins in water by salvi and tof-sims
Uploaded: 2016-02-15T15:00:00
Description: Watch this Scientific Journal Video about In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS at JoVE.com

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.</l...

<ol>
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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 ...

The overall goal of this procedure is to characterize adsorbed protein films in aqueous solutions, using the System for Analysis at the Liquid Vacuum Interface, and time-of-flight secondary ion mass spectrometry. This method can help answer key questions about biomolecules and the biointerface. For example, characterization of adsorbed proteins at a solid surface.The main advantages of this technique is that the biomolecules can be started in situ in the liquid environment, and in combination with molecular mapping. Though this method can provide insight into biomolecules at the solid-liquid interface, it can also be applied to other systems, including investigation into single cancer cells, drug interactions, and the attachment of biofilms. Into a five milliliter syringe, draw up two milliliters of a 70%ethanol solution.Connect the syringe to the inlet end of the SALVI device, and slowly inject one milliliter of the liquid over the course of 10 minutes. At the end of the injection, remove the syringe, and connect the inlet and outlet of the SALVI using a polyether-ether-ketone union. Keep the SALVI microchannel filled with the 70%ethanol solution at room temperature for four hours.Then, fill a second syringe with sterile deionized water. Connect it to the inlet of the SALVI and slowly inject the deionized water into the device over the course of 10 minutes. Remove the syringe and reconnect the inlet and the outlet of the device together.In order to immobilize a protein film, start by drawing up two milliliters of fibronectin solution into a syringe. Connect the syringe to the inlet of the SALVI device, and slowly inject one milliliter of the liquid over the course of 10 minutes. Then, remove the syringe, and again reconnect the inlet and outlet.Incubate the setup in a clean culture dish at room temperature for 12 hours. Following incubation, slowly inject one milliliter of sterile deionized water over the course of 10 minutes, and then reconnect the inlet to the outlet. To begin, lay the ToF-SIMS stage on a flat, clean surface.Put the SALVI device onto the stage with the silicon wafer and silicon nitride membrane facing upward. Fix the SALVI device to the stage using four metal clamping pieces, and screw the metal pieces holding the corner of the device into the metal plate by four screws. Next, 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 stage, so that it does not interfere with the primary ion beam during analysis. Next, mount the Faraday cup onto the stage using a screw.Then, open the ToF-SIMS loadlock door and set the stage horizontally onto the loading platform before closing the door. Turn on the vacuum pump and let the loadlock chamber pump down to around 10 to the minus sixth millibar. Then, move the SALVI device into the main chamber after about 30 minutes, once the vacuum is stabilized.Adjust the primary ion beam, and analyzer of the ToF-SIMS, to acquire a spatial resolution of approximately 400 nanometers by following the manufacturer's recommended protocol. Next, 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.Visual demonstration of this step is critical, as it is difficult to learn, because the sample is thicker than normal solid samples, and it's difficult to adjust the settings to find the best focus of the channel. Before each measurement, use a one kiloelectronvolt oxygen beam to scan on the silicon nitride window for about 15 seconds to remove surface contamination. Also, use an electron flood gun to compensate surface charging during all measurements.Next, select the positive or negative mode before starting data acquisition. Use the 25 kiloelectronvolt bismuth three plus as the primary ion beam in all measurements. For depth profiling, scan the primary ion beam on a round area with a diameter of about two micrometers with a resolution of 32 pixels by 32 pixels.To minimize the time required to punch through the silicon nitride membrane, use a long pulse width primary ion beam. After the silicon nitride membrane punch-through, keep the same current for about 200 seconds to obtain sufficient data for two-dimensional image reconstructions. For data collection, reduce the pulse width to 80 nanoseconds in order to acquire spectra with better mass resolution.Continue this acquisition for about another 200 seconds. Using the SALVI microfluidic interface, the primary ion beam can directly bombard on the hydrated fibronectin film. A number of water clusters and amino acid fragments are indicated by the ToF-SIMS mass spec data.This provides direct evidence of the property of water molecules surrounding and inside the hydrated protein molecules. Here, one can see the depth profile time series of the hydrated fibronectin film and of deionized water. The intensities of selected second ions are negligible before the silicon nitride membrane is punched through.Once the silicon nitride membrane is punched through, the intensities of the secondary ions increase significantly. The short pulse width process is used to obtain data with better mass resolution for image and spectrum reconstructions. The 2-D images from the hydrated fibronectin film and deionized water represent the counts of selected secondary ions from the sample.The brighter the image, the higher the secondary ion counts. These intuitive images give a clear comparison of selected secondary ions between different samples, which is helpful in data analysis. While attempting this procedure, it is important to remember that a microreactor needs to be leak-free before installing in the vacuum chamber.This technique paved the way for researchers in the field of biology and biointerfaces to explore protein adsorption, biofilm attachment, surface-assembled monolayers, and many other phenomena. After watching this video, you should have a good understanding of how to study various liquid samples using SALVI and the liquid ToF-SIMS.

Research

JoVE Journal

Chemistry

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 ...

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

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 ...

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. ...

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

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 ...

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 ...

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

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) ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

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 ...

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 ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

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 ...

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 ...