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

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.