Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

The overall goal of this experiment is to investigate the gold sorption behavior by S-layer proteins of the presented bacterial strain from an ecological and technological point of view. These methods can answer key question in environmental related science at such is fields of sorption processes and removal and recovery of metals. The main advantage of these techniques are that you can detect metal sorption and nanoparticle adsorption in another scale range.

By using QCM-D it is possible to detect this in an real-time measurement. To begin this procedure, equip the fluid cells with sensor dummies. Pump at least 20 milliliters per module of a 2%alkaline liquid cleansing agent in ultrapure water through the QCM-D and tube system.

Following this, pump a five-fold volume per module of ultrapure water through the system with a flow rate up to 300 microliters per minute per the manufacturer's protocol. Clean the silicon dioxide sensors outside the flow modules by incubation in 2%sodium dodecyl sulfate solution for at least 20 minutes. Then, rinse the sensors several times with ultrapure water.

After drying the crystals with filtered compressed air, place them in an ozone cleaning chamber for 20 minutes. Following this, repeat the cleaning procedure twice to remove all organic contents. To remove bound metals from the sensor surface, rinse the sensors with one molar nitric acid.

Then rinse the sensors several times with ultrapure water. Next, modify the sensors with three grams per liter of alternating layers of polyethylenimine and polystyrene sulfonate via dip coating using the layer by layer or LBL technique. Place the sensors inside the appropriate polyelectrolyte solution in deep-well plates and incubate these for 10 minutes at room temperature.

Then, remove the sensors from the polyelectrolyte solution and rinse them between every dip coating step intensively with ultrapure water. Once the external modification is complete, place the sensors inside the flow module. Equilibrate the sensors by rinsing them with ultrapure water before starting the experiments.

At this point, dissolve previously isolated and purifed Slp1 in formula urea for converting the polymers into monomers. Centrifuge the monomerized proteins at 15, 000 times g and 4 degrees Celsius for one hour to remove bigger protein agglomerates. Mix the solubilized and centrifuged Slp1 supernatant with recrystallization buffer to a final protein concentration of 0.2 grams per liter.

After successful protein recrystallization on top of the polyelectrolyte modified sensors inside the flow modules, rinse the coated sensors with recrystallization buffer intensively with a flow rate of 125 microliters per minute until stable values of frequency and dissipation shifts are detected. After successful Slp1 coating in the flow modules, rinse the obtained Slp1 layer intensively with 1.6 millimolar citrate buffer pH 5.0 until stable values of frequency and dissipation shifts are detected. Next, pump a previously prepared one millimolar nanoparticle solution to the flow modules with a flow rate of 125 microliters per minute.

Then, track the mass adsorption to the Slp1 layer. After completing the metal nanoparticle interaction rinse the layer with nanoparticle-free buffer to remove weak bound metals or nanoparticles. Using an atomic force microscope or AFM with an inverted optical microscope, record images in liquid using ultrapure water directly on the coated QCM-D sensors.

Rinse the sensors with ultrapure water after the QCM-D experiments and place them inside the AFM fluid cell. Use a cantilever with a resonance frequency of approximately 25 kilohertz in water and a stiffness of less than 0.1 newton per meter. Adjust the scanning speed between 2.5 and 10 micrometers per second.

Take images in dynamic contact mode while the cantilever is excited by a Piezo at its resonance frequency. Finally, determine the distance of the cantilever to the surface by the oscillation damping. The maximum metal binding capacities of gold three by suspended Slp1 show that it was stabily bound during the 24-hour incubation within the investigated pH range.

The immediate decrease in frequency indicates a rapid adsorption and a high affinity of the proteins to the polyelectrolyte modified surface. The dissipation also increases immediately which indicates an adsorption of viscoelastic molecules to the surface because of fast damping resulting in increasing dissipation values. Presynthesized gold zero nanoparticles were incubated with Slp1 and analyzed by SDS-PAGE which verified the QCM-D data showing that nanoparticles do not disturb the Slp1 structure.

The adsorption of presynthesized metallic gold zero nanoparticles is shown for QCM-D experiments in delta F-delta D plots and as thickness and mass profiles. The protein lattice of Slp1 recrystallized on polyelectrolyte modified sensors is shown here. The lattice constant was determined as 13 to 14 nanometers and the layer thickness was found to be 10 plus or minus two nanometers.

AFM studies verified that after the gold three interaction the Slp1 lattice remained completely intact which confirms the QCM-D results that predicted the stability of the coating. AFM verifies the adsorption of gold zero nanoparticles on the Slp1 lattice but due to the particle size, they do not follow the p4 symmetry of Slp1. Once mastered this technique can be done within one working day if it is performed properly.

Following these methods, other techniques like infrared spectroscopy or helium ion microscopy can be performed to answer further questions like specific binding sights or interaction with gold or gold nanoparticles. The presented technique paved the way for the uzrachem to study the interactions of biomolecules with metals and for example the adsorption and disorption behavior on different kind of surfaces. After watching this video, you should now have a good understanding of how to investigate interactions of metals with biological solventses and please when you are using this method don't forget to use goggles, lab coat and of course also gloves.