单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

The overall goal of the following experiment is to synthesize alkylate stabilized gold nanoparticles known as monolayer protected clusters or MPCs, and assemble these materials into thin film assemblies that are used as an absorption interface for azarin protein monolayer electrochemistry. This is achieved by first protecting gold colloids with organic ligands to create gold nanoparticles surrounded by an Alcan Pholate film. Next, the MPCs are anchored to gold and glass substrates and networked into thin multi-layer covalently linked films whose growth is tracked via optical and electrochemical measurements, as well as cross-sectional microscopy.

Then the electron transfer protein azarin is absorbed at the interface and cyclic vault telemetry is performed for protein monolayer electrochemistry. The results show that the MPC film interfaces created by this procedure allow for more optimized electrochemical analysis of adsorb protein based on the MPC film, providing a more homogeneous adsorption interface and fast electron transfer kinetics that lack traditional distance dependence. The main advantage of this technique over traditional protein electrochemistry is that electrodes modified when nanoparticle films provide an absorption interface that results in a more ideal electrochemical behavior as measured by cycl photometry.

Visual demonstration of this method is critical as the synthesis includes important visual cues while the film construction and characterization involves multiple intricate steps and analytical techniques. NPC films can be assembled on modified gold electrodes or glass slides by using a dip cycle method of alternating MPC layers and dile linking molecules. And consequently, these layers can be tracked electrochemically or optically to a desired film thickness.

To synthesize gold clusters under a fume hood with appropriate ventilation begin by dissolving 1.1 grams of tetra oxide ammonium bromide in 30 milliliters of toluene dissolve 0.38 grams of sodium borah hydride in around 20 milliliters of 18 mega ohms ultra purified water, and allow it to chill on ice for at least 30 minutes. Next, quantitatively transfer a solution of hydrogen tetra chloro orating water into the tetra ide ammonium bromide toluene solution using an additional five milliliters of ultra purified water. In order to phase transfer the aqueous gold solution to the non-aqueous solution, lightly cap and stir rigorously for 30 minutes.

So the burnt orange aqueous and clear non-aqueous phases are well mixed. Transfer both the clear aqueous and burnt orange non-aqueous phases into a separatory funnel. Discard the aqueous layer and decant the non-aqueous layer into a clean flask.

Add C six and a ratio of two to one to the hydrogen tetra chloro or rate containing non-aqueous solution. Stir for 30 minutes to form a gold one polymer as detected by a color change from reddish orange to a pale yellow, nearly colorless solution. Transfer the reaction mixture to an insulated ice bath and chill to zero degrees Celsius for at least 30 minutes with stirring quantitatively and quickly add the chilled sodium borohydride solution to the reaction mixture in order to reduce gold one to a metallic gold in the presence of thighs.

Upon addition, it will instantaneously form a thick black solution of monolayer protected gold clusters or MPCs. Stir the reaction overnight at zero degrees Celsius the following day. Transfer the reaction mixture into a separatory funnel.

Discard the aqueous layer into a waste beaker and rotary. Evaporate the non-aqueous toluene layer to near complete dryness, leaving a heavy black sludge in the flask. Precipitate the MPCs by adding aceto nitrile and allow the solution to sit overnight using a glass frit of medium porosity with rubber fittings and a sidearm flask with an aspirator.

Collect MPCs by vacuum filtration and rinse with a large amount of aceto nitrile. After assembling the sandwich cell electrochemically, clean the gold substrate by performing cyclic vol telemetry or CV in the potential windows from 0.2 to 0.9 volts 0.2 to 1.2 volts and 0.2 to 1.35 volts are 100 millivolts per second in a solution of 0.1 molar sulfuric acid and 0.01 molar potassium chloride. Measure the double layer charging current of the cleaned bare gold substrate by performing CV at standard conditions, which includes a potential window from 0.1 to 0.4 volts versus silver cy chloride scanned at 100 millivolts per second in potassium phosphate buffer or KPB.

Discard the buffer and rinse twice excessively with ultra purified water and ethanol. Expose the cleaned gold substrate to around 300 microliters of five millimolar C six solution in ethanol and allow it to sit overnight to form an ordered C six Sam. Discard the C six solution from the cell and rinse it twice excessively with ethanol and ultra purified water.

Measure the charging current of the SAM at standard conditions. Discard the KPB and rinse twice excessively with ultra purified water and ethanol. The charging current should be markedly decreased.

From that of the bare gold measurements, expose the SAM modified gold substrate to around 300 microliters of five millimole NDT solution in ethanol and allow it to sit for one hour to inter disperse NDT linking molecules within the C six Sam. Discard the NDT solution and rinse twice excessively and thoroughly with ethanol and ultra purified water than once with diam methane. Expose the gold substrate to an MPC solution of DIAM methane with agitation by slowly bubbling it with nitrogen gas for one hour.

This is the anchoring MPC layer of the film assembly. Discard the MPC solution and again rinse successively with di chloro methane ultra purified water and KPB. Measure the charging current of the MPC layer at standard conditions.

Discard the KPB and rinse excessively with ultra purified water and DIAM methane. Expose the gold substrate to around 300 microliters of a five millimolar NDT solution in DIAM methane with agitation by slowly bubbling it with nitrogen gas for 20 minutes. Discard the NDT and rinse thoroughly with DIAM methane to deposit the second MPC layer.

Re immerse the film assembly in the MPC solution of DIAM methane and allow it to sit with agitation by slowly bubbling it with nitrogen gas for one hour rinses before. Then measure the charging current of the MPC layer again at standard conditions and rinse again using the same procedure. Deposit additional MPC layers if desired.

After the networked MPC film is complete, rinse the film modified substrate with KPB to absorb AZ protein onto the MPC film assembly. Inject around 150 microliters of five to 10 micromolar AZ solution in KPB into the E chem sandwich cell and allow to sit capped and refrigerated for at least one hour. After the ECM cell returns to room temperature, thoroughly rinse it with KPB, refill the ECM cell with KPB and bubble the KPB with nitrogen gas for 10 minutes.

Perform monolayer electrochemical studies such as CV in the potential window from minus 0.25 volts to 0.25 volts. Scanned at 100 millivolts per second in KPB Rinse A 3M PT MS modified glass slide with DIAM methane and place it in a MPC solution of DIAM methane for one hour while agitating on a shaker at low speed. This completes the first MPC layer of the film assembly by anchoring MPCs to the ME captan end groups of the sane.

Rinse the slide thoroughly with Dior methane, dry it with nitrogen gas and take a UV vis spectrum of the slide After soaking the slide in NDT solution in diam methane. Place the slide in MPC solution for one hour while agitating on a shaker at low speed. This completes the second MPC layer of the film assembly.

Rinse the slide thoroughly with DIAM methane, dry with nitrogen gas and take a UV vis spectrum of the slide. The absorbance across the spectrum should be increasing as additional MPC layers are absorbed to the film assembly. To attach an MPC film assembled on a 3M PT MS modified glass slide onto a standard microscope slide.

Prepare the embed eight 12 epoxy resin and allow it to thicken for at least 12 hours. Fill a beam capsule with epoxy resin and invert it on top of the MPC film sample. Place pressure on the capsule so that a bubble rises to the top of the capsule, creating a seal between the epoxy resin and MPC film sample.

Allow it to polymerize for at least 18 hours at 60 degrees Celsius and then cool the mounted slides to room temperature. Heat the mounted slides for 20 seconds on a cast aluminum hot plate at 200 degrees Celsius in order to facilitate the removal of the block with the attached MPC on fast film. Cut the sample containing the film off the beam capsule block using a jeweler saw reed the removed portion in a silicon flat mold with the MPC film side up facing the interior of the silicon.

Well fill the silicon well with epoxy resin at room temperature and allow it to polymerize for at least 18 hours at 60 degrees Celsius. Then call the sample to room temperature. Cut thin sample sections of 60 to 80 nanometers on a Leica UCT ultra microtome by using a diamond knife to cut sections perpendicular to the knife's edge place sliced sections on form VAR carbon support film on 400 mesh copper grids and take TEM images of cross sections of MPC film assemblies shown.

Here are the results of double layer charging current monitoring of MPC film growth for a total of five dipping cycles involving alternating exposure to MPC and NDT solutions. Charging currents increase systematically with each dipping cycle. Adding layers of MPC to the film.

Here is a typical cyclic Volta Graham for AZ add absorb to an MPC film assembly collected using a potential window from minus 0.25 to plus 0.25 volts scanned at 100 millivolts per second in 4.4 millimolar potassium phosphate buffer. A representative UV vis spectral monitoring of a diol linked MPC film growth on a 3M PT MS modified glass slide is shown here. A dip cycle consists of exposure of the glass slide to NDT linker solution, followed by exposure to MPC solution.

Each subsequent dip results in growth in film thickness and a concurrent absorbance increase. This figure shows a transmission electron microscopy cross-sectional image analysis of a DIA linked MPC film assembly. The inset shows a typical TEM image of hexa methol functionalized MPCs used in the film assembly TEM analysis.

Using Image J determined the average gold core diameter of the MPCs to be around two nanometers. After watching this video, you should have a good understanding of how to synthesize, characterize, and assemble MPC golden narrow par of films that act as an interface for the absorption and subsequent electrochemical analysis of redox proteins. The film assembly process is readily adaptable by manipulating the MPC synthesis procedure as well as the inner particle linkage mechanism to accommodate the absorption of a diverse range of proteins.

Though this method can provide insight into the electrochemistry of protein absorption to modified synthetic platforms, it can also be applied to the development of electron transfer modeling systems, biosensing schemes, and synthetic biocompatible materials.