The overall goal of this procedure is to encapsulate cytochrome c in Silical sol-gels. To super critically process these gels to form bio aerogels, and to use these bio aerogels to rapidly recognize nitric oxide through a gas phase reaction. This method can help answer key questions in the biosensing and bio analytical fields, such as what conditions are necessary for cytochrome c to remain active when encapsulated in a highly porous aerogel?

The significance for this technique is that it is a simple procedure for encapsulating a protein in a matrix in which proteins have not often been successfully encapsulated. Though this method can provide insight into cytochrome c, it may also lead to procedural developments for encapsulating other proteins in aerogels with potential significance for future bio analytical devices. Make a solution with a Cytochrome c concentration in the range of 0.7 to millimolar.

As well as a diluted 0.105 millimolar cytochrome c solution, as described in the text protocol. Then, label a disposable 50 milliliter polypropylene beaker, Beaker A.Place the beaker on the pan of an analytical balance, and use a glass pasteur pipette to add 1.88 grams of tetra methoxy silane into the beaker. Zero the balance.

And then pipette 2.88 grams of methanol into Beaker A.Cover Beaker A with parafilm. Then, add a magnetic stir bar to a disposable 50 milliliter polypropylene beaker labelled Beaker B, and place on the pan of an analytical balance. Use a glass pipette to add 0.75 grams of water, and 3.00 grams of methanol before covering Beaker B with parafilm.

Begin stirring the contents of Beaker B on a stir plate inside a fume hood. Then, use a syringe to insert 5 microliters of 28.0 to 30.0%ammonium hydroxide solution through the parafilm cover into the mixture while stirring. As soon as this step is complete, add the contents of Beaker A to Beaker B.Stir the mixture for 20 minutes while covered in parafilm.

While the silica sol mixture is mixing, acquire eight to nine polypropylene scintillation vials and corresponding caps. Put plastic wrap over the cap end of the vial to create a flat surface for the gel to form on. And place the cap over it.

Making sure that the plastic wrap stays intact inside the cap. Line up the vials with the cap end down on the bench top, and the open bottoms facing up. Then cut parafilm pieces that will be used to cover each vial.

Upon completion of sol mixing, add 3 milliliters of the sol mixture to a clean disposable 50 milliliter polypropylene beaker. Use a glass pasteur pipette to very slowly drop 500 microliters of the 0.105 millimolar diluted cytochrome c solution down the side of the beaker into the 3 milliliter sol mixture over the course of approximately one minute. Make sure to gently swirl the mixture while adding the cytochrome c to avoid formation of large red clumps.

Pipette 0.5 milliliters of the resulting cytochrome c silica sol into each prepared mold. Also pipette 0.5 milliliters of the remaining plain silica sol into one or two molds to use as control samples during the super critical drying process. Cover the face up openings of the molds with parafilm, and put in the refrigerator overnight, or for at least 12 hours to produce sol gels.

Take the molds out of the refrigerator. Remove the parafilm from the top of one mold containing a cytochrome c sol gel, also remove the cap and plastic wrap from the bottom. After adding some ethanol from a wash bottle into the mold, use the circular disc end of a syringe plunger to carefully push the gel out of the mold and into a clean 20 milliliter glass scintillation vial containing approximately 5 milliliters of ethanol.

Repeat this gel removal procedure until all of the cytochrome c gels are added to the vial, and all of the silica gels are added to a separate vial. Then fill the vials to the top with ethanol. Cap and store between 2 and 8 degrees celsius.

Every four hours throughout the day, remove the gels from the refrigerator. Decap the ethanol off the gels, and replace with fresh ethanol. During an additional three days, submerge the wet sol gels in acetone, decanting and adding fresh acetone three times a day.

Cool a critical point drying apparatus to 10 degrees celsius, by setting the temperature of an attached circulator to eight degrees celsius. Once the apparatus has reached 10 degrees celsius, perform a leak test on the apparatus as described in the text protocol. After ensuring that the apparatus is leak free, carefully pour the wet gels from the scintillation vials, along with most of the acetone into the three long sections of the transfer boat.

Add more acetone if needed to ensure that all gels are completely submerged in acetone before sealing the boat inside of the apparatus. Open the fill valve of the apparatus to add carbon dioxide. Upon observing the carbon dioxide sinking of the bottom of the apparatus through the apparatus window, open the drain valve to release acetone for five minutes.

This seeping of the acetone to the bottom will occur before the apparatus is completely filled with carbon dioxide. Therefore, keep the fill valve open during the draining so that the apparatus will continue to fill. Then, close the drain valve.

Keep the fill valve cracked open slightly. In the beginning, the mixture draining out contains water, because the acetone is not anhydrous. Freezing, clogging and pressure buildup in the drain tube is possible.

If a clog is detected, close the drain valve until the clog melts. Five minutes later, open the drain valve for an additional five minutes and adjust the fill valve to be open enough so that the apparatus remains full during the whole draining time. After five minutes, close the drain valve and keep the fill valve cracked open.

Then, repeat this draining step one more time, five minutes later. Once the draining steps are complete, close the fill valve, and drain the liquid carbon dioxide, so that the level remains visible just above the prongs on the boat through the apparatus window. Next, set the temperature of the apparatus attached circulator to 40 degrees celsius to ensure that the liquid carbon dioxide rises above it's critical temperature and pressure.

After approximately 15 minutes, observe the transition from liquid to super critical fluid through the apparatus window as the liquid meniscus above the prongs of the boat disappears. Following at least 15 minutes of equilibration time, open the vent valve a small amount to begin release of the super critical fluid. Over the course of approximately 45 minutes, continue to incrementally open the vent valve wider and wider so that a steady but very low hiss of releasing fluid can be heard, and the pressure gauge is observed to slowly decrease to zero.

After the pressure of the apparatus has gone to zero, open the apparatus door. Remove the boat. And use forceps to place the newly dried aerogels in clean glass scintillation vials.

The cytochrome c silica aerogels are now ready to be used to detect the presence of nitric oxide gas, following characterization as described in the text protocol. Aerogels encapsulating 15 micromolar cytochrome c in 4.4 millimolar, 40 millimolar, and 70 millimolar potassium phosphate buffer are shown in comparison to a dime. Shown here, a representative spectra of cytochrome c in buffer compared to cytochrome c encapsulated in aerogels under a variety of conditions.

The cytochrome c soret peak at 409 nanometers remains essentially unshifted within aerogels, and gives a characteristic red shift when the bio aerogels are exposed to gas phase nitric oxide. After watching this video, you should have a good understanding of how to synthesize silica sol gels, encapsulate cytochrome c into these sol gels, and dry these composite sol gels into aerogels. Further information can be found in the text protocol on characterization of the bio aerogels, and on using the bio aerogels to detect gas phase nitric oxide.

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