Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Safety glasses or goggles, laboratory coat, and laboratory gloves should be worn at all times during the procedure. Never operate the critical point drying apparatus without safety glasses or goggles. All solutions containing tetramethoxysilane, methanol, ethanol, acetone, and ammonia should be processed within a fume hood.

1. Make Buffer and Cyt. c Solutions

1. To make ~750 ml of pH 7, 40 mM potassium phosphate buffer, first prepare 500 ml of 0.04 M potassium phosphate monobasic by weighing out 2.72 g of potassium phosphate monobasic and dissolving in water using a 500 ml volumetric flask.
2. Prepare 500 ml of 0.04 M potassium phosphate dibasic by weighing out 3.48 g of potassium phosphate dibasic and dissolving in water using a 500 ml volumetric flask.
3. Pour the dibasic salt solution into a large beaker with a stir bar and begin stirring the solution on a stir plate.
4. Slowly add portions of the monobasic salt solution to the dibasic salt solution while monitoring the pH with a pH electrode and meter until the pH is 7.00. Approximately 250-300 ml of the monobasic salt solution will be used.
5. Weigh out approximately 0.023 g of cyt. c and place in glass scintillation vial. Add 2,000 μl of prepared potassium phosphate buffer using a micropipette and then gently swirl the solution to mix until all of the solid, red cyt. c has dissolved into the solution and no particulate matter remains.
6. Take 20 μl of the prepared cyt. c solution and add to a 1-cm path length plastic cuvette. Add 3 ml of prepared buffer.
7. Take UV-vis spectrum from 300-700 nm using buffer in the reference cell. Use the cyt. c absorbance (A) at 409 nm, the extinction coefficient³¹ (ε) of 106,100 M^-1cm^-1, the cuvette path length (l), and the Beer-Lambert law to determine the concentration (c) of the solution (A = εlc).
8. Back calculate the concentration of the original prepared solution. The 2 ml prepared cyt. c solution is typically between 0.7 to 0.9 mM in concentration.
9. Dilute the original prepared cyt. c solution to 800 μl of 0.105 mM by pipetting 117 μl of 0.72 mM prepared cyt. c solution into a scintillation vial. Then add the balance of the 800 μl (683 μl in this case) of prepared buffer. Swirl to mix. The exact volumes will vary depending on the exact concentration of the original prepared cyt. c solution as the volume of cyt. c to pipet is calculated as (800 μl * 0.105 mM)/(original cyt. c concentration in mM).
10. Store original prepared and diluted cyt. c solutions at 2-8 °C in a refrigerator until ready to use for up to two weeks.

2. Synthesize Silica (SiO₂) Sol

1. Label a disposable 50 ml polypropylene beaker 'Beaker A'. Place beaker on the pan of an analytical balance and use a glass Pasteur pipette to add 1.88 g tetramethoxysilane into the beaker. Zero the balance and then pipette 2.88 g of methanol into 'Beaker A'.
2. Cover 'Beaker A' with Parafilm.
3. Label a disposable 50 ml polypropylene beaker 'Beaker B'. Add a magnetic stir bar and place on the pan of an analytical balance. Use a glass pipette to add 0.75 g water and 3.00 g methanol.
4. Cover 'Beaker B' with Parafilm.
5. Begin stirring the contents of 'Beaker B' on a stir plate inside a fume hood, then use a syringe to insert 5 μl of 28.0-30.0% ammonium hydroxide solution through the Parafilm cover into the mixture while stirring.
6. As soon as step 2.5 is complete, add the contents of 'Beaker A' to 'Beaker B'. Stir the mixture for 20 min while covered in Parafilm.

3. Prepare Gel Molds

Note: There is time to prepare the gel molds while the silica sol mixture is stirring in step 2.6.

1. Acquire 8-9 polypropylene scintillation vials (16 mm x 57 mm, volume size 6.5 ml, with bottoms sliced off) 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.
2. Line up the vials with cap-end down on the bench top and opened bottoms facing up.

4. Prepare Cyt. c-silica Sol-gels

1. Upon completion of sol mixing (step 2.6), add 3 ml of the sol mixture to a clean disposable 50 ml polypropylene beaker.
2. Use a glass Pasteur pipette to slowly drop 500 μl of the 0.105 mM diluted cyt. c solution (made in step 1.9) to the 3 ml sol mixture over the course of ~1 min. Make sure to gently swirl the mixture while adding the cyt. c to avoid formation of large red clumps. Assuming the volumes are additive, by diluting 500 μl of the 0.105 mM cyt. c solution to 3,500 μl, the cyt. c concentration now in the sol is, in theory, 15 μM.
3. Pipet 0.5 ml of the resulting cyt. c silica sol into each prepared mold. Also pipet 0.5 ml of remaining 'plain' silica sol into one or two molds to use as control samples during the supercritical drying process.
4. Cover the face-up openings of the molds with Parafilm and put in refrigerator (~2-8 °C) overnight or for at least 12 hr to produce sol-gels.
5. Take the molds out of the refrigerator. Remove the Parafilm from the top of one mold containing a cyt. c sol-gel; also remove the cap and plastic wrap from the bottom.
6. After adding some ethanol from a wash bottle into the mold, use the circular disk end of a syringe plunger to carefully push the gel out of the mold and into a clean 20 ml glass scintillation vial containing approximately 5 ml of ethanol.
7. Repeat this gel removal procedure (steps 4.5 and 4.6) until all of the cyt. c gels are added to the vial and all of the silica gels are added to a separate vial. If more than one concentration of cyt. c gel has been made, be sure to store like gels together within separate vials. Then fill the vials to the top with ethanol, cap and store between 2-8 °C.
8. Every four hours throughout the day, remove the gels from the refrigerator, decant the ethanol off the gels and replace with fresh ethanol.
9. During an additional three days, submerge the wet sol gels in acetone, decanting and adding fresh acetone three times a day.

5. Supercritically Dry Cyt. c-silica Sol-gels

1. Cool a critical point drying apparatus (see Figure 1) to 10 °C by setting the temperature of an attached circulator to 8 °C.
2. Once the apparatus has reached 10 °C, perform a leak test on the apparatus by filling a transfer boat with acetone and sealing it inside of the apparatus.
   1. Open the fill valve of the apparatus and add carbon dioxide until the apparatus is half-full.
   2. Close the fill valve and listen carefully for hissing at the doors and valves where O-rings or seals may be deteriorating.
   3. Replace any O-rings or seals if a leak is found.
3. After completing the leak test, open the drain valve to release the acetone and carbon dioxide into the drain of a fume hood. Then remove the transfer boat from the apparatus.
4. 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 transfer boat's three long sections (specimen baskets or gauze covers are not necessary). Delicately push and move the gels within the boat with forceps to ensure that all gels are completely submerged in acetone. Add more acetone if needed before sealing the boat inside of the apparatus.
5. Open the fill valve of the apparatus to add carbon dioxide, then open the drain valve to release acetone for five minutes as soon as the acetone mixing with the carbon dioxide is observed to be sinking to the bottom of the apparatus through the apparatus window. This seeping of the acetone to the bottom will occur before the apparatus is completely filled with carbon dioxide, so the fill valve should remain open to the extent necessary during the draining so that the apparatus will continue to fill even while the drain is open.
6. Close the drain valve. Keep the fill valve cracked open slightly.
7. Five minutes later, open the drain valve for five minutes again and adjust the fill valve to be opened enough so that the apparatus remains full during the whole draining time. Close the drain valve, keep the fill valve cracked open, then repeat this draining step one more time five minutes later.
8. Following these first three drainage steps, open the drain for 5 min at a time approximately every 40 min over the span of at least six hours to ensure complete replacement of acetone by liquid carbon dioxide within the gels. Always adjust the fill valve to be open enough during each draining so that the liquid level in the apparatus never drops below the top of the boat during draining.
9. Once 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 by looking through the apparatus window.
10. Set the temperature of the apparatus attached circulator to 40 °C to ensure that the liquid carbon dioxide rises above its critical temperature and pressure (T_c = 31 °C; P_c = 7.4 MPa).
11. After approximately 15 min, observe the transition from liquid to supercritical fluid through the apparatus window as the liquid meniscus above the prongs of the boat disappears. Allow at least 15 min of equilibration time, then open the vent valve a small amount to begin to release the supercritical fluid.
12. Over the course of approximately 45 min, 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.
13. 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.

6. Characterize Cyt. c-silica Aerogels with UV-visible and Circular Dichroism (CD) Spectroscopy

1. Prepare a cardboard platform to hold the aerogel monoliths in the beam path of the UV-Visible spectrophotometer or CD spectrometer.
   1. Cut out a 2.5 cm x 2.5 cm piece of lightweight cardboard (such as cardboard from a box of laboratory tissue), fold it in half, cut halfway up on the fold, then fold the two flaps, created by cutting, back.
   2. Cut out a 5 cm x 5 cm piece of lightweight cardboard, with a 1.5 cm x 1.5 cm square hole in the middle. Then use black electrical tape to decrease the size of the hold to 0.5 cm x 0.5 cm.
   3. Tape the flaps of the folded and cut cardboard against the 5 cm x 5 cm piece of cardboard so that a small bent surface is created for an aerogel monolith to sit on directly in front of the 0.5 cm x 0.5 cm hole (see Figure 2). Then tape the back piece of cardboard to the UV-visible spectrophotometer so the hole is in line with the beam path.
2. Measure the thickness of the gels, which will be used for path lengths, with a micrometer.
3. Place one gel on the cardboard platform and measure a spectrum from 300-800 nm with air in the reference compartment of the UV-visible spectrophotometer.
4. Fit a polynomial, A = aλⁿ, to the wavelength (λ) region where the absorbance (A) is mainly due to background scattering, ~700-800 nm. The a coefficient is typically fit to a number between ~1 x 10⁸ and 1 x 10⁶ and the n coefficient is typically fit to a number between ~2 and 3.
5. Calculate the scatter at other wavelengths, using the coefficients, a and n, obtained from the fit.
6. Subtract this calculated scatter background absorbance from the raw spectrum to obtain a scatter corrected spectrum.
7. Fit the scatter subtracted spectrum with a Gaussian curve in the region of 370 to 490 nm using appropriate software (GRAMS/AI 8.0) to determine the peak height, peak center, and peak width of the Soret peak of the aerogel.
8. Apply the Beer-Lambert Law using the measured thickness of the gel for path length (l), the extinction coefficient³¹ (ε) of 106,100 M^-1cm^-1 and the Soret peak height (A) to compute the concentration (c) of cyt. c in the aerogel (A = εlc).
9. Compare the calculated cyt. c concentration to the concentration theoretically in the gel (15 μM) to ascertain the viability of cyt. c within the aerogel. Typical percent viabilities are close to 100%, but it should be noted that these viabilities are just estimates because the calculation is based on the extinction coefficient of cyt. c in solution³¹ which is presumed to be slightly different than the extinction coefficient of cyt. c in the aerogels that is not known.
10. Run nitrogen in the CD instrument at least 5 min before turning the lamp on.
11. Tape the cardboard holder to the CD spectrometer so the hole is in line with the beam path.
12. Measure a continuous wavelength blank spectrum with nothing in the cardboard holder from 350-500 nm at 100 nm/min, taking an average of three scans.
13. Place one gel (thickness previously measured in step 6.2) on the cardboard platform and measure a spectrum from 350-500 nm at 100 nm/min, taking an average of three scans.
14. Repeat the UV-visible and CD measurements for all aerogel monoliths of interest.

7. Detect Presence of Nitric Oxide (NO) Gas with Cyt. c-silica Aerogels

CAUTION: Working with NO is dangerous and all NO gas should be handled in a fume hood or exhausted into a fume hood. Sustained exposure to NO is toxic to tissues as highly poisonous nitrogen dioxide and/or nitrogen tetroxide will form when NO comes in contact with air. Heat and corrosive fumes are also produced when NO comes in contact with water.

1. Place an 8 L nitric oxide cylinder (10% nitric oxide, 90% nitrogen) in a well-ventilated fume hood and adjust the pressure to 4 psi.
2. Connect tubing to both the nitric oxide cylinder and to a nitrogen cylinder (pressure set to 6 psi) and link up the ends of the tubing to a T-valve (see Figure 3a).
3. Choose an aerogel monolith for the experiment and measure the thickness (or path length) with a micrometer.
4. Place the aerogel (~3 mm thick) in a disposable cuvette with plastic cap and put the cuvette into the spectrophotometer. Cut the aerogel slightly if necessary to fit in the cuvette.
5. Insert two syringe needles into the plastic cap of the cuvette, one connected to the output of the T-valve, and one connected to a tube to serve as exhaust into the fume hood (see Figure 3b). Use Parafilm to seal the needles to the tubing and the cap to the cuvette.
6. Place an empty disposable cuvette in the reference cell.
7. Adjust the aerogel cuvette position to ensure that the aerogel lies in the beam path prior to starting the experiment.
8. Take an initial spectrum from 800 to 300 nm.
9. Monitor the difference between the absorbance at 414 nm and the absorbance at 408 nm while turning the T-valve to switch between nitrogen and the nitric oxide/nitrogen mixture at set time intervals making sure that at no time is the flow rate of nitrogen or nitric oxide/nitrogen mixture so high that the aerogel moves around in the cuvette.
10. Take a final spectrum from 800 to 300 nm, once the exposure cycles are finished.
11. Repeat the procedure with three to four monoliths to obtain an average sensing response.