Simplified Procedure for Encapsulating Cytochrome c in Silica

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Simplified Procedure for Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures while Retaining Gas-Phase Bioactivity Amanda S. Harper-Leatherman,* Mariam Iftikhar, Adela Ndoi, Steven J. Scappaticci, George P. Lisi, Kaitlyn L. Buzard, and Elizabeth M. Garvey Chemistry & Biochemistry Department, Fairfield University, 1073 North Benson Road, Fairfield, Connecticut 06824, United States S Supporting Information *

ABSTRACT: Cytochrome c (cyt. c) has been encapsulated in silica sol−gels and processed to form bioaerogels with gas-phase activity for nitric oxide through a simplified synthetic procedure. Previous reports demonstrated a need to adsorb cyt. c to metal nanoparticles prior to silica sol−gel encapsulation and processing to form aerogels. We report that cyt. c can be encapsulated in aerogels without added nanoparticles and retain structural stability and gas-phase activity for nitric oxide. While the UV−visible Soret absorbance and nitric oxide response indicate that cyt. c encapsulated with nanoparticles in aerogels remains slightly more stable and functional than cyt. c encapsulated alone, these properties are not very different in the two types of aerogels. From UV−visible and Soret circular dichroism results, we infer that cyt. c encapsulated alone self-organizes to reduce contact with the silica gel in a way that may bear at least some resemblance to the way cyt. c self-organizes into superstructures of protein within aerogels when nanoparticles are present. Both the buffer concentration and the cyt. c concentration of solutions used to synthesize the bioaerogels affect the structural integrity of the protein encapsulated alone within the dried aerogels. Optimized bioaerogels are formed when cyt. c is encapsulated from 40 mM phosphate buffered solutions, and when the loaded cyt. c concentration in the aerogel is in the range of 5 to 15 μM. Increased viability of cyt. c in aerogels is also observed when supercritical fluid used to produce aerogels is vented over relatively long times.

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INTRODUCTION Aerogels are high surface area, high porosity, sol−gel-derived materials that contain a pore-solid three-dimensional, nanoscale mesh network.1,2 After synthesis, the gel pores remain filled with water and alcohol, and the method of removing the liquid results in either a densified xerogel or a highly porous aerogel. Drying fluid-filled gels through supercritical solvent extraction creates aerogels, while xerogels form by evaporation of the liquid under ambient conditions. The supercritical drying procedure minimizes pore collapse caused by capillary forces acting on pore walls. Aerogels have gas-phase analyte response times close to open-medium diffusion rates due to the 80−99% open porosity of the materials.3 This porosity opens up tremendous available surface area within these materials making them excellent nanoscopic scaffolding for building efficient devices where surface reactions are important. Aerogels have been used to improve sensors, as well as electrodes for battery, supercapacitor, and fuel cell applications when chemical functionality is assembled within the aerogel nanoarchitecture.1,3−9 The porosity allows ample head space for chemical dopants to be incorporated into the pore-solid nanoarchitecture as well as space and pathways for analyte diffusion. Many proteins and enzymes have been successfully encapsulated and studied in sol−gels that have been kept wet or that have been dried ambiently to add biofunctionality to these materials.10−14 Typically, the sol−gel synthesis used to encapsulate biomolecules incorporates buffer to raise the pH during synthesis, and it incorporates less alcohol to decrease the possibilities of © 2012 American Chemical Society

protein denaturation. The heme proteins cytochrome c (cyt. c), hemoglobin, and myoglobin are some examples of proteins that have been thoroughly studied after sol−gel encapsulation.14−20 Researchers have studied how sol−gel encapsulation affects heme protein structure and function. UV−visible spectroscopy indicates minimal changes in encapsulated heme protein structural conformation.14 In the case of cyt. c, when the effects of sol−gel synthesis pH, buffer type and alcohol/ alkoxide ratio were studied, it was determined that the stability of cyt. c in tetramethoxysilane sol−gels is similar to the stability of cyt. c in solution under the same conditions.20 Size-specific pores form around cyt. c within the sol−gel network, and cyt. c is stabilized to thermal denaturation within the sol−gel.19 Heme proteins also retain oxidation/reduction, biocatalysis, and ligand binding functions within sol−gels.18,19,21 Ligand binding function involves heme proteins interacting and binding with specific target molecules. A conformational change takes place that can be detected by UV−visible absorbance, making it possible to develop sensors for the molecules of interest when these proteins are encapsulated in sol−gels. One such target molecule is nitric oxide (NO) that binds to myoglobin, hemoglobin, and cyt. c. Nitric oxide is a long-recognized toxic environmental pollutant, and has attracted much recent research interest due to its diverse role in the physiology of the human body, ranging Received: March 14, 2012 Revised: August 4, 2012 Published: August 27, 2012 14756

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from a role in the nervous, cardiovascular, and immune systems.22 Detection of NO was demonstrated using a manganese myoglobin encapsulated sol−gel which could specifically bind the molecule in the presence of oxygen.19 In addition, when cyt. c was encapsulated in a sol−gel and formed into a very thin film, optical NO sensing took place within minutes.23 While many biomolecules have been encapsulated in sol− gels kept wet or dried ambiently, examples of biomolecules encapsulated in supercritically dried aerogels are less common. Much bioaerogel research has focused on gelation in the presence of very robust proteins such as the enzyme, lipase.24−26 Other drying processes have been studied, such as recent work producing bioaerogels encapsulating a red fluorescent protein using an ionic liquid to reduce pore shrinkage, instead of the supercritical drying process.27 Research has also involved immobilizing biomolecules such as enzymes,28 bacteria,29 oligonucleotides,30 antibodies,31 and biomembranes32 onto preformed aerogels. In addition, synthetic conditions have been developed in which the heme protein, cyt. c, remains active in supercritically dried aerogels resulting in improved gas sensors due to the rapid gasphase responses of aerogels compared to xerogels.33−37 Initial attempts to encapsulate cyt. c from buffered solution into sol−gels that were then dried to form aerogels resulted in a loss of the Soret visible absorption at ∼409 nm arising from the metal−ligand coordination of the folded protein. To stabilize cyt. c in aerogels, a method taking advantage of the fact that the positively charged heme pocket of cyt. c will specifically adsorb to negatively charged citrate-stabilized metal nanoparticles was developed.38,39 It was found that, when gold (or silver) nanoparticles and cyt. c are added together in solution, spontaneous electrostatic and other weak interactions help to create loosely multilayered superstructures of protein (abbreviated as Au∼cyt. c superstructures in the case of gold nanoparticles). When these superstructures are encapsulated within aerogels, cyt. c retains its characteristic visible absorption, to a large extent.33−37 The functionality of cyt. c is retained within the Au∼cyt. c superstructures that are encapsulated within aerogels exhibited by the rapid gas-phase recognition of nitric oxide (NO), facilitated by the high porosity of the aerogel.33 Au∼cyt. c aerogels exhibit faster (150 s) response times to nitric oxide compared to cyt. c xerogels (200 s) even when they are 103 times as thick.33,23 Cyt. c is protected from protein denaturation during the harsh physical and chemical processing conditions necessary to form the aerogel by organization within the Au∼cyt. c superstructure. In addition, cyt. c remains viable during weeks of storage of the bioaerogel at ambient temperature and ambient atmosphere. While encapsulating Au∼cyt. c superstructures into aerogels results in relatively stable, functional cyt. c in the nanoarchitecture, we have explored alternative aerogel synthetic conditions to determine if cyt. c can be encapsulated alone without nucleating metal nanoparticles. Given that not all proteins will interact with metal nanoparticles to form multilayered superstructures, and given the fact that metal nanoparticle synthesis or purchase requires additional time and expense, it would be useful to elucidate a more general process of encapsulating proteins into aerogels. In this report, we demonstrate a simplified synthesis of aerogel nanoarchitectures that stabilize cyt. c to unfolding without the addition of gold or silver nanoparticles. We outline the synthetic conditions in which cyt. c retains its structure and the conditions where structural integrity is lost within aerogels, measured by UV−visible and circular dichroism spectroscopies. Cyt. c functionality in aerogels was also

explored through recognition of gas-phase nitric oxide. By studying the spectroscopy and gas-phase activity of cyt. c encapsulated alone in comparison to Au∼cyt. c superstructures encapsulated in aerogels, we have ascertained more clearly how the aerogel synthetic conditions affect the structural and functional characteristics of this protein. This has moved us one step closer to delineating a more general process of encapsulating other proteins into aerogels that may aid in the future development of biosensors or other bioanalytical devices.

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EXPERIMENTAL SECTION

Reagents. Cytochrome c (horse heart, Type VI; Sigma Aldrich) was used as received. The concentrations of cytochrome solutions were calculated from their visible spectra using an extinction coefficient of 106 100 M−1 cm−1.40 The extinction coefficient of 106 100 M−1 cm−1 is for the Soret peak of highly purified cyt. c at 410 nm.40 Our solutions, that are used as received, display Soret peaks at 409 nm. We are assuming the extinction coefficient of our solutions is the same as the fully purified cyt. c showing a Soret at 410 nm. Potassium Phosphate Monobasic (Mallinckrodt Chemical Works), Potassium Phosphate Dibasic (Fisher Scientific Company), Sodium Phosphate Monobasic (Kodak), Sodium Phosphate Dibasic (Baker), and all other reagents were used as received, unless otherwise noted; the water used in all experiments was 18 MΩ cm (Millipore Direct-Q). Preparation of Colloidal Gold∼Protein Superstructures. The preparation of Au∼cyt. c superstructures was similar to that previously described.33−37 An aliquot of the stock cyt. c solution (0.9 mM, in pH 7, 50 mM phosphate buffer) was added dropwise while stirring to an aliquot of colloidal gold sol (BBInternational; stored in the dark at 4 °C) to create a specific number ratio of protein to gold nanoparticles. For the 5700:1 cyt. c/Au ratio, 89 μL of cyt. c was added dropwise to 711 μL of 5 nm colloidal gold sol (first diluted with water to 1.3 × 1013 particles/mL from the stock solution of 5 × 1013 particles/mL). The mixture was stirred for a minimum of 10 min; no separation techniques (i.e., centrifugation or filtration) were employed in order to prevent shear forces from disrupting the soft superstructure. Preparation of Silica Sol. The preparation of base-catalyzed silica sol was similar to that previously described.33−37,41 Plastic containers were used throughout the synthesis. 1.89 g of tetramethoxysilane (TMOS, 98%, Sigma Aldrich) and 2.88 g of methanol (Fischer Scientific) were combined. In a second container, 0.75 g of water and 3 g of methanol were combined. Both containers were covered with Parafilm after their contents were added. While stirring the methanol and water, 5 μL of ammonium hydroxide (ACS reagent NH3 content 28−30%) was added via syringe. Immediately following the addition of the catalyst, the TMOS solution was added to the second container, and this mixture was stirred covered for 20 min. While the mixture was stirring, the gel molds were prepared. Each mold consisted of a small polypropylene tube with both ends open (13 mm diameter, × 55 mm) and a cap for one end. The caps were lined with plastic wrap to prevent the sol mixture from leaking. Preparation of Au(5‑nm)∼cyt. c−SiO2 Aerogels and cyt. c− SiO2 Aerogels. The synthesis of the bioaerogels was similar to that previously described.33−37 Once the sol mixture finished stirring, 3 mL of the mixture was pipetted into a new plastic beaker and 500 μL of either Au∼cyt. c superstructures or 500 μL of cyt. c (dissolved in phosphate buffer of either 4.4, 40, or 70 mM concentration) was added dropwise while lightly shaking the beaker. The concentration of cyt. c in the final gel was adjusted by adjusting the concentration of the cyt. c in the solution dropped into the sol. The final concentrations of cyt. c in the gels (based on what was loaded into the gels) ranged from 5 to 25 μM. Aliquots of ∼0.5 mL of the resulting gel mixture were poured into the molds and sealed with Parafilm. The gels were aged overnight at 4 °C, then transferred to glass scintillation vials, and rinsed with ethanol (200 proof, 99.5%, ACS reagent, Acros) three times over the course of one day. For the next three days, the wet sol gels were rinsed three times a day with acetone (Fisher Scientific). The wet sol−gels were loaded into a supercritical dryer (E3100 Series Critical Point Drying apparatus, Quorum Technologies). The acetone was replaced 14757

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with liquid carbon dioxide by flowing carbon dioxide through the dryer approximately ten times, for five minutes each, over the course of at least six hours. The liquid carbon dioxide was brought above its critical temperature and pressure (Tc = 31 °C; Pc = 7.4 MPa) and vented. Spectroscopic Characterization. The UV−visible spectroscopy of buffered protein solutions and supercritically dried aerogels was obtained with a Cary 50 UV−visible spectrophotometer or a UV-1800 Shimadzu UV spectrophotometer. The scattering background from the silica aerogel and encapsulated buffer of each aerogel monolith was modeled using a region of the spectrum (typically ∼700−800 nm) where the absorbance was mainly due to background scattering and not cyt. c. The polynomial, A = aλn, was fit to this region of the spectrum. The scatter at the other wavelengths was calculated using the coefficients, a and n, obtained from the fit, and this calculated scatter absorbance was subtracted from the raw spectrum to obtain a scatter corrected spectrum. The a coefficient was typically fit to a number between ∼1 × 108 and 1 × 106. The n coefficient was typically fit to a number between ∼2 and 3. See Supporting Information Figure S-1 for an example raw aerogel spectrum, modeled scattering absorbance, and scatter subtracted spectrum. Each scatter subtracted spectrum was then fit with a Gaussian curve in the region of 370 to 490 nm using GRAMS/AI 8.0 software. The CD spectra were obtained using a JASCO J-810 instrument. CD scans were from 500 to 350 nm with 100 nm/min scan rates. Porosimetry Characterization. Cyt. c−SiO2 aerogels encapsulating 10 μM cyt. c using encapsulated solution buffer concentration of 4.4, 40, or 70 mM were prepared as described above. The aerogels were pyrolyzed at 600 °C for eight hours, and sent to Micromeritics Analytical Services to obtain 40-point Nitrogen adsorption and 40-point desorption isotherms (20 Å to 3000 Å) on each of the three different samples using a degassing step at 150 °C prior to analysis. Optical Nitric Oxide Detection. A single aerogel monolith (∼3 mm thick) was placed in a disposable cuvette (Fisherbrand, methacrylate, 10 mm × 10 mm × 45 mm) with plastic cap, and an empty cuvette was used in the reference cell42 of the UV-1800 Shimadzu UV spectrophotometer with UVProbe v 2.33 software. The cuvette position was adjusted to produce an optimized spectrum prior to the experiment. Then, an initial spectrum from 800 to 300 nm was obtained. The difference between the absorbance at 414 nm and the absorbance at 408 nm, ΔA (A414 nm − A408 nm), was monitored with a cycle time of 4.5 s and 534 readings. The nitric oxide cylinder (Airgas, 10% nitric oxide, 90% nitrogen, 8 L cylinder) was kept in the exhaust hood. Both the nitric oxide and the nitrogen cylinders were connected to the cuvette in the spectrophotometer through a T-valve using Tygon tubing. Another Tygon tube released gas from the cuvette back to the exhaust hood. The pressure of the nitric oxide tank was kept at 4 psi and the nitrogen tank at 6 psi. The aerogel was exposed to the nitrogen gas for 2 min initially. The aerogel was then exposed to nitric oxide for 3.5 min, switched to nitrogen for 8 min, switched to nitric oxide for 3.5 min, switched to nitrogen for 7 min, switched to nitric oxide for 3.5 min, and then finally switched to nitrogen for 7.9 min. Once the exposure cycles were complete, a spectrum from 800 to 300 nm was obtained. Three to four gels of each type of bioaerogel were measured in this way, and average responses were obtained.

Figure 1. Visible spectra of 15 μM cytochrome c in (a) 50 mM phosphate buffer solution; (b) Au(5‑nm)∼cyt. c−SiO2 aerogel; (c) cyt. c−SiO2 aerogel (exposed to air); (d) cyt. c−SiO2 aerogel (exposed to nitric oxide for 3.5 min). These representative spectra of each type of gel are offset for clarity, and the dashed line denotes the position of the Soret peak of cyt. c in buffer. While each spectra is of 15 μM cyt. c, the gel thicknesses are only 0.2−0.5 cm compared to the 1 cm solution cuvette resulting in a higher solution absorbance.

in a fully functional form as this peak is associated with the Met 80 sulfur-heme iron ligation.43 Previous research33−37 showed that cyt. c generally retained spectroscopically measured structural integrity within dry aerogels when the protein was first associated with 5 or 10 nm gold or silver nanoparticles in about a 10 000:1::cyt. c:nanoparticle ratio before encapsulation in aerogels (example spectra from such an aerogel shown in Figure 1b). Our research shows that cyt. c also retains its Soret band when encapsulated within aerogels with no added nanoparticles (Figure 1c). Au∼cyt. c−SiO2 aerogels (Figure 1b) encapsulating Au(5‑nm)∼cyt. c in a 1:5700::gold nanoparticle:cyt. c ratio in which the overall concentration of cyt. c encapsulated in the aerogel was 15 μM had an average Gaussian-fitted Soret peak center at 408.4(±0.1) nm and an average fitted peak width of 23.4(±0.7) nm. This small blue-shift in the Soret peak center and peak widening indicates some small differences in protein conformation compared to cyt. c in solution. This subtle blue-shift in Soret peak center has also been observed when cyt. c is encapsulated in sol−gels kept wet.18,19 The concentration of cyt. c out of the 15 μM that remained active in the Au∼cyt. c−SiO2 aerogels on average was calculated to be 100(±9)% from the Gaussian-fitted Soret absorbance using an extinction coefficient of 106 100 M−1 cm−1.40 The calculation of percent activity of cyt. c in the aerogels is just an estimate, since the extinction coefficient of cyt. c in the aerogels is not known and is presumed to be slightly different due to some cyt. c being denatured in the aerogels. The calculation of percent activity of cyt. c in the aerogels is just an estimate, since the extinction coefficient of cyt. c in the aerogels is not known and is presumed to be slightly different due to some cyt. c being denatured in the aerogels. The cyt. c−SiO2 aerogels (Figure 1c) encapsulating an overall concentration of 15 μM unassociated cyt. c in aerogels had an average Gaussian-fitted Soret peak center at 407.4(±0.5) nm and an average fitted peak width of 24(±1) nm. This slight additional blue shift and peak widening of the Soret peak indicates slightly less structural stability of cyt. c encapsulated alone in aerogels versus cyt. c encapsulated within a Au∼cyt. c superstructure, although the difference is very small in comparison to the effects of denaturing agents that have been

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RESULTS AND DISCUSSION Structural Stability of Cyt. c in Aerogels Compared to Au∼cyt. c Superstructures in Aerogels. The visible Soret absorbance band appears at 409 nm with a peak width of 22 nm when cyt. c is dissolved in 50 mM buffered phosphate solution (Figure 1a). Highly purified cyt. c displays a Soret absorbance at 410 nm,40 and the small shift to 409 nm may be because cyt. c is being used as received. Since the Soret band is an indication of the integrity of the protein metal−ligand coordination, further changes in the position and width of the Soret band can be an indication of structural changes as protein environment is changed.20 Although not visible on the same scale as the Soret band in Figure 1, there is a band present at 695 nm in the solution spectra that is another common indicator that cyt. c is 14758

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studied in the past.44 Using the Gaussian-fitted Soret absorbance to calculate the concentration of cyt. c that remained active in the cyt. c−SiO2 aerogels indicated that 100(±10)% of the protein remained active on average. As noted above for the Au∼cyt. c aerogels, this calculation of percent activity in the cyt. c aerogels is just an estimate, since it is based on the extinction coefficient of cyt. c in solution.40 However, it should also be noted that, while this percentage may seem high, it is not completely unreasonable, as there is no evidence of any physical removal of cyt. c from the gels during any of the rinsing steps needed to process cyt. c−SiO2 sol−gels to form aerogels, as no detectable Soret peak is present in the rinses of cyt. c−SiO2 sol−gels. It should be pointed out that, before incorporating the cyt. c in the sol−gel as either unassociated cyt. c or as Au∼cyt. c superstructures, the cyt. c is in the same buffered solution of 50 mM phosphate buffer. In addition, the synthesis and processing of the gels was the same for both types of cyt. c gels as outlined in the Experimental Section. The cyt. c concentration, buffer conditions, gel synthesis and processing were within the range of conditions used in previous reports of Au∼cyt. c aerogels.33−37 These previous reports indicated that cyt. c did not remain active in aerogels without added nanoparticles, but since these reports focused on the parameters used for stabilizing Au∼cyt. c superstructures in aerogels, they did not give as much detail about the exact parameters used for the cyt. c aerogels that were synthesized.33−37 Our current work fills this gap by outlining the parameters necessary for stabilizing unassociated cyt. c in aerogels. Although gel scattering was not directly measured, large background absorbances in certain cases indicated some increased scattering in the gels presented in this report compared to previous reports that could be due to differences in room humidities when synthesizing both the Au∼cyt. c and cyt. c aerogels. Background scattering was modeled and subtracted as described in the Experimental Section, but there is potential for uncompensated scattering that could be one reason for higher than previously reported percent viabilities.33−37 Therefore, this is another reason why the percent viability calculations give a rough estimate of the viability of cyt. c in the gels. The Soret peak center, width and absorbance data suggest, based on this first comparison, that there is not a drastic difference between the viability of cyt. c encapsulated in cyt. c−SiO2 aerogels and the viability of cyt. c encapsulated in Au∼cyt. c−SiO2 aerogels. We do not have a clear picture of how the cyt. c proteins are arranged in the cyt. c−SiO2 aerogels since TEM micrographs cannot easily differentiate cyt. c from SiO2 aerogel, but the UV−visible results suggest that many of the cyt. c molecules are experiencing a buffer-like environment in the aerogels since the Soret peak of the cyt. c−SiO2 aerogels does not differ greatly from that in buffer. However, considering Soret circular dichroism spectroscopy of the cyt. c−SiO2 aerogels in combination with the UV−visible results suggests that there are proteins in at least two types of environments in the cyt. c−SiO2 aerogels. While the Soret CD spectrum of cyt. c in solution displays a positive and negative shape indicative of a folded tertiary protein, both Au(5‑nm)∼cyt. c−SiO2 aerogels and cyt. c−SiO2 aerogels display Soret CD spectra with single peaks centered roughly around the inflection point of the solution spectra signifying some protein unfolding (Figure 2).43 While much of the protein may be structurally intact according to the UV−visible results, at least some of the protein is unfolded according to the CD results.

Figure 2. Circular dichroism spectra of cyt. c in sodium phosphate buffered solution (solid), two representative spectra of cyt. c−SiO2 aerogels (dashed), and two representative spectra of Au(5‑nm)∼cyt. c−SiO2 aerogels (dotted).

The unfolding displayed by the CD spectra of Au∼cyt. c−SiO2 aerogels was previously reconciled33 with the folded protein displayed by the UV−vis spectra of these aerogels by explaining that gold nanoparticle-nucleated protein superstructures were forming in the aerogels in which the outermost layer of protein was damaged when coming in contact with the silica gel. It was explained that the boundary layer of protein in contact with the gel must be what circular dichroism probes, since the CD results are very similar to the unfolded protein CD spectra resulting from cyt. c binding to the surfaces of lipid membranes or micelles.45 The similarity between both the Soret CD spectra and the UV−vis spectra for cyt. c−SiO2 aerogels compared to the CD and UV−vis spectra for Au(5‑nm)∼cyt. c−SiO2 aerogels gives support to the idea that cyt. c may be associating in such a way within the cyt. c−SiO2 aerogels so that not all of the protein is in contact with the silica gel. However, since there is no clear nucleation point for forming organized structures like in the Au∼cyt. c superstructures, it can be hypothesized that, if structures exist, they are more loosely organized and of varying sizes and shapes. To be certain, further experiments are necessary to determine the position of protein relative to other protein within these aerogels. Ligand Binding Function of Cyt. c in Aerogels Compared to Au∼cyt. c Superstructures in Aerogels. To learn more about the viability of cyt. c in aerogels with no added nanoparticles, we studied the response of these aerogels to nitric oxide to see if the ligand binding functionality of cyt. c is retained in these gels. Cyt. c−SiO2 aerogels displayed a Soret band wavelength shift in the presence of nitric oxide indicative of gas-phase binding of nitric oxide (Figure 1d). In addition, characteristic sideband changes were observed (see Supporting Information Figure S-2 for a closer look at the sideband region of the Figure 1 spectra). Past Au∼cyt. c aerogel research showed that, when Au∼cyt. c superstructures in aerogels were exposed to nitric oxide, the Soret peak shifted from 408 to 414 nm.33 This change was reversible when Au∼cyt. c−SiO2 aerogels were subsequently exposed to argon. To test the reversibility of the NO ligand binding of cyt. c− SiO2 aerogels, we monitored the change in the 414 nm absorbance in comparison to the 408 nm absorbance as we toggled the gas flow over the cyt. c aerogels from nitrogen to nitric oxide. Initially, we exposed the aerogels to nitrogen and zeroed the difference between the 414 nm absorbance and 14759

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the 408 nm absorbance, ΔA (A414 nm − A408 nm). Then by monitoring ΔA, we observed the 414 nm absorbance increase in comparison to the 408 nm absorbance as nitrogen atmosphere was replaced with nitric oxide (Figure 3a, solid red line).

this report, while 10 nm gold nanoparticles were used in the prior report. In addition, it is possible that the exact NO pressure used was different in this report than in the prior report. One difference between the NO response of the two types of aerogels is that the Au∼cyt. c aerogels lose more of the original absorbance at 408 nm then the cyt. c aerogels after the first exposure to NO, since the ending response after returning to nitrogen is higher. However, upon each additional exposure to NO, the response becomes more and more reversible indicating that, while much cyt. c remains in the NO bound state after the initial exposure to NO, an equilibrium is reached in which a certain amount of cyt. c is able to switch back and forth between association and nonassociation with NO. In contrast, the cyt. c aerogels lose an equal amount of the original 408 nm absorbance after each exposure to NO indicating that a small, but equal, amount of cyt. c remains bound to NO after each exposure to NO. This trend is shown more clearly when the average change in absorbance, ΔA (A414 nm − A408 nm), at the end of each nitrogen cycle for cyt. c−SiO2 aerogels (red circles) and Au(5‑nm)∼cyt. c−SiO2 (blue triangles) aerogels are plotted and connected with lines (Figure 3b). This result, indicating somewhat less reversible ligand binding of cyt. c−SiO2 aerogels compared to Au(5‑nm)∼cyt. c−SiO2 aerogels, correlates with the slightly less stable protein in cyt. c−SiO2 aerogels as indicated by the small blue-shift in the Soret peak center and widened peak width (Figure 1). The subtle differences in the nitric oxide responses that indicate a somewhat less stable protein in cyt. c−SiO2 aerogels compared to Au(5‑nm)∼cyt. c−SiO2 aerogels can be explained considering the arrangement of protein in the Au∼cyt. c aerogels and the proposed arrangement of protein in the cyt. c aerogels. In Au∼cyt. c aerogels, much exposure to NO involves interaction with cyt. c that is stable within nanoparticlenucleated superstructures and that is likely to reversibly bind with NO. The arrangement of cyt. c molecules in cyt. c aerogels is not known for sure as discussed above, but it can be hypothesized that, if loosely organized structures of cyt. c exist in the aerogels, these would potentially be less well-organized, leading to cyt. c that is less able to reversibly bind with NO. Despite the response differences, when the responses for both the aerogels encapsulating cyt. c and the aerogels encapsulating Au∼cyt. c are zeroed after the first cycle of NO to nitrogen, the responses look very similar (Figure S-3). Much of the differences between the two types of aerogels seems to stem from the difference in response to the first exposure of NO. In addition, while some cyt. c has bound NO irreversibly, an average of 73(±14)% of the cyt. c in the Au∼cyt. c aerogel and 78(±17)% of the cyt. c in the cyt. c aerogel is still structurally intact based on the Soret absorbance at ∼408 nm after the three NO cycles are complete (see Figure S-4 for representative spectra of each type of bioaerogel before and after the nitric oxide cycles). Effects of Synthetic Variations on Cyt. c Viability in Aerogels. To explore the necessary parameters needed for cyt. c to remain active in aerogels, we varied the cyt. c concentration in the aerogels, and we varied the buffer strength used to make the cyt. c solutions that were encapsulated in the aerogels. Past research on encapsulating cyt. c in sol−gels kept wet has shown that methanol concentration in the sol, buffer type, and pH have all affected the structure and function of cyt. c in the gels.20 We remained close to the optimized synthetic parameters for cyt. c encapsulation in wet sol−gels and for Au∼cyt. c encapsulation in aerogels. However, we chose to make subtle changes in encapsulated buffer concentration, since some past

Figure 3. (a) Monitoring the shift (ΔA = A414 nm − A408 nm) in the Soret intensity of cyt. c (solid red) and Au∼cyt. c (dashed blue) encapsulated in SiO2 composite aerogel nanoarchitectures as gas flow is toggled between nitrogen (where Soret peak maximum is at ∼408 nm) and nitric oxide (where Soret peak maximum is at ∼414 nm). Each curve is an average of 3−4 trials, with two of the cyt. c−SiO2 trials monitored at ΔA = A414 nm − A407 nm since the initial Soret peak maximum was at 407 nm for these trials. (b) The trend in the average change in absorbance, ΔA (A414 nm − A408 nm), is shown more clearly by only plotting the data points at the end of each nitrogen cycle for cyt. c−SiO2 aerogels (red circles) and Au(5‑nm)∼cyt. c−SiO2 (blue triangles) with lines connecting these points.

Upon purging with nitrogen, the ΔA, and therefore the 414 nm absorbance, decreased, but did not completely diminish. Multiple exposure cycles between nitrogen and nitric oxide resulted in a quasi-reversible response. We compared the response of aerogels encapsulating cyt. c to that of Au(5‑nm)∼cyt. c aerogels (1:5700::gold nanoparticle:cyt. c ratio) with the same concentration of cyt. c loaded into the aerogels as the unassociated cyt. c aerogels (15 μM). The Au∼cyt. c aerogels displayed a slightly bigger response to NO than the cyt. c aerogels did, but change in response with time as exposure cycles between nitric oxide and nitrogen were completed was very similar (Figure 3a, dashed blue line). Each type of aerogel exhibited a ΔA (A414 nm − A408 nm) response that did not return to the same baseline in nitrogen at which it started after being in nitric oxide. In other words, the 414 nm NO-bound cyt. c absorbance was still dominating the 408 nm absorbance even after minutes of nitrogen exposure. Some of the cyt. c must not release the NO from the heme iron after returning to nitrogen in both types of aerogels. The Au∼cyt. c aerogel NO results are slightly different than what was observed in a prior report.33 However, 5 nm gold nanoparticles are used in 14760

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Since the signal increase is most proportional to cyt. c concentration in the range of 5 to 15 μM, it appears that this range is the most ideal for encapsulating stable cyt. c. Similar trends in absorbance/path length were observed when increasing concentrations of cyt. c were encapsulated using 70 mM and 4.4 mM phosphate buffered solutions (Figure S-5 and S-6, respectively) with the 5 to 15 μM range giving the most linear responses. While aerogels encapsulating concentrations greater than 25 μM cyt. c using 4.4 mM and 40 mM phosphate buffered solutions did display Soret peaks, they also exhibited greater scattering in the UV−vis signal, making the peak determination less reliable, with Soret peaks completely engulfed by scattering at 50 μM encapsulated cyt. c. When we compare the visible spectra of aerogels loaded with 15 μM cyt. c made by encapsulating cyt. c dissolved in 4.4 mM, 40 mM, or 70 mM phosphate buffer, it is clear that average absorbance/path length is relatively constant for this concentration of cyt. c even as buffer strength is varied (Figure 5a).

studies have indicated that cyt. c reaction kinetics are affected by ionic strength in wet gels due to the fact that at low ionic strength cyt. c can adsorb to the negatively charged silica walls.46 We also hypothesize that buffer strength should affect stability of cyt. c within cyt. c−SiO2 aerogels because of the electrostatic forces that would be involved in forming loosely organized superstructures of protein that may be stabilizing cyt. c in the aerogels. In addition, the buffer strength has the potential to change the physical characteristics of the aerogel scaffolding that could affect cyt. c encapsulation. The viability of cyt. c in Au∼cyt. c aerogels has been shown to be affected by the cyt. c concentration in the gels.35 Therefore, we chose to vary the cyt. c concentration in the cyt. c−SiO2 aerogels as well. When cyt. c is encapsulated from 40 mM phosphate buffered solutions, the Soret absorbance/path length increases as cyt. c concentration that is loaded into the aerogel increases in the range of 5 μM to 25 μM (Figure 4a) as would be expected for

Figure 5. (a) Averaged UV−visible spectral absorbance of aerogels divided by gel path length for gels encapsulating 15 μM cyt. c in 70 mM (black) (average of 4 spectra), 40 mM (red, dotted) (average of 8 spectra), and 4.4 mM (green, dashed) (average of 9 spectra) potassium phosphate buffer. (b) Sample aerogels encapsulating 15 μM cyt. c in 4.4 mM, 40 mM, and 70 mM potassium phosphate buffer from left to right.

Figure 4. (a) Averaged UV−visible absorbance of aerogels divided by gel path length for gels encapsulating different concentrations of cyt. c, while buffer concentration remains 40 mM. Cyt. c concentration loaded into the gel increases from the lowest to the highest peak from 5 μM (black), 10 μM (red), 15 μM (green), 20 μM (orange), and 25 μM (blue). (b) The average Gaussian fitted Soret peak absorbance divided by gel path length as encapsulated cyt. c concentration (that which is loaded into the gel) increases, while buffer remains 40 mM.

However, at the highest buffer strength, the signal-to-noise ratio is greatly decreased due to decreased gel translucency (Figure 5b). Similar trends were apparent for 5 μM, 10 μM, 20 μM, and 25 μM cyt. c aerogels made from cyt. c solutions containing 4.4, 40, or 70 mM buffer (Figures S-7 − S-10). The average scatter subtracted spectra of aerogels made by loading cyt. c concentrations of 5, 10, 15, 20, or 25 μM (each an average of 2−13 spectra) and encapsulated solution buffer concentration of either 4.4, 40, or 70 mM were each fitted with a Gaussian curve to determine the average Soret peak absorbance/ path length, peak center, and peak width. A summary of these results is tabulated in Table 1 showing more clearly how Soret absorbance/path length did not vary substantially as the buffer concentration for cyt. c solution encapsulated in aerogel was

stable protein encapsulated in the aerogels. The response is proportional to a point. Above 15 μM, there is some rollover in the response. Since we are plotting the average absorbance of many gels in each of the Figure 4 plots, we chose to divide each spectrum by the path length, or thickness, of each gel, and then average all of these path length corrected spectra for each loaded cyt. c concentration to compensate for the effects of small differences in pathlengths. 14761

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26 (±2) 23.0 (±0.8) 22.5 (±0.5) na 23 (±2)

22.9 (±0.3) 22.4 (±0.6) 24.1 (±0.4) 22.6 (±0.6) 22.7 (±0.7) 23.6 (±0.4) 23.2 (±0.7)

405.9 (±0.1) 406.9 (±0.3) 408.0 (±0.1) na 405.8 (±0.2) 407.0 (±0.5) 406.8 (±0.7) 405.2 (±0.2) 406.9 (±0.1) 407.3 (±0.3) 405.4 (±0.2) 406.4 (±0.2) 407.1 (±0.3) 405.9 (±0.3) 407.1 (±0.1)

Table 2. Surface Area, Pore Volume, and Pore Size from Nitrogen Physisorption Data for Pyrolyzed Cyt. c−SiO2 Aerogels Encapsulating 10 μM Cyt. c as Encapsulated Cyt. c Buffer Concentration Is Varied buffer concentration loaded into gel (mM) 2

BET surface area (m /g) t-Plot micropore area (m2/g) t-Plot micropore volume (cm3/g) BJH adsorption cumulative volume of pores between 1.7 and 300 nm (cm3/g) BJH adsorption average pore diameter (nm)

4.4

40

70

376 87 0.036 1.4

289 78 0.033 1.3

203 69 0.031 2.3

14

17

46

24 (±3)

As buffer strength increased, surface area, micropore area, and micropore volume decreased, and cumulative pore volume and average pore diameter increased. Therefore, in addition to the low 4.4 mM buffer strength potentially not providing enough resistance to pH changes during the sol−gel synthesis, or causing more adsorption of cyt. c to negatively charged silica walls, the high surface area and low average pore diameter of these gels may physically lead to more unfavorable interaction of cyt. c with the silica gel leading to less cyt. c stabilization in these gels. It is also possible that the high cumulative pore volume of the gels made using 70 mM cyt. c solutions could lead to larger, less organized cyt. c structures in the gels leading to less cyt. c stabilization. Out of the three buffer strengths compared, cyt. c is most stabilized to unfolding in aerogels when encapsulated in 40 mM buffer, since the average Soret peak centers and widths at all cyt. c concentrations are closest to these parameters for cyt. c in solution (see Table 1). The mid buffer strength may be the optimal strength for stabilizing cyt. c in aerogels, because it provides a high enough resistance to pH changes without completely electrostatically shielding cyt. c proteins from each other. In addition, the lower surface area of 40 mM gels compared to 4.4 mM gels may lead to less unfavorable interactions between

22.5 (±0.2) 21.6 (±0.4)

70 40

3.3 (±1) 4.0 (±1)

4.4 70

na 2.3 (±1)

40 4.4

2.2 (±0.8) 2.5 (±0.3)

70 40

2.3 (±0.7) 2.3 (±0.3)

4.4 70

1.6 (±0.2) 1.6 (±0.4)

40 4.4

1.2 (±0.1) 0.5 (±0.1)

70 40 4.4

0.59 (±0.02) 1.0 (±0.4)

varied. In contrast to absorbance/path length, the Soret peak center was blue-shifted and the peak width was generally wider for the lowest buffer strength of 4.4 mM used to encapsulate cyt. c in aerogels meaning that there may be a disruption in the Met 80 coordination to the heme iron when using lower buffer concentrations to encapsulate cyt. c.43,45 The low buffer strength may not provide enough resistance to pH changes during the sol−gel synthesis or may result in more adsorption of cyt. c to silica walls.46 Cyt. c that is encapsulated in aerogels from 70 mM buffered solutions results in average Soret peak centers that are a little lower than the solution value of 409 nm (see Table 1), and some widening of the peaks occurs. This widening may be exacerbated by the decrease in peak signal-to-noise at the high buffer concentration, revealing an inherent limitation in using visible spectroscopy to probe the protein structure of cyt. c encapsulated in aerogels using high buffer strengths. Or it may be possible that the higher ionic strength is shielding the proteins from each other, decreasing protein association that may help to stabilize the protein in the aerogel. Additional insight into the variation of cyt. c properties in aerogels when encapsulated using different buffer strengths was obtained through porosimetry analysis. The surface area, pore volume, and pore size of cyt. c−SiO2 aerogels loaded with 10 μM cyt. c made by encapsulating cyt. c dissolved in 4.4 mM, 40 mM, or 70 mM phosphate buffer were compared (see Table 2).

Soret absorbance/ path length (cm‑1) Soret peak center (nm) Soret peak width (nm)

25 20 buffer concentration loaded into gel (mM)

15 10

2.6 (±0.3)

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Cyt. c concentration loaded into gel (μM)

Table 1. Average Gaussian Fitted Soret Peak Absorbance Divided by Gel Path Length, Peak Center, and Peak Width for Different Concentrations of Encapsulated Cyt. c as Encapsulated Cyt. c Buffer Concentration Is Varied in Scatter Corrected Cyt. c−SiO2 Aerogels

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c−SiO2 aerogels and cyt. c−SiO2 aerogels indicates that cyt. c viability is similar in both types of aerogels, although the protein is slightly more stable when encapsulated as Au∼cyt. c superstructures. Although we do not know the exact configuration of cyt. c within cyt. c−SiO2 aerogels, the spectroscopy results suggest that the encapsulated cyt. c may associate with other cyt. c in the aerogels to reduce contact with the silica gel in a way that may be analogous to the way metal-nucleated cyt. c superstructures form in aerogels. By varying the concentrations of both the encapsulated cyt. c and the phosphate buffer, we found that optimized protein structure in the aerogel resulted when encapsulating 40 mM phosphate buffered solutions that produced loaded aerogel cyt. c concentrations in the range of 5 to 15 μM. Venting supercritical fluid too quickly to produce bioaerogels also decreased the amount of viable protein in the gels. The increased protein structural stability and increased NO ligandbinding reversibility of Au(5‑nm)∼cyt. c−SiO2 aerogels compared to cyt. c−SiO2 aerogels should be taken into account when developing biosensors based on these bioaerogels. However, the results of this study have helped to clarify the necessary conditions for cyt. c to remain active in aerogels and have shown how cyt. c structure and function are affected by different aerogel synthetic parameters. This information will be useful for future studies aimed at incorporating other proteins into aerogels for the development of biosensors or other bioanalytical devices.

cyt. c and silica in these gels. In contrast to the effects of buffer, there does not appear to be a clear trend in Soret peak center shift or increase in peak width when comparing different concentrations of cyt. c encapsulated from one buffer strength (Table 1). These results indicate that within the cyt. c concentration range of 5 to 25 μM, the encapsulated buffer strength is most important to protein viability within the aerogels. In addition to studying the effects that encapsulated cyt. c concentration and encapsulated buffer concentration had on the stability of cyt. c in aerogels, we also studied the effects the supercritical drying method used to make the aerogels had on the stability of cyt. c in the aerogels. As stated in the Experimental Section, we use a critical point drying apparatus to replace the acetone in soaked sol−gels with liquid carbon dioxide, that is then brought to the critical point and released as a supercritical fluid through a gas vent. If the supercritical fluid is released too quickly, aerogels can break from the fast pressure change. So, it is important to allow the pressure to release over the course of a few minutes. We hypothesized that releasing the pressure over a longer period of time may result in more cyt. c remaining structurally active in the aerogels, since a fast pressure release may be detrimental to the cyt. c in the aerogel in addition to causing gel breakage. Releasing the pressure over 7 min compared to 45 min resulted in similar Soret peak centers (407.1(±0.1) nm and 407.1(±0.3)) and peak widths (23.1(±0.5) nm and 23.6(±0.6) nm) for the long and short release times, respectively) (Figure 6). However, the Soret peak absorbance/path length was

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ASSOCIATED CONTENT

* Supporting Information S

Additional UV−vis spectra and additional NO ligand-binding response data of cyt. c −SiO2 and Au(5‑nm)∼cyt. c −SiO2 aerogels. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: aharper@fairfield.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support for this work was provided by a Cottrell College Science Award from the Research Corporation for Science Advancement, Fairfield University College of Arts & Sciences and Fairfield University Chemistry & Biochemistry Department. We thank Molly E. Graffam and Bayan H. Abunar for assistance with synthesis of aerogels for porosimetry analysis. We are very grateful to Dr. Alanna Schepartz of Yale University for use of the circular dichroism spectrometer. Thank you to Michael Leopold, Deon Miles, Matthew Kubasik, Kraig Steffen and the anonymous reviewers for helpful suggestions during the revision of this article. We also very gratefully acknowledge Jean Marie Wallace for much helpful insight and advice in regards to this article and this general research area.

Figure 6. Averaged UV−visible spectral absorbance divided by gel path length for cyt. c−SiO2 aerogels encapsulating 10 μM cyt. c in 50 mM phosphate buffer in which the supercritically dried aerogels were made by either releasing supercritical carbon dioxide over 45 min (solid, black (average of 9 spectra)) or 7 min (dashed, red (average of 4 spectra)).

decreased when the pressure was released over 7 min versus 45 min indicating that less cyt. c may remain active in aerogels produced through fast release of supercritical fluid. The cyt. c in the aerogels appears to be sensitive to large pressure changes resulting in some cyt. c losing all structural integrity, but the cyt. c that remains appears to retain the same structural integrity as cyt. c in aerogels in which the supercritical fluid is released more slowly, since the peak center and peak width do not vary much.

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REFERENCES

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CONCLUSIONS We have shown that cyt. c encapsulated in aerogels without added nanoparticles retains much structural integrity and retains response to nitric oxide resulting in a simplified synthesis of these bioactive aerogels. The similarity of UV−visible and Soret circular dichroism spectroscopy results for Au(5‑nm)∼cyt. 14763

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