Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium. The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.


Introduction
The use of silica as a structural support for industrial catalysts is well established, allowing for the improved catalyst activity, stability and processability, 1 hence potentially reducing the operating cost. These benefits are compounded in the case of enzyme immobilization, as storage within a silica pore system can confer significant benefits on the enzyme lifetime over its free counterpart. Accordingly, much effort has been expended in finding the best method to attach enzymes to silica species, with multiple reviews comparing investigations using different methods of immobilization on siliceous solid supports. 2,3,4 Enzymes are typically attached via physisorption or covalent bonding, in addition to encapsulation within a porous material. 5 However, there are significant drawbacks related to each method: physisorption relies on transient surface interactions between the silica and biomolecule, which can very easily be weakened by the reaction conditions leading to the unacceptable enzyme leaching. The much stronger covalent attachment usually results in lower activity due to the reduced conformational freedom of the active species. Encapsulation can result in reduced activity due to the enzyme inaccessibility or diffusional limitations.
This specific silica speciation data is of particular interest when comparing different titration efficiencies for the precipitation reaction -I.E. how the final reaction pH and the rate at which this is reached affects the polymerization of monomeric silica to an 'oligomer' and its subsequent coagulation to solid silica. By modifying the amount of acid added in stage 2.4 slightly, under-or over-titration of the reaction mixture can be performed ( Figure 5). By measuring the silica speciation again for these two cases, a clear difference can be seen in the reaction completion (Figure 4) despite only minor changes to the titration profile of the reaction (Figure 5).
Although no difference is present between the consumption of monomeric species for the three reaction cases (remaining between 29 -33%), there is a clear difference in the amount of oligomeric silica species which precipitate in each case. This is in agreement with traditional theory on sol-gel silicas -in the 'undershoot' case the pH is held higher for longer, allowing for individual particles to grow and hence aiding efficient coagulation; in the 'overshoot' case the coagulation is induced much faster due to the rapid titration, hence fewer of the silica species have grown to a sufficient size to coagulate and remain trapped in the colloid phase. 16 Given the importance of titration upon silica formation, a priori knowledge of the appropriate titration volume is essential. Although not extractable from the reaction stoichiometry due to the complex protonation behavior of the amine additives and change in silica surface acidity on coagulation, highly reliable empirical relationships between system contents, concentrations and titer volumes are readily generated (Figure 1).
Once coagulation has been completed, material surfaces can be readily modified through the use of acid elution, as has recently been reported by the authors elsewhere. 13 This allows for fine-tuning of material properties such as composition, porosity, and chemical activity of additive (Figure 6a and b).
In this study, BSA was used as an exemplar encapsulant enzyme, however, the techniques described here can be used for multiple enzymes 17,18 . The procedure followed for protein detection is the Bradford assay protocol, 19 using the supernatants stored from each centrifugation cycle. The amount of protein in the supernatant is calculated using a calibration curve created from known amounts of BSA dissolved in the supernatant of a sample with zero protein content (Control sample). The amount of protein encapsulated into silica will be calculated by subtraction of the detected protein in supernatants from the initial amount of protein added. The only reagent needed for the assay is the Bradford Reagent (either procured or made according to standard recipes).
There are three types of assay format, depending on the sample volume, the expected amount of protein to be detected and the measurement method used. Herein, the followed format is specified for a spectrophotometer, requires disposable cuvettes of macro and of micro size and can detect from 10 µg/mL to 1.4 mg/mL of protein.
In Figure 7 the amount of protein detected after each wash (step 4.3) is shown as a % of the initial protein amount (which was 50 mg). Around 50% of BSA was detected in the supernatant after the first centrifugation, which relates to ~50% immobilization efficiency. As there was no BSA detected in the following washes, BSA (or any other enzyme) could be securely encapsulated during silica synthesis with no leaching -this is a significant advantage of this method. In order to confirm the presence of BSA in the silica produced, Fourier Transform Infrared Spectroscopy (FTIR) analysis was performed. The presence of the characteristic bands of amide I and II in the area of 1,500/cm and 1650/cm (Figure 8) in the samples prepared in the presence of BSA, but not in the control samples (no BSA) confirmed the presence of BSA in the solids.
In addition to the method of enzyme addition described above (BSA added during neutralization of reaction mixture), there are other possible variations e.g.,BSA addition during mixing of the silicate and the additive solutions, prior to neutralization or enzyme added to the silicate or additive solution before their mixing and neutralization. Some of these possibilities were explored further and the immobilization efficiencies (mass of BSA immobilized as a percentage of enzyme added to the reaction system, calculated based on the Bradford assay) and the amount of BSA in the final silica were measured (concentration of BSA in silica as a percentage of the total composite weight produced, see Figure  9). It was clear that when BSA was added to the unreacted reagents (cases A-C in Figure 9) there were no considerable differences in the immobilization efficiency or the amount of BSA in the resulting composite. However, when BSA is added during silica formation (case D in Figure  9), immobilization efficiency and the amount of BSA in the final product were both significantly lower. Despite these differences, the average amount of silica produced remained unchanged (between 85-90 mg). These observations can be explained on the basis of the ionization (or isoelectric point) of BSA, silicate/silica and the additive. The different methods of addition allow for different interactions between the enzyme and silica precursors. As the pH at the time of the addition of the enzyme changes, the ionization of each species will determine intermolecular interactions, which in turn will control the immobilization efficiency.

Discussion
In the current work, we present a method for rapidly precipitating bioinspired silica materials and encapsulation of biomolecules therein. We demonstrate critical steps within the procedure, namely the amount of reaction-initiating acid to be added, and timing of addition of the biomolecule encapsulant. We show the effect of acid addition amount on both reaction progression and yield (Figure 4 and Figure 5, respectively), and demonstrated a method for tight control over synthesis conditions, allowing for consistency despite this sensitivity. Regarding active species encapsulation, although straightforward in terms of procedure, encapsulation is shown to be sensitive to the conditions of the experiment (order of addition, pH of addition, environmental conditions), however, consistency in material properties is again achievable.
The synthesis conditions can be modified through the use of different additives, many of which have been published elsewhere, 15 providing a range of morphologies and porosities. Further, post-synthetic techniques to modify and chemically tailor bioinspired silica materials have been reported such as mild purification 13 and surface amine decoration. 20 Finally, due to the mild, aqueous nature of the synthesis, in situ encapsulation is possible for a wider range of substrates than those demonstrated here, ranging from enzymes 17,18 to whole cells, 21 metal salts, 22 active pharmaceutical ingredients, 23 and quantum dots. 24 Unlike other organic-mediated silica syntheses (such as the MCM-41 or SBA-15 family of materials), the polyfunctional nature of bioinspired additives cannot produce ordered pore structures, nor highly monodisperse particle-size distributions characteristic of Stöber-type silica. 25 This is due to the lack of well-defined micellization behavior of bioinspired additives (outside of special cases) 26 coupled with their increased catalytic activity over monofunctional amine-containing additives. 26 On the other hand, this polyfunctional additive nature enables the use of shorter reaction times and milder temperature & pressure compared to other organic-mediated silica syntheses. This also leads to the possibility of room-temperature additive elution as described above, which has yet to be achieved for these other silica families due to the specifics of their surface chemistry. 27,28,29 Consequently, bioinspired silica materials have been shown to be both more economical and practical to produce at a larger scale, leading to easier commercialization and development. 14 In summary, bioinspired silica synthesis represents a rapid, facile method for producing active species supports or gas sorbent media. Through tight control of pH during and after the reaction, a wide array of silica-amine composites can be synthesized with varying properties, which is further complemented by the possibility of in situ encapsulation of an array of different organic, inorganic, or bio-organic materials. Although independent post-synthetic modification of bioinspired additive and encapsulant concentration has yet to be achieved, these methods represent a promising step towards environmentally benign chemical processes.

Disclosures
The authors declare no competing financial interest.