The main advantages of this method are that it is fast, easy, environmentally friendly and allows for tailoring of materials. This method can provide insight into how bioinspired silica is formed, the available ways of incorporation of biomolecules and the post-synthetic material treatment. Generally, individuals new to this method will struggle because silica formation is rapid and a specific amount of acid needs to be added quickly in order to obtain the optimum results.
We first had the idea of this method when we realized how mild it was, such that the conditions were compatible with enzymes. We thought of using this method to enhance enzyme performance and stability. Visual demonstration of this method is critical as the acid neutralization step is the most important for the final material properties.
First, weigh 318.2 milligrams of sodium silicate pentahydrate into a 180-milliliter plastic container and dissolve in 20 milliliters of deionized water. In a second container, weigh 58.1 milligrams of pentaethylene hexamine or PEHA and dissolve in 20 milliliters of deionized water. Subtract this amount of water from the volume of deionized water to be used for the dissolution of sodium silicate pentahydrate.
To perform in situ encapsulation during synthesis, dissolve a predetermined mass of protein in five milliliters of deionized water and place the solution in the refrigerator for total dissolution. Check occasionally on the dissolution progress, preferably without stirring. Combine the sodium silicate pentahydrate and PEHA solutions in one of the containers and add sufficient deionized water to make the final solution volume 41.5 milliliters.
Place the freshly-prepared mixture on the top of a stir plate and add a stir bar to provide consistent mixing. Then, suspend a pH probe in the stirring solution and record the initial pH. Begin the synthesis by adding a predetermined quantity of one-molar hydrochloric acid and observe the immediate evolution of turbidity.
The first critical step is the rapid addition of acid to initiate silica formation. The pH needs to be lowered to seven as fast as possible. If encapsulation is taking place, add the encapsulant solution as soon as the acid addition is complete.
Then record the pH after five minutes to determine the reaction completion. After the reaction has reached completion, modify composition of produced silica by adding more hydrochloric acid to the mixture until the desired pH between two and seven has been reached and allow the suspension to stabilize for approximately one minute. Following this, decant the bioinspired silica suspension into 50-milliliter centrifuge tubes, then centrifuge the suspension at 5, 000 g for 15 minutes.
After centrifugation, remove the supernatant and store for further analysis. Refill the centrifuge tubes with deionized water and resuspend the silica using a vortex mixer. In the case of biomolecules incorporation, a critical step is to give the supernatants from bioinspired silica washing in order to be further analyzed.
After repeating the centrifugation and resuspension steps twice, remove the supernatant and scrape the silica into ceramic crucibles. Dry the silica in an oven overnight at 85 degrees Celsius. In a five-milliliter plastic vial, dilute 300 microliters of previously-prepared molybdenum blue reagent with three milliliters of deionized water.
Then, add 10 microliters of salycic acid test solution and shake to mix. After 15 minutes, add 1.6 milliliters of previously-prepared para-aminophenol sulfate-reducing agent to reduce the yellow silicomolybdate complex to its blue isomer. Following this, measure the sample absorbance at 810 nanometers in a UV-vis spectrophotometer and calculate silicon concentration against a calibration curve.
Using disposable pipette tips, add a pre-determined amount of Bradford reagent and sample in each assigned cuvette. Mix each cuvette by inverting three times and allow to develop for 10 minutes. Next, measure the absorbance at 595 nanometers using pure supernatant as a blank.
Calculate the original absorbance of each cuvette by subtracting the control sample absorbance from each measurement. Now, create a calibration curve for each set of experiments by plotting measured absorbance against concentration of BSA to avoid random fluctuations that might affect the assay's sensitivity. Finally, determine the protein content for each sample during resuspension to monitor possible protein loss.
The yield of the silica-forming reaction is typically 58 plus or minus 6.5%The molybdenum blue spectroscopic method detected the amount of unreacted monomeric silicate species as well as those species which reacted to form polysilicates or oligomers but have not managed to reach sufficient size to coagulate. Once coagulation is complete, material surfaces can be readily modified through the use of acid elution which allows for fine-tuning of material properties such as composition, porosity, and chemical activity of additive. 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 could be securely encapsulated during synthesis with no leaching. The FTIR spectrum of the silica showed characteristic amide bands in the samples with BSA but not in the control samples. When BSA was added to the unreacted reagents, there were no considerable differences in the immobilization efficiency or the amount of BSA in the resulting composite.
When BSA was added during silica formation, immobilization efficiency and the amount of BSA in the product were significantly lower. Once mastered this technique can be done in less than five minutes if it's performed properly. While attempting this procedure, it's important to remember to be accurate with weights and volumes of reactants and quickly add the acid in one dose.
Following this procedure, other methods like FTIR, SCM, porosimetry, and enzyme assays can be performed in order to answer additional questions about the material properties and morphology as well as the activity of the biomolecules. After its development, this technique paved the way for researchers in the field of bioinspired silica formation to explore a fast, easy, controllable, and environmentally-friendly method of synthesizing silica with the ability of in situ capsulation of biomolecules. After watching this video, you should have a good understanding of how to synthesize silica using a bioinspired method, encapsulate biomolecules in situ, measure encapsulation efficiency, and perform post-synthetic elution of the additives.
Don't forget that working with amines, acids, biomolecules can be extremely hazardous and precautions such as PPE and spillage prevention should always be taken while performing this procedure.
