Proof of Concept
Abstract
HESTIA’s proof of concept can be divided into three parts. First, we produced a sustainable insulation material by manufacturing cellulose hydrogels and aerogels and characterizing their insulative properties. Second, we expressed proteins designed to bind the insulation material. Lastly, we showed that the properties of our insulation material were enhanced by the presence of binding proteins by testing uncoated and coated aerogels.
Click on the diagram below to read more about each part.
Producing a Sustainable and Biodegradable Thermally Insulative Material
We produced cellulose hydrogels and aerogels, studied and improved their properties.
Producing a cellulose aerogel
Many trials were necessary to produce and improve the aerogel. They were first produced through critical point drying, then through freeze drying. Freeze-drying proved less resource-consuming and more accessible, which fit our vision of HESTIA better. Using the comments provided by EMPA’s material science experts, the sol-gel chemistry was improved. By experimenting with the solvent exchange, baths of 70% ethanol followed by water solvent exchange produced the most structurally sound and robust aerogel. After production the porosity and density were studied. For the most optimized aerogels, a density of 0.149 g/cm3 and a porosity of 73.38% were observed, the details of these experiments can be found in the Results.
The density and porosity obtained after several trials matched a description of a porous material. The values were compared with the samples produced in the litterature1 and compatible with our application. After successfully producing cellulose aerogels, we continued our proof of concept by testing its insulative properties.
Testing cellulose aerogel insulative properties
The thermal conductivity tests were performed with an in house testbench to find out the thermal insulative properties of the aerogel. The detailed method can be found in Measurement and the resulting experiment can be found in the Results. Within the limits of our in-house apparatus thermal conductivity assay, we showed that the produced macro-porous aerogel had comparable insulative properties to an EPS.
The porous aerogel curve for the increase of heat in the non heated second chamber is comparable to the EPS curve suggesting our produced cellulose aerogel is an insulation material that has reduced heat conductivity.
Expressing proteins binding to the insulation material
After expressing and purifying our constructs, we showed how the CBD-Cellulose interaction could be used to link proteins to an aerogel.
CBD characterization with nitrocellulose and our 03a GFP fusion (mSA-GFP-CBD) construct
In HESTIA’s design the CBD serves as linkage between the aerogel and the proteins. To prove CBD attachment, we used 03a GFP fusion (mSA-GFP-CBD) construct and observed results through fluorescence. We verified the cellulose binding properties of our constructs by performing a drop-plot using a nitrocellulose membrane followed by PBS washes.
After washing, we observed a more important degradation of fluorescence in the case of control (GFP without CBD vs GFP with CBD). suggesting any CBD-containing constructs would be able to bind cellulose in the long term. A statistical analysis confirmed the observation by suggesting a larger decrease in fluorescence after PBS washes for the sfGFP control than for the GFP fusion recombinant protein.
CBD-Aerogel linkage with our 03a GFP fusion (mSA-GFP-CBD) construct
The next step was to show proper linkage of the CBD containing constructs to actual the aerogel. For that we proceeded with the control 03a (mSA-GFP-CBD) construct and observed results through fluorescence.
The following key experiment was performed : hydrogels were soaked with water, liquid sfGFP and liquid purified 03a (mSA-GFP-CBD). They were then sent to freeze drying. After freeze drying, the samples were observed under a UV light.
From the difference in fluorescence, we were able to prove a linkage of our CBD containing constructs to our aerogels.
Enhancing the Properties of the Insulative Material with the Expressed Proteins
After successfully coating the aerogel with the designed proteins, we tested the properties of our new material. Additionally, we proved the hydrophobicity of our silk biofilm.
Liquid 01a Silk fusion (mSA-silk-CBD) and 03a GFP fusion (mSA-GFP-CBD) test
To show an enhancement of desired properties on our newly coated aerogels, we worked with our 01a Silk fusion (mSA-silk-CBD) and 03a GFP fusion (mSA-GFP-CBD) constructs.
In this experiment, we first coated hydrogels with proteins (either the silk fusion protein (01a), the GFP fusion protein (03a), the GFP control protein or water). Using the Keyence microscope, we filmed the application and the absorption of 4 to 5 drops of water on our coated aerogels. Then we measured the delay time it takes for the water to be absorbed, and computed the mean for each aerogel.
The details of the performed one-way ANOVA test and the computation of the Tuskey HSD for pairwise-comparison can be found in our Notebooks (link to Notebooks). The results show that there is a significant difference in time for water to be absorbed between the silk fusion protein and the other groups, while there is no significant difference among the other groups (dH20, GFP and GFP fusion protein 03a). Therefore we can conclude that aerogels absorb water more slowly when they are coated with the silk fusion protein, suggesting that the silk coating provides hydrophobicity to the aerogel.
Creation and hydrophobicity testing of a Silk Biofilm
In HESTIA’s design, a hydrophobic silk biofilm would give the aerogel protection against water degradation. Using the purified 01a silk fusion (mSA-silk-CBD) construct (A), we managed to create a silk biofilm and test its hydrophobicity. As a control, we created a biofilm of the 03a GFP fusion (mSA-GFP-CBD) construct (B) and compared their properties.
The GFP fusion protein biofilm presented more cracks and inhomogeneous structure than the Silk fusion biofilm. After successfully creating biofilms, we tested their hydrophobicity with a Keyence microscope and evaluated the water angle.
We obtained an angle of 55.2° for the silk (C) and 27.3° for the GFP biofilm (D). This meant that the silk fusion protein biofilm (01a) had a higher contact angle than the GFP biofilm. Moreover, by measuring the time it takes for three water droplets to be absorbed, we obtained a mean of 466,33 s for the silk biofilm (E), and 28,33 s for the GFP biofilm (F). With these values, we concluded that it takes more time for a drop of water to be evaporated or absorbed from the silk biofilm. These two results suggested that our biofilm helps protect the aerogel from water droplets.
Outlook
HESTIA’s proof of concept was built upon three main bricks : (1) the production of a sustainable and thermally insulative cellulose aerogel, (2) the expression of protein constructs able to bind the material, and (3) the enhancement of the material through binding of the proteins. After being studied and proven, they provide a solid foundation for HESTIA. To consolidate our proof of concept, future experiments should focus on : showing linkage between a silk biofilm and an aerogel, creating a silk biofilm with a linked modular protein (see Design) and proving an enhancement of both hydrophobicity and fire retardancy on a fully coated aerogel.
References
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Preparation, Properties, and Applications of Natural Cellulosic Aerogels: A ReviewEnergy and Built Environment, vol. 1, no. 1, pp. 60-76