Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.
Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly α-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable β-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.
Due to their combination of strength and extensibility, spider silk fibers can absorb more kinetic energy than most other natural or synthetic fibers 1. Furthermore, unlike most synthetic polymeric materials silk materials are nontoxic and biocompatible and cause no allergic reaction when incorporated 2,3. Putative health risks can be prevented by using spider silk. These features make spider silk highly attractive for a variety of medical and technical applications. Since spiders can’t be farmed due to their cannibalistic behavior, biotechnological methods have been developed to produce spider silk proteins, both cost-efficiently and in sufficient quantities 4.
The recombinant silk protein eADF4(C16) has been engineered based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4). eADF4(C16) has a molecular weight of 48kDa 5 and is soluble in various solvents (hexafluoroisopropanol (HFIP) 6, formic acid 7 and aqueous buffers) 8. eADF4(C16) can be processed into different morphologies such as films 9, capsules 8, particles 10, hydrogels 11, coatings 7, fibers 12 and nonwoven meshes 6. Due to their chemical stability, the latter provide high potential in filter applications.
Here, we present a protocol to fabricate air filter devices including a nonwoven mesh of electrospun recombinant spider silk proteins. Electrospinning or electrostatic spinning is a technique typically employed for producing polymer fibers with diameters in the range of 10 nm -10 μm 13, and nonwoven meshes have already been investigated for filter applications 14. In the past, electrospinning has been successfully applied for processing of regenerated 15 as well as recombinantly produced 16 spider silk proteins. Typically a high electric voltage (5-30 kV) is applied to a syringe and a counter electrode (0-20 kV) placed in a distance of 8-20 cm. The strong electrostatic field induces repulsive forces within the charged solution. If the surface tension is exceeded, a Taylor cone is formed, and a thin jet erupts from the tip 17,18. After formation, bending instabilities occur within the jet causing further stretching as the solvent evaporates, and a solid fiber is formed. Finally, the fiber is randomly deposited on the counter electrode as a nonwoven mesh 19. Fiber properties like diameter and surface topology (smooth, porous) are mainly dependent on solution parameters such as concentration, viscosity, surface free energy and the solvent’s intrinsic electrical conductivity and permeability 20. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers with diameters from 80-1,100 nm depending on the protein concentration in the solution. eADF4(C16) electrospun from HFIP displays a predominantly α-helical secondary structure and the fibers are water soluble 6. In order to stabilize the silk fibers, β-sheet structures have to be induced by subsequent treatment with ethanol. In contrast to previously established post-treatment methods 21, in this study eADF4(C16) nonwovens have been treated with ethanol vapor in order to preserve the morphology of the silk fibers. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD) as described in the literature 22. FSD is a signal-processing tool that allows resolution of FTIR spectra consisting of several overlapping bands. Thereby, the indistinct bands of the broad amid I region can be narrowed using a high pass filter to receive a deconvoluted spectrum with improved peak resolutions.
In order to evaluate the efficiency of filter substrates complemented with silk nonwoven meshes, air permeability tests were performed using an Akustron device according to standard protocols. Deposition rates were measured using a Palas universal particle sizer.
New filter devices should allow lowering the overall energy consumption in air filtration at constant or higher filter efficiencies. Here, such devices were created using nonwovens made of spider silk. Due to its low surface tension and high volatility, HFIP has been chosen as a suitable solvent for the electrospinning process. Furthermore, aqueous silk solutions have been tested in previous experiments, but no fibers could be generated. Here, it would be crucial to use additives in order to lower the surface tension and thereby improve the spinning properties of the solution. The most critical step is to adjust the conditions and the used material concentration and solvent of the spinning solution, spinning height, voltage and extrusion velocity. During performance, for instance clogging of the tip can be prevented by supplying the needle tip with moisture in form of water vapor, but any kind of additions in the electrospinning setup might subsequently disturb the sensitive process and electric field. Essential process parameters (concentration, voltage, distance, humidity) were individually determined carrying out separate experimental series (data not shown). Taking all parameters into consideration it is crucial to take care of a continuous taylor cone and spinning process to create uniform fibers.
The filter efficiency is one of the most important parameters of filter materials. This parameter is mainly influenced by the structure of the filter material. Wovens inherit uniform pore sizes and subsequently consistent air permeability. It is crucial to create homogeneous nonwoven meshes on these template materials to fill the pores and to generate a zero-defect filter. The filter efficiency in our filters shows a direct dependence on the spinning duration (of the silk proteins), and, therefore, on the number of nonwoven mesh layers. The gaps between single fibers are consistently filled, enabling the retention of smaller particles.
In this work we introduced a method to produce a novel filter material with spider silk nonwoven meshes, showing high filter efficiency. Therefore, these filters are promising candidates for future usage in air filtration systems.
The authors have nothing to disclose.
We gratefully acknowledge the technical and scientific support of Anja Lauterbach (Lehrstuhl Biomaterialien), Lorenz Summa (Sandler AG) and Armin Boeck (B/S/H/G). SEM-imaging was performed by Johannes Diehl (Lehrstuhl Biomaterialien). Funding was derived from BMBF (01RB0710).