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).

1. Spinning Dope Preparation
<ol>
 <li>Take lyophilized eADF4(C16) protein.</li>
 <li>Weigh 20 mg of eADF4(C16) in a 1 ml reaction vessel by using a high precision scale.</li>
 <li>Add 200 &mu;l of hexafluoroisopropanol (HFIP) and seal the vessel with Parafilm. 
 Note: HFIP is highly volatile and deleterious. As it can cause harm in the respiratory track, work under a safety hood, pipette carefully, and cap the tube.</li>
 <li>Vortex the suspension for 1 min and further shake it to clear the solution. To ensure, that the entire amount of protein is completely dissolved, wait over-night.</li>
</ol>
2. Electrospinning
<ol>
 <li>Prepare the electrospinning device (Figure 1): place the filter material on top of the counter electrode and preset the voltage of both the electrode (-22.5 kV) and the counter electrode (+2.5 kV). Set the volume flow to 315 &mu;l/hr. 
 Note: while electrospinning, toxic HFIP will be evaporated. Make sure your electrospinning device is connected to a fume hood.</li>
 <li>Take a commercially available 20 G needle and grind the sharp tip with a hand grinder to a residual length of 30 mm. Connect the needle to a 1 ml syringe. 
 Note: A plane needle tip is required in order to generate a well-defined Taylor cone.</li>
 <li>Load the entire spinning dope (200 &mu;l) into the syringe. Overlay the dope with 100 &mu;l of air, in order to allow the complete solution to be extruded during the spinning process. 
 Note: In order to avoid clogging of the needle, make sure that there are no particles (aggregates or impurities) in the spinning dope. Work under a fume hood!</li>
 <li>Attach the filled syringe to the syringe pump of the electrospinning device and carefully press the piston onto the syringe until a droplet appears at the tip of the needle. Lock the piston.</li>
 <li>Set the distance between the tip of the needle and the counter electrode to 8-20 cm.</li>
 <li>Start the syringe pump and remove the (usually dried out) droplet from the opening of the needle. Activate all safety installations of the electrospinning device immediately and start the high voltage source as soon as a new droplet appears. Electrospinning of the spinning dope will start subsequently. Use a stopwatch to control spinning duration. 
 Note: In order to avoid drying out of the solution and thus clogging of the needle, it is necessary to immediately start the spinning process after removing the dried droplet.</li>
 <li>Since electrospinning of recombinant spider silk proteins depends on humidity and temperature, an adaptation of process parameters towards individual lab conditions might be necessary (Figure 2). 
 Note: To prevent the droplet from drying (Figure 2B) enable a sufficient flow rate. If there is a low humidity in the surrounding atmosphere, adjust the relative humidity or raise the flow rate. Lower the voltage until a proper Taylor cone occurs (Figure 2A). When there is no solution at the tip (Figure 2C), raise the flow rate and lower the voltage until a droplet occurs. Then adjust voltage in order to establish a regular and stable Taylor Cone (Figure 2A).</li>
 <li>After 30 sec / 60 sec / 90 sec of electrospinning switch off the syringe pump. In order to avoid falling droplets, wait 10 sec before turning off the high voltage source to release the residual pressure in the syringe.</li>
 <li>Steps 6 to 8 can be performed on different types of filter materials, such as polyamide, polypropylene and polyester nonwoven meshes, as well as black paper for comparison.</li>
 <li>To produce a nonwoven mesh for subsequent stability experiments, use black paper instead of filter material and perform steps 5 to 7. After 5 min of electrospinning, stop the process as described in step 8.</li>
</ol>
3. Post-treatment of Silk Nonwoven Meshes
<ol>
 <li>Pre-heat an oven to 60 &deg;C.</li>
 <li>Place the filter substrates with eADF4(C16) nonwoven meshes vertically and with a minimum distance of 2 cm in a lockable glass container. The container should have two openings that will be used to subsequently introduce ethanol and water. 
 Note: When fixing the filter materials, make sure that the area necessary for permeability experiments is not damaged by the clamps.</li>
 <li>Connect two 60 ml syringes, one filled with ethanol and one filled with water, with silicone tubes pointing into the interior bottom of the post-treatment container (Figure 3). 
 Note: In order to be able to remove the liquid from the container after the treatment, place the openings of the pipes as close as possible to the bottom.</li>
 <li>Place the post-treatment container in the oven and add 60 ml of ethanol by extruding the syringe. Use a stopwatch to control treatment duration.</li>
 <li>After 90 min of ethanol vapor treatment, remove the ethanol with the syringe from the glass, and add 60 ml of water from the second syringe.</li>
 <li>Wait for another 90 min, then remove the water and switch off the oven. In order to avoid droplets by condensation, leave the container in the oven until it has completely cooled down.</li>
</ol>
4. Analysis of Spider Silk Nonwoven Meshes
<ol>
 <li>Prepare the silk nonwoven meshes for stability tests on black paper or any other removable support. Cut two frames out of cardboard and adjust double-sticky adhesive tape. Press one frame onto the silk nonwoven mesh deposited on black paper and use a scalpel to cut off the excess of silk fibers (keep the excess fibers for subsequent SEM-imaging). Carefully remove the frame in order to detach the nonwoven from the paper. Repeat this step with the second frame (Figure 4).</li>
 <li>Practical dip test: Cut a piece (1 cm2) of each, the post-treated and non-treated silk nonwoven mesh and dip it into deionized water. The non-treated silk nonwoven mesh will immediately dissolve, while the treated nonwoven mesh will be stable (Figure 5). After dipping, dry the dipped sample and prepare it for SEM imaging.</li>
 <li>Fourier transform infrared spectroscopy (FTIR)-measurement and subsequent Fourier self-deconvolution (FSD): In order to gain information about the structural changes of the silk proteins upon post-treatment of the nonwoven meshes, FTIR can be applied using the parameters: Transmittance-mode, scan from 800 to 4,000 cm-1, 60 accumulations are measured and averaged for each spectrum, one reference is measured per spectrum. For quantitative analysis of the data, FSD can be employed (Figure 6 and Figure 7). Thereby, the curves are reduced to the data area between 1,590 and 1,705 cm-1 and a baseline correction is performed. A local least square fit is calculated according to the peak positions taken from previous studies (1611, 1619, 1624, 1630, 1640, 1650, 1659, 1666, 1680, 1691, 1698 cm-1) 22.</li>
 <li>SEM imaging: SEM can be applied to investigate fiber diameters and the morphology of silk fibers on different filter substrates and to analyze the influence of the post-treatment on the fiber morphology (Figure 8). Use magnifications of 5,000x to 25,000x in order to get sufficiently detailed images.</li>
</ol>
5. Determination of Air Permeability
<ol>
 <li>Place a proper fitting part of the filter material on the measuring area of an Akustron air permeability device. Note:: If you use another type of air permeability device, make sure that it fulfills the requirements of DIN 53 887, DIN 53 120, ISO 9237 and ASTM D 737-96 standards.</li>
 <li>Employ an Akustron air permeability device (or any other as depicted in 5.1) . If necessary calculate the normed data [l / (m2 x sec)].</li>
 <li>Repeat steps 1 and 2 at least 10 times with different parts of your sample and calculate the arithmetic middle (Figure 9). 
 Note: Measuring air permeability requires contact of the device and the nonwoven mesh. Thus, a careful handling of the samples is essential to prevent rupture of the delicate silk nonwoven mesh.</li>
</ol>
6. Determination of Filter Efficiency
<ol>
 <li>Use a proper machine with pressure control and particle counter, such as a Universal particle sizer (Palas GmbH, Karlsruhe, Ger).</li>
 <li>Place the filter samples in the device and measure particle retention (Figure 10). Aerosol: Di-ethyl-hexyl-sebacat (DEHS); particle sizes: 0.3-3 &mu;m; duration: 30 sec; liquid velocity: 2,350 cm/sec; air flow: 3,400 m3/hr. 
 Note: Handle the sample with care and do not touch the surface to prevent destruction of the nonwoven mesh and avoid any pollution. Be sure to create enough samples of equal quality for performance measurements.</li>
</ol>

<ol>
	<li>Gosline, J. M., Guerette, P. A., Ortlepp, C. S., Savage, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+design+of+spider+silks:+From+fibroin+sequence+to+mechanical+function.">The mechanical design of spider silks: From fibroin sequence to mechanical function.</a> J. Exp. Biol. 202, 3295-3303 (1999).</li><li>Vollrath, F., Basedow, A. B. P., Engstr&#246;m, W., List, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+tolerance+to+spider+silks+and+protein+polymers+in+vivo.">Local tolerance to spider silks and protein polymers in vivo.</a> In Vivo. 16, 229-234 (2002).</li><li>Allmeling, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+silk+fibres+in+artificial+nerve+constructs+promote+peripheral+nerve+regeneration.">Spider silk fibres in artificial nerve constructs promote peripheral nerve regeneration.</a> Cell Prolif. 41, 408-420 (2008).</li><li>Huemmerich, D., Helsen, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+structure+elements+of+spider+dragline+silks+and+their+contribution+to+protein+solubility.">Primary structure elements of spider dragline silks and their contribution to protein solubility.</a> Biochemistry. 43, 13604-13612 (2004).</li><li>Huemmerich, D., Slotta, U., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+and+modification+of+films+made+from+recombinant+spider+silk+proteins.">Processing and modification of films made from recombinant spider silk proteins.</a> Appl. Phys. a-Mater. 82, 219-222 (2006).</li><li>Leal-Egana, A., Lang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+Fibroblasts+with+Different+Morphologies+Made+of+an+Engineered+Spider+Silk+Protein.">Interactions of Fibroblasts with Different Morphologies Made of an Engineered Spider Silk Protein.</a> Adv. Eng. Mater. 14, 67-75 (2012).</li><li>Wohlrab, S., Spiess, K., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Varying+surface+hydrophobicities+of+coatings+made+of+recombinant+spider+silk+proteins.">Varying surface hydrophobicities of coatings made of recombinant spider silk proteins.</a> J. Mater. Chem. 22, 22050-22054 (2012).</li><li>Hermanson, K. D., Huemmerich, D., Scheibel, T., Bausch, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+microcapsules+fabricated+from+reconstituted+spider+silk.">Engineered microcapsules fabricated from reconstituted spider silk.</a> Adv. Mater. 19, 1810-1815 (2007).</li><li>Spiess, K., Wohlrab, S., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+and+functionalization+of+engineered+spider+silk+films.">Structural characterization and functionalization of engineered spider silk films.</a> Soft Matter. 6, 4168-4174 (2010).</li><li>Slotta, U. K., Rammensee, S., Gorb, S., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineered+spider+silk+protein+forms+microspheres.">An engineered spider silk protein forms microspheres.</a> Angew. Chem.-Int. Edit. 47, 4592-4594 (2008).</li><li>Schacht, K., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+hydrogel+formation+of+a+recombinant+spider+silk+protein.">Controlled hydrogel formation of a recombinant spider silk protein.</a> Biomacromolecules. 12, 2488-2495 (2011).</li><li>Exler, J. H., Hummerich, D., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amphiphilic+properties+of+spider+silks+are+important+for+spinning.">The amphiphilic properties of spider silks are important for spinning.</a> Angew. Chem.-Int. Edit. 46, 3559-3562 (2007).</li><li>Sundaray, B., Subramanian, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+continuous+aligned+polymer+fibers.">Electrospinning of continuous aligned polymer fibers.</a> Appl. Phys. Lett. 84, 1222 (2004).</li><li>Van Hulle, S. W. H., Bjorge, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+assessment+of+electrospun+nanofibers+for+filter+applications.">Performance assessment of electrospun nanofibers for filter applications.</a> Desalination. 249, 942-948 (2009).</li><li>Zhou, S. B., Peng, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+a+novel+electrospun+spider+silk+fibroin/poly(D,L-lactide)+composite+fiber.">Preparation and characterization of a novel electrospun spider silk fibroin/poly(D,L-lactide) composite fiber.</a> J. Phys. Chem. B. 112, 11209-11216 (2008).</li><li>Stephens, J. S., Fahnestock, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+electrospinning+and+solution+casting+protocols+on+the+secondary+structure+of+a+genetically+engineered+dragline+spider+silk+analogue+investigated+via+fourier+transform+Raman+spectroscopy.">Effects of electrospinning and solution casting protocols on the secondary structure of a genetically engineered dragline spider silk analogue investigated via fourier transform Raman spectroscopy.</a> Biomacromolecules. 6, 1405-1413 (2005).</li><li>Greiner, A., Wendorff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+a+fascinating+method+for+the+preparation+of+ultrathin+fibers.">Electrospinning: a fascinating method for the preparation of ultrathin fibers.</a> Angew. Chem.-Int. Edit. 46, 5670-5703 (2007).</li><li>Smit, E., Buttner, U., Sanderson, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+yarns+from+electrospun+fibers.">Continuous yarns from electrospun fibers.</a> Polymer. 46, 2419-2442 (2005).</li><li>Teo, W. E., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+electrospinning+design+and+nanofibre+assemblies.">A review on electrospinning design and nanofibre assemblies.</a> Nanotechnology. 17, 89-106 (2006).</li><li>Greiner, A., Wendorff, J. H., Yarin, A. L., Zussman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biohybrid+nanosystems+with+polymer+nanofibers+and+nanotubes.">Biohybrid nanosystems with polymer nanofibers and nanotubes.</a> Appl. Microbiol. Biotechnol. 71, 387-393 (2006).</li><li>Spiess, K., Lammel, A., Scheibel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+spider+silk+proteins+for+applications+in+biomaterials.">Recombinant spider silk proteins for applications in biomaterials.</a> Macromolecular Biosciences. 10, 998-1007 (2010).</li><li>Hu, X., Kaplan, D., Cebe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+beta-sheet+crystallinity+in+fibrous+proteins+by+thermal+analysis+and+infrared-+spectroscopy.">Determining beta-sheet crystallinity in fibrous proteins by thermal analysis and infrared- spectroscopy.</a> Macromolecules. 39, 6161-6170 (2006).</li></ol>

We have nothing to disclose.

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.

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 &mu;m 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 &alpha;-helical secondary structure and the fibers are water soluble 6. In order to stabilize the silk fibers, &beta;-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.

air filter devices including nonwoven meshes of electrospun recombinant spider silk proteins

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 &alpha;-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 &beta;-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.

Electrospinning of recombinant spider silk solutions with concentrations of 10% w/v from HFIP resulted in smooth fibers with diameters ranging from 80 to 120 nm, allowing the formation of nonwoven meshes. Post-treatment with ethanol vapor did not lead to conspicuous morphological changes, which was, therefore, established as a proper way of silk nonwoven post-treatment (Figure 8). Structural changes were detected using FT-IR and subsequent FSD of amid I bands was performed to analyze single contribution peaks (Figure 6). It could be shown that post-treatment leads to an increase in &beta;-sheet structures, while the content of &alpha;-helical und random coil structures decreases (Figure 7). This result can be practically proven by dipping a post-treated nonwoven into water (Figure 5). Even after one week, no dissolution of the nonwoven mesh will occur.
The spinning duration is the most important parameter concerning the application of silk nonwovens in filter materials due to the pressure drop based on the increasing density of electrospun fibers. Extended spinning durations und thus a higher number of fiber layers result in an exponential decrease of air permeability. This effect could be detected for all different filter substrate materials before and after post-treatment (Figure 9). Likewise, the filtering efficiency of the silk-containing filter materials of sub-micrometer particles increases (Figure 10). While short spinning durations (30 sec) gain low filter efficiencies, higher spinning durations (90 sec) lead to higher efficiencies.
<img alt="Figure 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/50492/50492fig1highres.jpg" src="/files/ftp_upload/50492/50492fig1.jpg" /> 
 Figure 1. High electric voltage (0-30 kV) is applied to a syringe filled with a silk solution, and a counter electrode (0-20 kV) is placed in a distance of 8-20 cm. This setup leads to a strong electrostatic field, inducing 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. 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 in the form of a nonwoven mesh.
<img alt="Figure 2" src="/files/ftp_upload/50492/50492fig2.jpg" /> 
 Figure 2. Photographs of a regular Taylor cone (A), a dried droplet (B), and the setup without droplet (C).
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50492/50492fig3highres.jpg" src="/files/ftp_upload/50492/50492fig3.jpg" /> 
 Figure 3. Schematic procedure during vapor post-treatment. In the first step, the chamber is filled with ethanol, and the sample is steamed at 60 &deg;C for 90 min. In order to soften the nonwoven meshes for subsequent handling, ethanol is removed and the fibers are steamed with water vapor for 90 min at 60 &deg;C. <a href="https://www.jove.com/files/ftp_upload/50492/50492fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/50492/50492fig4.jpg" /> 
 Figure 4. Photograph of a cardboard frame with attached silk nonwoven meshes to be used for post-treatment.
<img alt="Figure 5" src="/files/ftp_upload/50492/50492fig5.jpg" /> 
 Figure 5. Electrospun and subsequently post-treated nonwoven in dry state (A) and under water (B).
<img alt="Figure 6" fo:content-width="6in" fo:src="/files/ftp_upload/50492/50492fig6highres.jpg" src="/files/ftp_upload/50492/50492fig6.jpg" /> 
 Figure 6. Fourier self-deconvoluted absorbance spectrum of an amide I band of an untreated (A) and a post-treated (B) spider silk nonwoven mesh. The solid line displays the absorbance band resulting from the single contribution peaks (dotted lines) as derived after deconvolution. The assignment of the respective curves was based on previously published values from the literature 22. <a href="https://www.jove.com/files/ftp_upload/50492/50492fig6large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/50492/50492fig7highres.jpg" src="/files/ftp_upload/50492/50492fig7.jpg" /> 
 Figure 7. Secondary structure content of non-treated and post-treated eADF4(C16) nonwoven meshes.
<img alt="Figure 8" fo:content-width="6in" fo:src="/files/ftp_upload/50492/50492fig8highres.jpg" src="/files/ftp_upload/50492/50492fig8.jpg" /> 
 Figure 8. SEM images of electrospun eADF4(C16)-fibers on different filter substrates: Polyamide (PA), Polyester (PE), Polypropylene (PP) and pure eADF4(C16) fibers before (S1) and after (S2) post-treatment with ethanol vapor. <a href="https://www.jove.com/files/ftp_upload/50492/50492fig8large.jpg" target="_blank">Click here to view larger figure</a>.
<img alt="Figure 9" fo:content-width="4.75in" fo:src="/files/ftp_upload/50492/50492fig9highres.jpg" src="/files/ftp_upload/50492/50492fig9.jpg" /> 
 Figure 9. Air permeability tests, before (A) and after post-treatment (B) of the silk nonwoven meshes with ethanol vapor, increasing spinning times lead to more nonwoven layers subsequently lowering the air permeability.
<img alt="Figure 10" fo:content-width="5in" fo:src="/files/ftp_upload/50492/50492fig10highres.jpg" src="/files/ftp_upload/50492/50492fig10.jpg" /> 
 Figure 10. Filter efficiency of di-ethyl-hexyl-sebacat aerosol on electrospun spider silk nonwoven meshes on polyamide filter materials at different spinning durations, influencing the silk layer quantity, after post-treatment with ethanol.

Watch this Scientific Journal Video about Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins at JoVE.com

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

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 ...

air-filter-devices-including-nonwoven-meshes-electrospun-recombinant

Research

JoVE Journal

Bioengineering

16.0K Views.  University of Bayreuth. סיבי משי עכביש להציג תכונות מכאניות יוצאות דופן. תכנון הנדסי Araneus diadematus fibroin 4 (eADF4) למשתלב ארוגים באמצעות electrospinning. הנה, את eADF4 משתלב ארוגים המשמשים כדי לשפר את הביצועים של מכשירי סינון אוויר.

Video: Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.