Name: microfluidic dry-spinning and characterization of regenerated silk fibroin fibers
Uploaded: 2017-09-04T14:00:00.000+00:00
Duration: 8 min 28 s
Description: Watch this Scientific Journal Video about Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers at JoVE.com

This work is sponsored by the National Natural Science Foundation of China (21674018), the National Key Research and Development Program of China (2016YFA0201702 /2016YFA0201700), and the "Shuguang Program" supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (15SG30), DHU Distinguished Young Professor Program (A201302), the Fundamental Research Funds for the Central Universities, and the 111 Project (No.111-2-04).

CAUTION: Please consult all relevant material safety data sheets before use. Several of the chemicals used in preparing the molding are acutely toxic. Please use personal protective equipment (safety glasses, gloves, lab coat, full length pants, and closed-toe shoes).
1. Microfluidic Spinning of RSF Aqueous Solution
<ol>
	<li>Preparation of RSF aqueous spinning dope4,15,16
	<ol>
		<li>Degumming of silkworm cocoon
		<ol>
			<li>Degum the Bombyx mori cocoons twice in Na2CO3 aqueous solutions (0.5 wt% in water) at 100 &#176;C for 30 min each, and then wash the silk with deionized water to remove the sericin.</li>
		</ol>
		</li>
		<li>Dissolving of degummed silkworm cocoon
		<ol>
			<li>Dry the degummed cocoon silks in air; then dissolve the degummed cocoon silks in 9.0 M LiBr aqueous solution with a ratio of 1:10 (w/v) at 40 &#176;C for 2 h. For example, add 10 mL LiBr per 1 gram silk (Figure 1a).</li>
		</ol>
		</li>
		<li>Centrifuging and filtering
		<ol>
			<li>Dilute the RSF solution 1.5 times by deionized water. Centrifuge and filter to remove impurities. Centrifuge the RSF solution in 250 mL bottles at 4 &#176;C for 10 min at 1,234.8 x g. Filter the RSF solution using a 20 &#181;m filter and a vacuum pump. Double filter paper bed is preferable considering the experimentation effect.</li>
		</ol>
		</li>
		<li>Dialyzing
		<ol>
			<li>Dialyze the RSF solution in deionized water at 5 &#176;C for 3 days using a cellulose semipermeable membrane (MWCO: 14,000 &#177; 2,000). The total volume of RSF solution is about 1 L, loaded in 4 dialysis bags. Put these dialysis bags in the bucket, which is filled with 10 L reverse osmosis (RO) deionized water. 
			NOTE: The pH value of deionized water should be higher than 6, to avoid gelation during the concentration process. The pH of deionized water did not require adjustment for this protocol.</li>
		</ol>
		</li>
		<li>Concentrating
		<ol>
			<li>Condense the RSF aqueous solution to 20 wt% by forced airflow at 5 &#176;C. Add 3 M CaCl2 aqueous solution into the RSF solution to 1.0 mmol/g Ca2+ final concentration; then concentrate by forced air flow to 38-47 wt%.</li>
			<li>Weigh one drop of RSF solution on a glass slide and then dry it for 2 h in an oven at 105 &#176;C. 
			NOTE: The weight percent of the remaining solid compared to the weight of the drop before drying is the total concentration of protein and CaCl2. The RSF concentration is derived after deduction of the mass of CaCl2. At least four repeated measurements were performed. Our previous studies showed that the concentration of Ca2+ greatly affected the rheological properties and spinnability of the RSF aqueous solutions. Meanwhile, the addition of Ca2+ prompted the limited formation of &#946;-sheet and the aggregation of RSF17. In a native spinning dope, CaCl2 is considered to play an important role during the storage of spinning dope to avoid gelation before spinning.18</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of microfluidic chip8,19
	<ol>
		<li>Preparing photomask
		<ol>
			<li>Design the micro-channel in a CAD program. Print the CAD file to produce a high-resolution transparency19.</li>
		</ol>
		</li>
		<li>Preparing the mold
		<ol>
			<li>Cleaning the glass slide
			<ol>
				<li>In a chemical hood, boil the glass slide in the mixed solution of concentrated sulfuric acid and 30 vol% hydrogen peroxide solution (10:1) for 20 min on a hot plate. 
				CAUTION: Sulfuric acid and hydrogen peroxide vapors are extremely toxic.</li>
			</ol>
			</li>
			<li>Washing the glass slide
			<ol>
				<li>Wash the glass slide using deionized water, and blow dry with high purity nitrogen.</li>
			</ol>
			</li>
			<li>Coating film
			<ol>
				<li>Coat the SU-8 photoresist on the glass slide by a custom-built coating device with a gap of 100 &#181;m between the bottom surface of the coating bar and the upper surface of glass.</li>
			</ol>
			</li>
			<li>Spin coating
			<ol>
				<li>Spread the photoresist on the glass slide to form a uniform film using a spin coater at 40.3 x g for 30 s. The thickness of the uniform film is about 85 &#181;m.</li>
			</ol>
			</li>
			<li>Solidification
			<ol>
				<li>Solidify the photoresist in an oven with a temperature controlling program. Elevate the temperature from room temperature to 65 &#176;C at 2 &#176;C/min and hold at 65 &#176;C for 2 min. Continue to heat up from 65 &#176;C to 95&#176;C and hold at 95 &#176;C for 15 min. Turn off the oven and cool down naturally to room temperature in the oven.</li>
			</ol>
			</li>
			<li>Ultraviolet light exposure
			<ol>
				<li>Expose the side of glass slide with the photoresist to ultraviolet light for 12 s using the transparency as a photomask by photolithography19. 
				NOTE: The wavenumber of the ultraviolet light is 365 nm and the exposure energy is 273.6 mJ/cm2. 
				CAUTION: Take proper safety measure while working with UV light and the oven.</li>
			</ol>
			</li>
			<li>Solidify the photoresist as described in step 1.2.2.5.</li>
			<li>Developing
			<ol>
				<li>Clean the photoresist ultrasonically in developer solution for 30 s. Wash the glass slide by isopropanol and developer, alternating between the two, until there is no precipitation on the glass slide.</li>
			</ol>
			</li>
			<li>Solidify the photoresist in an oven with a temperature controlling program. Elevate the temperature from room temperature to 170 &#176;C at 2 &#176;C/min and hold at 170 &#176;C for 30 min. Turn off the oven and cool down naturally to room temperature in the oven.</li>
		</ol>
		</li>
		<li>Soft lithography
		<ol>
			<li>Pour 8.8 g liquid polydimethylsiloxane (PDMS) pre-polymer over the mold and cure for 30 min at 65 &#176;C, and 15 min at 80 &#176;C. Liquid PDMS pre-polymer consists of PDMS and curing agent (typically at 10:1 (w/w)).</li>
		</ol>
		</li>
		<li>Punching
		<ol>
			<li>Punch a hole through the PDMS replica at the beginning of the channel by drill. The diameter of the drill is 1.2 mm.</li>
		</ol>
		</li>
		<li>Sealing
		<ol>
			<li>Seal the PDMS replica with the channel to a flat PDMS layer without pattern by an oxygen plasma treatment on the surfaces of the two PDMS layers. 
			NOTE: The overall preparation process of the chip takes about 72 h.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fabrication of RSF fiber
	<ol>
		<li>Injection of spinning dope
		<ol>
			<li>Inject the RSF spinning dope into the microchannel at 2 &#181;L/min by a syringe pump.</li>
		</ol>
		</li>
		<li>Environment regulation
		<ol>
			<li>Adjust the relative humidity to 40 &#177; 5 RH% or 50 &#177; 5 RH% using a humidifier. At 40 &#177; 5 RH%, as-spun fiber solidified more quickly than 50 &#177; 5 RH%.</li>
		</ol>
		</li>
		<li>Production of RSF fiber
		<ol>
			<li>Touch the RSF drop by a pinpoint at the outlet of the microfluidic channel, draw RSF fiber into air, and then reel it onto a roller through a 10-cm air gap at a speed of 3 cm/s (Figure 1b).</li>
		</ol>
		</li>
		<li>Store the RSF fibers in a sealed desiccator for 24 h.</li>
		<li>Fiber post-treatment
		<ol>
			<li>Draw the as-spun fibers 4 times at 0.9 mm s-1 in 80 vol% ethanol solution by a custom-built machine, and then keep the drawn fiber fixed and immerse the fibers in the solution for 1 h. Because of this treatment, the length of the fiber was changed from originally 15 mm to 60 mm.</li>
		</ol>
		</li>
		<li>Preparing the sample for characterization
		<ol>
			<li>Fix the post-treated fibers on a paper frame with a 10-mm gauge length. At least 20 fibers are need for measurement, including in the tensile test, SEM, FTIR, and WAXS. The diameters of the post-drawn fibers range from 5 to 10 &#181;m. Figure 1 shows the schematic of the fiber production and WAXD characterization. The mechanical properties of the RSF fibers were examined by a material testing system at (25 &#177; 2) &#176;C and (45 &#177; 5)% relative humidity. The extension rate and gauge length were 2 mm/min and 1 cm, respectively.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Synchrotron Radiation Characterization of Crystalline Structure of RSF Fiber
<ol>
	<li>Synchrotron radiation characterization4,13,17
	<ol>
		<li>Adjustment of beamline
		<ol>
			<li>Adjust the wavelength of the X-rays and spot size to 0.07746 nm and 3 x 2 &#181;m2, respectively. 
			NOTE: The protocol is performed using the BL15U1 beamline at the Shanghai Synchrotron Radiation Facility.</li>
		</ol>
		</li>
		<li>Location of the X-ray spot
		<ol>
			<li>Find the location of the X-ray spot. 
			NOTE: The location of the X-ray spot is adjusted by the laboratory technician at the Synchrotron Radiation Facility.</li>
		</ol>
		</li>
		<li>Testing of standard sample: Cerium dioxide (CeO2)
		<ol>
			<li>Test a standard sample CeO2 powder. The CeO2 powder was characterized to calculate the circle center and the distance from the sample to the detector.</li>
		</ol>
		</li>
		<li>Sample preparation
		<ol>
			<li>Fix the RSF filaments in parallel with each other on the paper frame with a 10 mm gauge length. Glue the paper frame on the testing stage. 
			NOTE: Keep the fiber horizontal.</li>
		</ol>
		</li>
		<li>SR-WAXD testing
		<ol>
			<li>Open the shutter
			<ol>
				<li>Close the door of the beamline station after ensuring that there are no persons in the room. Open the shutter of X-ray beam source.</li>
			</ol>
			</li>
			<li>Focusing
			<ol>
				<li>Move the fiber slightly until in focus. Adjust the location of fiber along the x, y, z direction remotely via a software (Figure 1c).</li>
			</ol>
			</li>
			<li>Sample exposure
			<ol>
				<li>Move the fiber up and down remotely via a software, until it is on the X-ray spot. Press the start button on the software to expose the fiber to the X-ray beamline for 20 s (Figure 1c).</li>
			</ol>
			</li>
			<li>Background diffraction
			<ol>
				<li>Test the diffraction of the air background with the exposure time of 20 s. Move the fiber away from the X-ray spot remotely via a software. Press the &#34;start&#34; button on the software to expose the X-ray beamline to &#34;air medium&#34; for 20 s.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>SR-WAXD data processing13
	<ol>
		<li>Software calibration
		<ol>
			<li>Process the WAXD data using FIT2D (V12.077). Calibrate the circle center and the distance of sample-to-detector using the diffraction data of the CeO2 powder.</li>
		</ol>
		</li>
		<li>2-D diffraction pattern
		<ol>
			<li>Subtract the air background from the fiber diffraction pattern using FIT2D (V12.077).</li>
		</ol>
		</li>
		<li>Crystallinity and crystallite size
		<ol>
			<li>Integrate the diffraction intensity as a function of scattering angle 2&#952;. Perform deconvolution of the intensity integration by automated peak separation software (Version 4.12)12. The degree of crystallinity, xc, was estimated from the relation: 
			<img alt="Equation" src="/files/ftp_upload/56271/56271eq1.jpg" /> 
			, where Ic is the sum of the integrated intensities of the crystalline peaks and Ia is the integrated intensity of the amorphous halo4,11. The integration of the WAXD patterns showed the peak width of the [200], [020], [002] reflections. The FWHM of these peaks were used to determine the crystallite size along the a, b, and c axes by using Scherrer&#39;s formula4.</li>
		</ol>
		</li>
		<li>Determination of the crystallite orientations
		<ol>
			<li>Calculate the orientations of the crystallite and amorphous according to azimuthal integrations of (020) and (210) peaks11. The crystalline orientation can be calculated quantitatively according to Herman&#39;s orientation. Here, both (020) and (210) peaks were fitted with two Gaussian functions. The narrower one is for crystalline orientation and the other broader one is for the oriented amorphous material13. Here, the crystallite orientations of RSF fibers were compared using the full width at half maximum (FWHM) of azimuthal integrations (002) peaks.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

During the dialysis of the RSF solution, the pH value is critical for the following concentration process. If the pH value of the deionized water is smaller than 6, the RSF solution will be easier to gel during the concentration process. To avoid gelation, CaCl2 is added to the RSF solution. The concentration of CaCl2 is 1 mmol per weight of RSF.
Our previous work demonstrated the possibility of microfluidic dry-spinning of an RSF aqueous solution8...

Spider and silkworm can produce outstanding silk fiber from aqueous protein solution at ambient temperature and pressure. Shearing and extensional flow can induce the formation of liquid crystal texture in the silk gland1. In recent years, there has been a great interest in mimicking the spinning process of the spider in order to produce high strength artificial fibers. However, large quantities of spider silk protein cannot be produced efficiently and economically by farming spiders due to cannibalism. Substantial amounts of silkworm silk can be obtained easily by farming. Otherwise, the silkworm and spider have a similar spinning process and amino acid composition. Therefore, silkworm silk fibroin is selected as a substitute to spin artificial animal silk by many researchers.
Spider and silkworm extrude protein solution through their spinning duct into fiber in air. The high stress forces generated along the spinning duct most likely stretch the silk fibroin molecules to a more extended conformation2. Artificial silk fibers have been spun using conventional wet spinning and dry-spinning processes3,4, which do not take into account the fluid forces generated in the spinning duct.
First, microfluidic approaches were used to investigate the assembly of silk protein5,6. Then, microfluidic fabrication of RSF was studied via modeling the shearing and extensional forces7,8. Young&#39;s modulus and diameter of RSF fibers can be tuned by microfluidic wet spinning, but the tensile strength of drawn fiber was less than 100 MPa7. Finally, high strength RSF fibers were successfully prepared using the microfluidic dry-spinning method, but the diameter of the fiber is only 2 &#181;m8. Recently, microfluidic wet spinning was successfully used in the production of high strength recombinant spider silk fiber. The post-spinning drawing in air improved the surface and internal defects of artificial fiber9.
In this study, the improved microfluidic spinning process for RSF fiber is introduced. It aims to mimic the spinning process of silkworm silk, including the spinning dope, shearing forces, and dry-spinning process. This spinning method not only can produce high strength artificial silk fiber, but also can adjust the diameter of the fiber. Firstly, the RSF spinning dope was sheared and elongated in a biomimic channel with a second order exponential decay. Secondly, the influences of relative humidity (RH) on the fiber morphology and properties were studied in the microfluidic dry-spinning process10. Compared to the conventional spinning spinneret, our microfluidic system is highly biomimetic and can be used to produce high strength fiber from solutions at ambient temperature by the dry or wet spinning method.
Due to the high-resolution, high-brightness, and high-energy of the synchrotron radiation microfocus X-ray, it can be used to characterize the microstructure of a single fiber with a diameter of several micrometers4,11,12,13,14. Here, SR-WAXD technique was used to calculate the crystallinity, crystallite size and crystalline orientation of RSF fibers.

<table><tbody><tr><td>&lt;em&gt;B. mori&lt;/em&gt; Cocoons</td><td>Farmer in Tongxiang, Zhejiang Province, China</td><td></td><td></td></tr><tr><td>Sodium carbonate, anhydrous, 99.8%</td><td>Shanghai Lingfeng Chemical Reagent Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Lithium bromide, 99.1%</td><td>Shanghai China Lithium Industrial Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Calcium chloride, anhydrous, 96.0%</td><td>Shanghai Lingfeng Chemical Reagent Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Ethanol, anhydrous, 99.7%</td><td>Sinopharm Group Chemical Reagent Co.,Ltd., China</td><td>10009218</td><td>Analytically Pure</td></tr><tr><td>SU-8 photoresist</td><td>MicroChem Corp., USA</td><td></td><td></td></tr><tr><td>Developing solution</td><td>MicroChem Corp., USA</td><td></td><td></td></tr><tr><td>Sylgard 184</td><td>Dow Corning, USA</td><td></td><td></td></tr><tr><td>Isopropanol</td><td>Shanghai Lingfeng Chemical Reagent Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Concentrated sulfuric acid</td><td>Pinghu Chemical Reagent Factory, China</td><td></td><td>Analytically Pure</td></tr><tr><td>30 vol% hydrogen peroxide</td><td>Shanghai Jinlu Chemical reagent Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Acetone</td><td>Shanghai Zhengxing Chemical Reagent Factory, China</td><td></td><td>Analytically Pure</td></tr><tr><td>Oxygen plasma treatment</td><td>DT-01, Suzhou Omega Machinery Electronic Technology Co., Ltd., China</td><td></td><td></td></tr><tr><td>Syringe pump</td><td>&amp;nbsp;KD Scientific, USA</td><td></td><td>KDS 200P</td></tr><tr><td>Humidifier</td><td>SEN electric</td><td></td><td></td></tr><tr><td>Driller</td><td>Hangzhou Bo Yang Machinery Co., Ltd., China</td><td></td><td>bench drilling machine Z406c</td></tr><tr><td>Material testing system</td><td>Instron, USA</td><td></td><td>Model: 5565</td></tr><tr><td>PeakFit</td><td>Systat Software, Inc., USA</td><td></td><td>Version 4.12</td></tr></tbody></table>

microfluidic dry-spinning and characterization of regenerated silk fibroin fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

High strength RSF fibers were successfully produced by using the microfluidic spinning method. The stress-strain curves and SEM images of the stretched RSF fibers C44R40 are shown in Figure 2. At least 10 fibers were measured in the tensile test. Stress-strain curves were chosen according to the average value of the breaking stress and strain of fibers. The WAXD data of the fibers are shown in Figure 3. The crystallinity and crys...

Watch this Scientific Journal Video about Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers at JoVE.com

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

microfluidic-dry-spinning-characterization-regenerated-silk-fibroin

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

9.7K Views.  Donghua University. A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented. 

Video: Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Bioengineering

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 ...

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.

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 ...

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, ...

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures.

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...

Biology

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

Neuroscience

Formation and Attachment of FN-Silk Membranes to Inserts of Cell Culture 

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

Author Spotlight: Enhancing In Vitro Cell Culture Models with Recombinant Functionalized Spider Silk Membranes

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...

Electrospinning of Silk Biomaterials

Silk fibers have been processed and used to create fabrics and threads for centuries. However, the solubilizing of silk fibers, thereby turning it into a versatile pre-polymer solution is a much newer technology. Solubilized silk can be processed in many different ways to create a biocompatible material with controllable mechanical properties. 
This video introduces the processing of silk from silk worm cocoons, and shows how the silk solution can be used to create a fiber mat via electrospinning. Several applications of this technique, such as its use as a structural material in engineered tissue scaffolds, are then introduced.

Silk Fibrin Extraction from Bombyx mori and Electrospinning

Silk fibers have been processed and used to create fabrics and threads for centuries. However, the solubilizing of silk fibers, thereby turning it into a ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

Summary

Explore More Videos

Chapters in this video

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Synthetic Spider Silk Production on a Laboratory Scale

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

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Procedure for Fabricating Biofunctional Nanofibers

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Electrospinning of Silk Biomaterials