Name: Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers (Video) | JoVE Journal
Uploaded: 2017-09-04T14:00:00.000+00:00
Duration: 8 min 28 s
Description: Learn microfluidic dry-spinning of regenerated silk fibroin fibers to study humidity effects on structure and tensile properties. This biomimetic method mimics natural silkworm spinning for advanced biomaterials research.

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5 minutes

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 °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 °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 °C for 10 min at 1,234.8 x g. Filter the RSF solution using a 20 µm 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 °C for 3 days using a cellulose semipermeable membrane (MWCO: 14,000 ± 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 °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 °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 β-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 µm 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 µm.</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 °C at 2 °C/min and hold at 65 °C for 2 min. Continue to heat up from 65 °C to 95°C and hold at 95 °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 °C at 2 °C/min and hold at 170 °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 °C, and 15 min at 80 °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 µL/min by a syringe pump.</li>
		</ol>
		</li>
		<li>Environment regulation
		<ol>
			<li>Adjust the relative humidity to 40 ± 5 RH% or 50 ± 5 RH% using a humidifier. At 40 ± 5 RH%, as-spun fiber solidified more quickly than 50 ± 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 µm. 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 ± 2) °C and (45 ± 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 µm2, 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 "start" button on the software to expose the X-ray beamline to "air medium" 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 2θ. 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="Chirality equation \(X_c = \frac{I_c}{I_c + I_a}\), analytical chemistry formula." src="/files/ftp_upload/56271/56271eq1.jpg" data-altupdatedat="1788250015000" data-alt="Equation"> 
			, 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'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'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>

<ol><li>Asakura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Some+observations+on+the+structure+and+function+of+the+spinning+apparatus+in+the+silkworm+Bombyx+mori%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">Some observations on the structure and function of the spinning apparatus in the silkworm Bombyx mori.</a> Biomacromolecules. 8 (1), 175-181 (2007).</li>
<li>Vollrath, F., Knight, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liquid+crystalline+spinning+of+spider+silk%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Liquid crystalline spinning of spider silk.</a> Nature. 410 (6828), 541-548 (2001).</li>
<li>Zhou, G. Q., Shao, Z. Z., Knight, D. P., Yan, J. P., Chen, X. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/adma.200800582/abstract">Silk Fibers Extruded Artificially from Aqueous Solutions of Regenerated Bombyx mori Silk Fibroin are Tougher than their Natural Counterparts.</a> Adv Mater. 21 (3), 366-370 (2009).</li>
<li>Sun, M. J., Zhang, Y. P., Zhao, Y. M., Shao, H. L., Hu, X. C. <a target="_blank" href="http://pubs.rsc.org/en/content/articlelanding/2012/jm/c2jm32576d">The structure-property relationships of artificial silk fabricated by dry-spinning process.</a> J Mater Chem. 22 (35), 18372-18379 (2012).</li>
<li>Martel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Silk+Fiber+Assembly+Studied+by+Synchrotron+Radiation+SAXS%2FWAXS+and+Raman+Spectroscopy%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Silk Fiber Assembly Studied by Synchrotron Radiation SAXS/WAXS and Raman Spectroscopy.</a> J Am Chem Soc. 130 (50), 17070-17074 (2008).</li>
<li>Rammensee, S., Slotta, U., Scheibel, T., Bausch, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assembly+mechanism+of+recombinant+spider+silk+proteins%5BTitle%5D)+AND+%22P+Natl+Acad+Sci+USA%22%5BJournal%5D)">Assembly mechanism of recombinant spider silk proteins.</a> P Natl Acad Sci USA. 105 (18), 6590-6595 (2008).</li>
<li>Kinahan, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tunable+silk%3A+using+microfluidics+to+fabricate+silk+fibers+with+controllable+properties%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">Tunable silk: using microfluidics to fabricate silk fibers with controllable properties.</a> Biomacromolecules. 12 (5), 1504-1511 (2011).</li>
<li>Luo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tough+silk+fibers+prepared+in+air+using+a+biomimetic+microfluidic+chip%5BTitle%5D)+AND+%22Int+J+Biol+Macromol%22%5BJournal%5D)">Tough silk fibers prepared in air using a biomimetic microfluidic chip.</a> Int J Biol Macromol. 66, 319-324 (2014).</li>
<li>Peng, Q. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombinant+spider+silk+from+aqueous+solutions+via+a+bio-inspired+microfluidic+chip%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Recombinant spider silk from aqueous solutions via a bio-inspired microfluidic chip.</a> Sci Rep. 6, (2016).</li>
<li>Peng, Q. F., Shao, H. L., Hu, X. C., Zhang, Y. P. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S100200711500091X">Role of humidity on the structures and properties of regenerated silk fibers.</a> Prog Nat Sci-Matter. 25 (5), 430-436 (2015).</li>
<li>Sampath, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+diffraction+study+of+nanocrystalline+and+amorphous+structure+within+major+and+minor+ampullate+dragline+spider+silks%5BTitle%5D)+AND+%22Soft+Matter%22%5BJournal%5D)">X-ray diffraction study of nanocrystalline and amorphous structure within major and minor ampullate dragline spider silks.</a> Soft Matter. 8 (25), 6713-6722 (2012).</li>
<li>Martel, A., Burghammer, M., Davies, R. J., Riekel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermal+Behavior+of+Bombyx+mori+silk%3A+Evolution+of+crystalline+parameters%2C+molecular+structure%2C+and+mechanical+properties%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">Thermal Behavior of Bombyx mori silk: Evolution of crystalline parameters, molecular structure, and mechanical properties.</a> Biomacromolecules. 8 (11), 3548-3556 (2007).</li>
<li>Pan, H., et al. <a target="_blank" href="http://pubs.rsc.org/en/Content/ArticleLanding/2014/TB/c3tb21148g">Nanoconfined crystallites toughen artificial silk.</a> J Matter Chem B. 2 (10), 1408-1414 (2014).</li>
<li>Zhang, C., et al. <a target="_blank" href="http://pubs.rsc.org/is/content/articlepdf/2012/ra/c6ra22544f">Microstructural evolution of regenerated silk fibroin/graphene oxide hybrid fibers under tensile deformation.</a> Rsc Adv. 7 (6), 3108-3116 (2017).</li>
<li>Wei, W., et al. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0928493111001950">Bio-inspired capillary dry spinning of regenerated silk fibroin aqueous solution.</a> Mat Sci Eng C-Mater. 31 (7), 1602-1608 (2011).</li>
<li>Jin, Y., Zhang, Y. P., Hang, Y. C., Shao, H. L., Hu, X. C. <a target="_blank" href="https://www.researchgate.net/publication/259437154_A_simple_process_for_dry_spinning_of_regenerated_silk_fibroin_aqueous_solution">A simple process for dry spinning of regenerated silk fibroin aqueous solution.</a> J Mater Res. 28 (20), 2897-2902 (2013).</li>
<li>Jin, Y., Hang, Y. C., Zhang, Y. P., Shao, H. L., Hu, X. C. <a target="_blank" href="http://www.tandfonline.com/doi/abs/10.1179/1432891714Z.000000000397">Role of Ca2+ on structures and properties of regenerated silk fibroin aqueous solutions and fibres.</a> Mater Res Innov. 18, 113-116 (2014).</li>
<li>Koh, L. D., et al. <a target="_blank" href="https://www.researchgate.net/profile/Enyi_YE/publication/272199400_Structures_Mechanical_Properties_and_Applications_of_Silk_Fibroin_Materials/links/569c445708ae6169e5627d01.pdf">Structures, mechanical properties and applications of silk fibroin materials.</a> Prog Polym Sci. 46, 86-110 (2015).</li>
<li>McDonald, J. C., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Poly%28dimethylsiloxane%29+as+a+material+for+fabricating+microfluidic+devices%5BTitle%5D)+AND+%22Accounts+Chem+Res%22%5BJournal%5D)">Poly(dimethylsiloxane) as a material for fabricating microfluidic devices.</a> Accounts Chem Res. 35 (7), 491-499 (2002).</li>
<li>Knight, D. P., Vollrath, F. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1689793/">Liquid crystals and flow elongation in a spider's silk production line.</a> P Roy Soc B-Biol Sci. 266 (1418), 519-523 (1999).</li>
</ol>

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

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...

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 ...

The overall goal of this experiment is to observe the effect of relative humidity on structure and tensile properties of regenerated silk fibroin fibers using a microfluidic dry-spinning method. This method can help answer key questions in the biomimetic spinning, such as shear forces and the dry spinning process. The main advantage of this technique is that the microfluidic channel is designed to mimic the spinning duct of a silk worm.This method can provide insight into the dry spinning process of silk fibroin. This can also be used to the wet spinning process of recombining spider silk. Generally, new individuals new to this method will struggle.Because, it needs two weeks to prepare the spinning dope and also to insure it has good spinning ability. Visual demonstration of this method is very critical. Because, if you are lack of experience, the spinning dope is very difficult to make, and the spinning time is very essential for you to make.Start by degumming the Bombyx mori cocoons in an aqueous solution containing 0.5%sodium carbonate. Incubate the submerged cocoons at 100 degrees Celsius for 30 minutes. Then, replace the sodium carbonate solution and repeat the incubation a second time.When finished, wash the silk with deionized water to remove the sericin. Dry the degummed cocoon silks in air, and then submerge them in 9.0 molar lithium bromide at a weight to weight ratio of 1:10. Incubate the silks at 40 degrees Celsius for two hours until the silks are completely dissolved.Next, dilute the regenerated silk fibroin solution 1.5 times in 250 milliliter bottles using deionized water. Centrifuge the solution, and then use a vacuum pump to filter it through a double filter paper bed, with a 20 micron mesh size to remove any impurities. Place 250 milliliters of the filtered regenerated silk fibroin solution into cellulose semi-permeable membrane dialysis bags, and dialyze each bag in 10 liters of reverse osmosis deionized water at five degrees Celsius for three days.Following dialysis, use forced air flow to condense the solution at five degrees Celsius down to 20%of the original weight. Next, add a three molar aqueous solution of calcium chloride to the condensed solution to achieve a final concentration of 1.0 millimoles per gram of calcium ions. Again, concentrate the regenerated silk fibroin solution by forced air flow to 44%of its starting weight.Begin by boiling a glass slide for 20 minutes in a mixed solution of concentrated sulfuric acid and 30%hydrogen peroxide solution in a 10:1 mixture. When finished, wash the glass slide using deionized water, and then blow dry it using high-purity nitrogen. Next, place the glass slide in a custom-built coating device with a gap of 100 micrometers between the bottom surface of the coating bar and the upper surface of the glass.Fill the gap with SU-8 photoresist and then spin coat the photoresist at 40.3 times G for 30 seconds to form a uniform film about 85 micrometers thick. Next, place the slide with the photoresist in an oven with a temperature-controlling program. Elevate the temperature from room temperature to 65 degrees Celsius at a rate of two degrees Celsius per minute, and hold the temperature at 65 degrees Celsius for two minutes.Then, continue to heat the slide from 65 degrees Celsius to 95 degrees Celsius at a rate of two degrees Celsius per minute, and hold the temperature at 95 degrees Celsius for 15 minutes. At this point, turn off the oven and let the slide cool down naturally to room temperature in the oven. Next, place the slide with the photoresist under a UV light and align a photomask containing the proper microchannel above the slide.Expose the side of the slide to UV light for 12 seconds. Following exposure, place the slide back into the oven, and heat as before. After baking, clean the photoresist ultrasonically in developer solution for 30 seconds.Then, use isopropanol and developer alternately to wash the glass slide until there is no precipitation on the glass slide. Solidify the photoresist in an oven with a temperature-controlling program. Elevate the temperature from room temperature to 170 degrees Celsius at two degrees Celsius per minute, and hold at 170 degrees Celsius for 30 minutes, and then turn off the oven and cool down naturally to room temperature in the oven.Next, place the cured SU-8 mold into an aluminum box, and pour 8.8 grams of PDMS pre-polymer over the mold to a height of about five millimeters. Cure the PDMS for 30 minutes at 65 degrees Celsius and 15 minutes at 80 degrees Celsius. Once cured, use a 1.2 millimeter diameter drill bit to drill a hole though the PDMS replica at the beginning of the channel.Then, place the PDMS channel side up along with a second flat PDMS layer into a plasma coater, and treat with oxygen plasma. Seal the PDMS layer with the channel to a flat PDMS layer immediately after removing them from the plasma chamber. The microchannel is now ready to use.Inject the regenerated silk fibroin spinning dope into the microchannel at two microliters per minute using a syringe pump. Adjust the relative humidity in the electrospinning area to 40 or 50%relative humidity using a humidifier. Then, turn on a roller so that the surface is moving at three centimeters per second.Once the humidity has stabilized, touch the regenerated silk fibroin drop and draw a silk fiber into the air. Then, place it onto the roller through the 10 centimeter air gap. The roller will draw the remaining silk fibers.When finished, store the fibers in a sealed desiccator for 24 hours. The next day, post-treat the fibers by drawing them out four times at 0.9 millimeters per second in 80%ethanol using a custom-built machine. Keep the drawn fiber fixed, and immerse them in 80%ethanol solution for one hour.Finally, fix the post-treated fibers on a paper frame with a 10 millimeter gauge length. Shown here is an SEM fiber of the final stretched regenerated silk fibroin fiber. These fibers had an average diameter of nine microns.When measured under tension, the fibers formed under the condition of 40%relative humidity had a slightly higher ultimate tensile strength and maximum strain, but had similar Young's modulus compared with the fibers formed under 50%relative humidity. After watching this video, you should understand how to dry spin a silk fiber from an aqueous silk solution with a bio-inspired microfluidic chip.

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Research

JoVE Journal

Chemistry

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 Film Culture System for in vitro Analysis and Biomaterial Design

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.

Synthetic Spider Silk Production on a Laboratory Scale

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.

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

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

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.

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

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.

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

Being comprehensively utilized, sum frequency generation (SFG) vibrational spectroscopy can help to reveal chain conformational order and secondary structural change happening at polymer and biomacromolecule interfaces.

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Three-dimensional (3D) microstructured composite beams are fabricated through the directed and localized infiltration of nanocomposites into 3D porous microfluidic networks. The flexibility of this manufacturing method enables the utilization of different thermosetting materials and nanofillers in order to achieve a variety of functional 3D reinforced nanocomposite macroscopic products.

Manufacturing of Three-dimensionally Microstructured Nanocomposites through Microfluidic Infiltration

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Learn microfluidic dry-spinning of regenerated silk fibroin fibers to study humidity effects on structure and tensile properties. This biomimetic method mimics natural silkworm spinning for advanced biomaterials research.

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

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