Name: microfluidic dry-spinning and characterization of regenerated silk fibroin fibers
Uploaded: 2017-09-04T14:00:00
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>
		<...

<ol>
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P., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+Fibers+Extruded+Artificially+from+Aqueous+Solutions+of+Regenerated+Bombyx+mori+Silk+Fibroin+are+Tougher+than+their+Natural+Counterparts.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure-property+relationships+of+artificial+silk+fabricated+by+dry-spinning+process.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+Fiber+Assembly+Studied+by+Synchrotron+Radiation+SAXS/WAXS+and+Raman+Spectroscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+mechanism+of+recombinant+spider+silk+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+silk:+using+microfluidics+to+fabricate+silk+fibers+with+controllable+properties.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tough+silk+fibers+prepared+in+air+using+a+biomimetic+microfluidic+chip.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+spider+silk+from+aqueous+solutions+via+a+bio-inspired+microfluidic+chip.">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.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+humidity+on+the+structures+and+properties+of+regenerated+silk+fibers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+diffraction+study+of+nanocrystalline+and+amorphous+structure+within+major+and+minor+ampullate+dragline+spider+silks.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+Behavior+of+Bombyx+mori+silk:+Evolution+of+crystalline+parameters,+molecular+structure,+and+mechanical+properties.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoconfined+crystallites+toughen+artificial+silk.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructural+evolution+of+regenerated+silk+fibroin/graphene+oxide+hybrid+fibers+under+tensile+deformation.">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.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+capillary+dry+spinning+of+regenerated+silk+fibroin+aqueous+solution.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Ca2++on+structures+and+properties+of+regenerated+silk+fibroin+aqueous+solutions+and+fibres.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures,+mechanical+properties+and+applications+of+silk+fibroin+materials.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(dimethylsiloxane)+as+a+material+for+fabricating+microfluidic+devices.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+crystals+and+flow+elongation+in+a+spider's+silk+production+line.">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>

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>B. mori 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>&#160;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 ...

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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