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. The geometry of the microfluidic channel was a simplified single-stage exponential function. For spider and silkworm, spinning dopes were draw down through a two-stage exponential regression spinning duct before fiber formation1,20. Here, the geometry of the microfluidic channel was designed by mimicking the second order exponential decay function of the silkworm spinning duct1. The width of the micro&#64258;uidic channel decreases from an initial width of 2,065 &#181;m to the terminal width of 265 &#181;m, and the length of the elongation channel is 21.5 mm. In the previous article, the diameter of the drawn RSF fiber was 2 &#181;m. Thus, a bundle of RSF threads had to be used for the mechanical test and structure characterization8.
The experiment shows that RSF concentration and relative humidity affect the diameter and microstructure of the RSF fibers in the dry spinning process. The RSF fiber spun at 40% RH shows a larger diameter and more crystalline structures than the fiber spun at 50% RH. However, the fiber spun at 50% RH has a higher crystalline orientation than that spun at 40% RH. The results might be related to evaporation rates of water at different humidities. A higher evaporation rate of water at 40% RH improves the intramolecular interactions and facilitates the fast phase transition of silk fibroin from sol-gel to solid silk fiber. A lower evaporation rate of water at 50% RH leads to a higher content of residue water in the solidified fiber. As a small molecular lubricant, the water facilitates the silk fibroin orientation and makes the partially solidified fiber to be stretched to finer fibers. This process helps us understand how water influences the formation of silk fibers during native spinning process.
The mechanical properties of post-treated RSF fibers are better than those of degummed silks4. After post-treatment, crystallinities of the fibers were drastically increased. The FWHM of the post-treated RSF fiber is smaller than that of as-spun fiber. It indicates that post-treatment improves the orientation of crystallites along the fiber axis. However, the complexity of the post-treatment process limits the mass production of RSF fibers with high strength.
Compared to a conventional spinneret, the microfluidic channel is well suited to mimic the geometry of a natural silk gland. Meanwhile, the microfluidic spinning was used to produce recombinant spider silk with outstanding mechanical properties9. Shearing and elongational sections were integrated in the microfluidic spinning chip to induce assembly and orientation of the protein molecules and fibrils. Therefore, the microfluidic spinning is promising in the production of high-performance animal silks, as well as other synthetic fibers from solution. However, the microfluidic spinning method can only produce single filaments and it cannot afford high production of artificial fibers.

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 crystalline orientation were calculated according to the WAXD data. For sample designation, we use C and R to present the concentration of RSF in the spinning dope and the relative humidity, respectively. For example, the fibers spun from 44 wt% RSF spinning dope at 40 &#177; 5% RH was designated as C44R40, which was post-drawn at a draw ratio of 4. Other fibers were renamed as C44R50, C47R40, and C47R50 according to the same description.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56271/56271fig1.jpg" /> 
Figure 1: Schematic of the fiber production and structure characterization. (a) Preparation of RSF solution, (b) microfluidic spinning process of RSF fibers, (c) synchrotron radiation experimental setup of RSF single fiber. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56271/56271fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56271/56271fig2.jpg" /> 
Figure 2: Stress-strain curves of post-treated RSF fibers. Insert shows SEM image of C44R40. Scale bar = 10 &#181;m. This figure has been modified from10. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56271/56271fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56271/56271fig3.jpg" /> 
Figure 3: SR-WAXD data of post-treated RSF fibers. (A) Two dimensional WAXD patterns of post-treated RSF single fibers: (a) C44R40, (b) C44R50, (c) C47R40, (d) C47R50, and (B) degummed B. mori silk; (C) One dimensional WAXD data of post-treated RSF fibers and degummed B. mori silk, which was performed at peak deconvolution in (D). This figure has been modified from reference10. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56271/56271fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

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

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

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&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Manufacturing of Three-dimensionally Microstructured Nanocomposites through Microfluidic Infiltration

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite in&#64257;ltration of 3D interconnected microfluidic networks. The manufacturing of the reinforced beams begins with the fabrication of microfluidic networks, which involves layer-by-layer deposition of fugitive ink filaments using a dispensing robot, filling the empty space between filaments using a low viscosity resin, curing the resin and finally removing the ink.&#160;Self-supported 3D structures with other geometries and many layers (e.g.&#160;a few hundreds layers) could be built using this method. The resulting tubular micro&#64258;uidic networks are then infiltrated with thermosetting nanocomposite suspensions containing nanofillers (e.g.&#160;single-walled carbon nanotubes), and subsequently cured. The infiltration is done by applying a pressure gradient between two ends of the empty network (either by applying a vacuum or vacuum-assisted microinjection). Prior to the infiltration, the nanocomposite suspensions are prepared by dispersing nanofillers into polymer matrices using ultrasonication and three-roll mixing methods. The nanocomposites (i.e.&#160;materials infiltrated) are then solidified under UV exposure/heat cure, resulting in a 3D-reinforced composite structure. The technique presented here enables the design of functional nanocomposite macroscopic products for microengineering applications such as actuators and sensors.

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.

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

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.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

Microfluidic-based Synthesis of Covalent Organic Frameworks (COFs): A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface

Covalent Organic Frameworks (COFs) are a class of porous covalent materials which are frequently synthesized as unprocessable crystalline powders. The first COF was reported in 2005 with much effort centered on the establishment of new synthetic routes for its preparation. To date, most available synthetic methods for COF synthesis are based on bulk mixing under solvothermal conditions. Therefore, there is increasing interest in developing systematic protocols for COF synthesis that provide for fine control over reaction conditions and improve COF processability on surfaces, which is essential for their use in practical applications. Herein, we present a novel microfluidic-based method for COF synthesis where the reaction between two constituent building blocks, 1,3,5-benzenetricarbaldehyde (BTCA) and 1,3,5-tris(4-aminophenyl)benzene (TAPB), takes place under controlled diffusion conditions and at room temperature. Using such an approach yields sponge-like, crystalline fibers of a COF material, hereafter called MF-COF. The mechanical properties of MF-COF and the dynamic nature of the approach allow the continuous production of MF-COF fibers and their direct printing onto surfaces. The general method opens new potential applications requiring advanced printing of 2D or 3D COF structures on flexible or rigid surfaces.

Microfluidic-based Synthesis of Covalent Organic Frameworks COFs: A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface

We present a novel microfluidic-based method for synthesis of covalent organic frameworks (COFs). We demonstrate how this approach can be used to produce continuous COF fibers, and also 2D or 3D COF structures on surfaces.

Covalent Organic Frameworks (COFs) are a class of porous covalent materials which are frequently synthesized as unprocessable crystalline powders. The ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...