Tensan Silk-Inspired Hierarchical Fibers forSmart Textile ApplicationsWenwen Zhang,†,‡,# Chao Ye,‡,# Ke Zheng,‡ Jiajia Zhong,§ Yuzhao Tang,§ Yimin Fan,*,†

Markus J. Buehler,*,⊥ Shengjie Ling,*,‡,⊥ and David L. Kaplan*,¶

†Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Jiangsu Key Lab of Biomass-Based GreenFuel & Chemicals, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China‡School of Physical Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, China§Shanghai Advanced Research Institute (Zhangjiang Lab), Chinese Academy of Sciences, Shanghai, 201210, China⊥Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,United States¶Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States

*S Supporting Information

ABSTRACT: Tensan silk, a natural fiber produced by the Japanese oak silkmoth (Antherea yamamai, abbreviated to A. yamamai), features superiorcharacteristics, such as compressive elasticity and chemical resistance, whencompared to the more common silk produced from the domesticated silkworm,Bombyx mori (B. mori). In this study, the “structure-property” relationshipswithin A. yamamai silk are disclosed from the different structural hierarchies,confirming the outstanding toughness as dominated by the distinct mesoscalefibrillar architectures. Inspired by this hierarchical construction, we fabricatedA. yamamai silk-like regenerated B. mori silk fibers (RBSFs) with mechanicalproperties (extensibility and modulus) comparable to natural A. yamamai silk.These RBSFs were further functionalized to form conductive RBSFs that weresensitive to force and temperature stimuli for applications in smart textiles.This study provides a blueprint in exploiting rational designs from A.yamanmai, which is rare and expensive in comparison to the common and cost-effective B. mori silk to empower enhanced material properties.KEYWORDS: silk fiber, hierarchical structure, biomimetic spinning, fiber sensor, smart textile

Animal silks have received considerable attention due totheir outstanding portfolio of mechanical properties,combining high modulus, strength, and extensibility.1,2

Many studies have pursued an understanding of the interplaybetween the structure and resulting mechanical properties ofsilks, with a goal toward the transfer of these insights intoartificial material designs.3,4 For example, a series of spinningtechniques (e.g., wet spinning,5 dry spinning,6 microfluidicspinning,7 and biomimetic spinning8) have been pursed to spinregenerated fibers with silk-like structures. However, asignificant challenge remains to replicate the mechanicalproperties of animal silks within artificial materials. Inparticular, there are no regenerated silk fibers that are able tocombine high strength, modulus and toughness in fiber formlike the properties displayed by native animal silks. Forexample, biomimetic fibers made of recombinant spidroins9

have reached a toughness of 189 ± 33 MJ m−3 with the sametoughness of natural spider dragline fiber (160 MJ m−3),10 afiber produced by the major ampullate gland of spiders as alifeline and used for the construction of the orb web outer rim

and spokes,11 yet their modulus and strength were only half ofthe spider silk 4 ± 1 GPa and 370 ± 59 MPa, respectively.Thus, the properties were much lower than that of the nativespider silk fibers (10 GPa in strength and 1.1 GPa intoughness).10

This mismatch between natural and regenerated fibers isassociated with continued limitations in understanding thefundamentals in these individual systems; in particular thecontributions of mesostructures (i.e., microfibrils and nano-fibrils) on the mechanical properties of the macroscale fibers.12

In classical silk structural models, such as the two-phase cross-linking network model,13 the mean field theory-based order/disorder fraction model,14,15 and the Maxwell model,16,17 silksare simplified to a uniform polymer-like fiber, and theirmechanical properties are directly related to secondary

Received: April 1, 2018Accepted: June 22, 2018Published: June 22, 2018

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structures. As a result, most artificial spinning methods inspiredby these models attempt to utilize processes that control thecontent of β-sheets along with molecular orientation.6

However, computational modeling has revealed that meso-structures in silks are critical for the toughness of the fibers andlead to mechanical advantages that the synthetic fibers do notdisplay. For instance, silk fibers often have defects (e.g.,cavities, cracks, surfaces, tears) that can reach several hundrednanometers in size.18 Defects usually are seeds for failure inpolymeric materials due to the localized stress concentra-tions,19 while silks can protect against these defects throughconfinement of the diameter of nanofibrils in the 20−80 nmrange. In such diameters, the failure stresses and strains of thedefective silk fibers (the crack size is 50% the width of thefibers) converge toward defect-free fibers.20

Accordingly, the first aim of this study was to experimentallyunderstand the role of hierarchical structures in the mechanicalperformance of animal silks. However, different silks showconsiderable complexity in their construction, mechanicalproperties, and functions due to the diversity of silk types andthe species as sources of these proteins.21−23 There are morethan 30 000 known species of spider, and 113 000 species ofLepidoptera insects produce silks.24 Thus, it is unrealistic tocharacterize each silk. Here, Antherea yamamai (A. yamamai)silk (refers to tensan silk)25,26 was selected (Figure 1A and B),because such silk could be considered as a bridge(intermediate) to understanding the “structure−property-function” relationships of the broader silk family. This silkhas a primary structure of its protein similar to that of Arachnidspider dragline silk (Nephila clavipes). Both proteins containlarge central repetitive regions consisting of poly(alanine)domains alternating with glycine-rich regions.21−23 On theother hand, this silk fiber is produced and used by “wild”silkworms to construct cocoons (with the same function as themost of the silks produced by Lepidoptera insects). Moreover,the β-sheet content of A. yamamai silk is intermediate betweenthat of B. mori silk and Nephila spider dragline silk, while themechanical properties of the A. yamamai silk are alsointermediate between those of the other two silks.25,26

A more critical motive of this study was to constructregenerated functional fibers following fiber design strategiesused by the A. yamamai silk moth. Owing to the strong bindinginteractions and high mechanical properties of silk fibroin, avariety of high-performance and functional biomaterials27

based upon silks which show extremely high mechanicalstrength28,29 and high conductivity30 have been obtained.Compared with widely used B. mori silk fibers, A. yamamai silkfibers present more ingenious mesostructures that areconstructed with highly organized microfibrils and nanofibrils.The synergistic effects of these mesoscale structures andinterfaces give rise to the superior extensibility and toughnessof A. yamamai silks. With the above motivations, theadvantages in natural designs can be directly transferred intothe engineered fibers, providing a blueprint on the design ofextraordinary features and functions, by building on thisapproach that is emulated from both silkworms and spiders.

RESULTS AND DISCUSSIONFigure 1C presents a typical stress−strain curve of A. yamamaisilk, which features ductile mechanical behavior within thetriphasic region: an elastic phase (from 0 to yield point), aplastic deformation region (after yield point) and a strainhardening region. The ductile failure is also present in spiderdragline silk (Figure 1C)31 but in sharp contrast to the brittlefracture of Bombyx mori (B. mori) silk, which shows almost fullelastic response until the failure at a stain of 12% (Figure S1).After normalizing the strength and stiffness of all A. yamamaisilk fibers by density (Figure 1D), the values are close to that ofspider dragline silk instead of B. mori silk, despite all silkspresent superior strength than most other natural andengineered materials.32 This difference in mechanical behaviorbetween A. yamamai and B. mori silks drove us to furtherinvestigate the structure of the A. yamamai silk. We firststudied the secondary structure (conformation) of the A.yamamai silk because many studies have confirmed that theconformation of silk fibroin (i.e., β-sheet, random coil, andhelical structure) plays a vital role in the mechanical propertiesof the silks. Different characterization techniques, such as X-raydiffraction (XRD),33,34 nuclear magnetic resonance

Figure 1. Mechanical properties of A. yamamai silkworm cocoon silk fibers. (A,B) Alien saturnid orginating from Asia, A. yamamai adult andsecond instar larva. The inset image is the A. yamamai cocoon. (C) Stress−strain curve of A. yamamai silk, B mori silk, and Nephila edulisdragline silk. (D) Comparison of the specific strength and specific stiffness of A. yamamai silk with other natural and synthetic materials. Aand B reproduced with permission from ref 25. Copyright 2014, Pensoft Publishers. The inert picture in panel B were reproduced withpermission from ref 26. Copyright 2015, Korean Society of Sericultural Science. The stress−strain curve of Nephila edulis dragline silk usedata from ref 31. Ashby plot of natural and synthetic materials are adapted from ref 32. Panels A and B are reproduced and adapted from ref25, CC-BY-3.0.

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(NMR),35−38 Raman spectroscopy,35−41 and Fourier transforminfrared spectroscopy (FTIR),42,43 have been used to evaluatethe conformation of animal silks, including spider dragline silk,B. mori silk, A. yamamai silk, or the silks produced by otherAntherea genus, such as Antherea pernyi (A. pernyi, also knownas Chinese tussah silk).43 However, the contents ofconformations obtained from the various techniques aredifferent.42 Thus, it is difficult to establish “conformation-property” paradigms for A. yamamai silk based on previouslyreported data.Herein, synchrotron FTIR microspectroscopy42 was applied

to evaluate the silk fibroin conformations in a single fiber of A.yamamai silk. The deconvolution of the amide III bandprovided an estimation of β-sheet structure in the degummedfiber at 40 ± 10% (Figure 2C), comparable with that ofdegummed B. mori silk fibers (38 ± 4%). We also comparedthe orientation of each conformation in A. yamamai silks. S-FTIR microspectra of A. yamamai silk obtained from differentinfrared polarization angles and their related polar plots areshown in Figure S2 and Figure 2, respectively. Both figuresconfirmed the high orientation of each conformation in the A.yamamai silk. For example, the coupled absorption β-sheetpeaks, such as (1222 and 965 cm−1) became progressivelysmaller from Ω 0° (beam parallel to the fiber axis) to Ω 90°(perpendicular to the fiber axis) and their related polar plotshowed considerable order absorbance parallel to the fiber axisdirection with a molecular order parameter (Smol) of 0.95. Thesimilar plot patterns and orientation parameters have beendetected in a range of animal silks, including B. mori(orientation factor of 0.9244) and A. pernyi silk (orientationfactor of 0.9343). These results demonstrate that A. yamamaisilk has a similar conformation (both content and orientation)with B. mori silk, opposite to the differences in mechanicalperformance, where A. yamamai silk displayed ductile failurewhile B. mori silk was brittle.The reason for the above contradiction can be inferred from

the structural difference at the mesoscale because at themacroscale both silks consist of sericin and silk fibroinfilaments (Figure 3). Therefore, polarized light microscopyand scanning electron microscopy (SEM) were used tocompare the mesostructures between A. yamamai (Figure 3)and B. mori silks (Figure S3). The brilliant color under cross-polarized light confirmed the high-orientation of both kinds offibers (Figure 3B and Figure S3A). However, comparison ofthe images in Figure 3B and 3C show that there are highlyaligned fibrillar bundles on the A. yamamai silk surface, only auniform and smooth morphology exists on the surface of the B.mori silk fibers (Figure S3B). To detect the structural details ofthe A. yamamai microfibrils, we exfoliated the surface layer ofthe degummed A. yamamai silk fiber. As shown in the surfaceSEM image (Figure 3D), these microfibrils appeared as ribbon-like structures with a width of 500−1000 nm and were highlyoriented along the fiber axis. In addition, the weak adhesionbetween microfibrils and the numerous pores with a width of∼200 nm were detected from the cross-sectional SEM image(Figure 3E).Although several studies have reported that microfibrils in

silks are composed of thinner nanofibrils,45−53 detailedinformation about such mesostructures remains unknowndue to the lack of an effective experimental approach to isolateor retain these building blocks for characterization. Traditionaldissolution processing, such as LiBr/H2O,

54 N-methylmorpho-line N-oxide (NMMO)/H2O,55−59 and CaCl2/EtOH/

Figure 2. Infrared dichroism of single A. yamamai silk fiber. (A)Polar plot of the absorbance of the characteristic peaks in SFTIRmicrospectra in the 1500−800 cm−1 region from single A. yamamaisilk fibers. The symbols represent individual experimental datapoints, while the curves are fitted using eq 1. The peaks at 1454 to1405 cm−1 are not sensitive to the conformation change. The peakat 1242 cm−1 is attributed to random coil, while the peaks at 1373,1222, 1054, and 965 cm−1 are assigned to the β-sheet. In these β-sheet peaks, 1222 and 965 cm−1 have parallel dichroism, while1373 and 1054 cm−1 have perpendicular dichroism. (B) Polar plotof the relative intensity of different component from thedeconvolution of amide III band in S-FTIR microspectra of singleA. yamamai silk fibers. The peaks at 1222, 1242, and 1265 cm−1 inamide III band are assigned to β-sheet, random coil and α-helix,respectively. More details of the assignments of these peaks can befound in ref 43. (C) Deconvolution results of the amide III band ofthe A. yamamai silk fibers.

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H2O,60−63 directly dissolve the silk fibers into silk fibroin

molecules or chains. The use of an ultramicrotome allowsslicing silk fibers into thin sections for topological character-ization;13 this method only detects surface morphologies onthe sections and is unable to provide detailed 3D structures ofthe nanofibrils.13 Herein, we modified our recently established“partial dissolution-mechanical isolation” strategy64 to exfoliateA. yamamai silk at the single nanofibril level. Briefly, 0.1 g ofdegummed A. yamamai silk fibers were immersed in 20 mL of0.075 mM sodium hypochlorite solution and incubated atroom temperature to partially dissolve the fibers to microfibrils.After 30 min, ultrasonification processing (120 μm amplitudeand 20 kHz frequency, at intervals of 10 s for 1 h) was applied

to further isolate microfibrils into single nanofibrils. Atomicforce microscopy (AFM) identified these isolated nanofibrilswith a diameter of 5 nm (Figure S4), similar to the diameter ofsingle B. mori nanofibrils.65,66

On the basis of the structural characterization, consideringstructural scales from secondary structure to fiber, weidentified significant differences between A. yamamai and B.mori silks at microfibril scale. Previous computational modelingresults67−69 suggested that microfibrils may contribute to thetoughness of the silks through restricted fibril shearing,controlled slippage, stress transfer, as well as energydissipation, while direct experimental evidence for the featuresin silks was lacking. Therefore, we measured the cross-sectional

Figure 3. Hierarchical structure of A. yamamai silk fiber: (A) schematic of the hierarchical structure of A. yamamai silk; (B,C) polarized lightmicroscopy image of A. yamamai silk; (D) cross-sectional SEM image of A. yamamai silk after tensile fracture; (E) cross-sectional SEMimage of A. yamamai silk fiber. The fiber was embedded in epoxy resin and further broken in liquid nitrogen.

Figure 4. Comparison of mechanical performance between A. yamamai silk and Nylon-66 fiber: (A) SEM images of tensile fracturemorphologies of A. yamamai silk and B. mori silk; (B) stress−strain curve of original and notched A. yamamai silk fiber; (C,D)microphotograph of notched A. yamamai silk fiber before (C, optical microscopy image) and after (D, SEM image) the tensile fracture; (E)stress−strain curve of original and notched Nylon-66 fiber; (F,G) microphotograph of notched Nylon-66 fiber before (F, optical microscopyimage) and after (G, SEM image) the tensile fracture. The red arrows in panels C and F indicate the notch of the fibers. These imagesconfirmed that the notched width was smaller than the half width of the fiber used in testing. The red arrows in panels D and G display thecrack propagation direction after tensile failure. (The CSA of the original fibers was measured by SEM observation and estimated by ImageJsoftware. The CSA of the notched fibers was the effective area sections which were not cut off after the incision.).

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morphologies (Figure 4A) of A. yamamai fibers after tensilefailure, where microfibrillar slipping and pulling were indeedfound. For comparison, a smooth fracture surface was detectedin B. mori silk fibers, which agree well with the brittle fractureobserved in mechanical tests (Figure 1C and Figure S1B).In addition, mesoscale spring-models20 disclosed that the

fibrillar structure could inhibit the transverse growth of cracksthrough longitudinal splitting when fiber was stretched, so wealso tested the mechanical properties of notched silk fibers tocompare the response with that of the adjacent un-notched(intact) fibers (Figure 4B−G and Figure S5). In notched fibers,only the un-notched part bears the tensile stress; thus in ourmeasurements, we calculated the tensile stress of the fibers byusing the cross-sectional area of the un-notched part. As shownin Figure 4B−D, when the notch width was smaller than thehalf width of the fiber (Figure 4C), the notched fibersexhibited the same stress−strain curves as the un-notchedfibers (Figure 4B); only failure to strain was reduced; featuringtypical ductile fracture behavior. Cross-sectional SEM images(Figure 4D) of notched A. yamamai fibers after tensile fractureconfirmed the ductile failure behavior, where the apparentmicrofibril splitting and pulling were observed, and the crackwas shifted around 90 deg during tensile processing (thedirection indicated by the red arrows). However, the stress−strain curves of notched nylon-66 fibers (Figure 4E), a fiberwith similar chemical structure with protein fibers but withoutthe microfibrils (Figure 4F), was significantly different fromthe unnotched fiber. Linear crack propagation was observed inthe notched fiber (Figure 4G) which was fractured beforereaching the yield point (strain of 13%).Although the A. yamamai moth has been cultivated in Japan

for more than 1000 years, it is still rare and expensive (moreexpensive than gold at the same weight). Thus, an alternativeapproach to the practical use such silk is to mimic thehierarchical structures by using other widely available silkfibers, such as the B. mori silks. However, conventionalspinning techniques are challenging to produce regeneratedfibers with a hierarchical structure. Inspired by liquid crystaland dry spinning features from the spider and silkworm

spinning, we previously reported a dry spinning approach tospin regenerated silk fibers with a polymorphic structure.5 Inthis method, degummed B. mori silk fiber was initially dissolvedto microfibril features in solution using hexafluoroisopropanol(HFIP) as a solvent and then spun into regenerated fibers bydirect extrusion or reeling in the air. With the aim ofproduction of regenerated B. mori silk fibers (RBSF) with A.yamamai silk-like hierarchical structures, we modified thisbiomimetic spinning route to weaken the degree to which theB. mori silk was dissolved. Thus, this modified approach wassuitable for generating regenerated fibers with longer micro-fibril alignment.In the particular spinning protocol, the degummed B. mori

silk fiber/HFIP (1/20 wt/wt) system was still incubated at 60°C, but the time was reduced to 3 days. After this process, thesingle silk fibers were partially dissolved into highly elongatedmicrofibrils with lengths above centimeters. During spinning inthe air, the microfibrils adhered together due to the dissolvedsilk fibroin, to obtain A. yamamai silk-like structures (Figure5A−D). No differences in the secondary structure between theA. yamamai silk-like RBSFs and the as-spun RBSFs producedfrom silk fibroin/HFIP solution (without hierarchicalstructure) were detected from FTIR spectra (Figure S6).These RBSFs exhibited ductile mechanical behavior with atriphasic stress−strain region (the red curve in Figure 5E), verysimilar to the A. yamamai silk. The average strain to failure ofRBSFs reached 23 ± 4%, approximately twice that of B. morisilk (13 ± 4%), and comparable to A. yamamai silk (26 ± 7%).More remarkably, the average modulus of these regeneratedfibers was 14 ± 3 GPa, 2 times higher than A. yamamai silks.To confirm that the ductile mechanical behavior of theseRBSFs was due to the weak interface and connections betweenmicrofibrils, we increased the interface adhesion betweenmicrofibrils in RBSFs by adding 4 wt % silk fibroin/HFIPsolution into the microfibril pulp before spinning. The tensilestress−strain curves of such RBSFs indeed showed B. mori silk-like brittle failure, with no yield plateau region (the blue curvein Figure 5E).

Figure 5. Structure and mechanical properties of RBSFs: (A) SEM image of RBSFs; (B,C) surface and cross-sectional structure of RBSFs;(D) polarized light microscopy image of RBSFs; (E) stress−strain curve of RBSFs with (red curve) and without (blue curve) weak fibrillarinterface. The inserts are cross-sectional SEM images of the RBSFs after tensile fracture.

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Conductive fibers have received a great deal of interest forelectronic, optoelectronic, and energy storage-related fields.70

Silks as a robust textile fiber have not been extensively used inthis fields because it remains challenging to produceconductive silk fibers while retaining the useful mechanicalproperties. Wet-spinning71 and dry-spinning techniques72 havebeen pursued to address this issue, while the content ofconductive fillers was insufficient to reach conductivepercolation thresholds. On the basis of the inspiration thattoughness of the RBSFs can be enhanced by the organizationof high fibrillar orientation, as well as weak fibrillar interfaces,we designed conductive and ductile RBSFs using a yarn-spinning technique. In this route, degummed single B. mori silkfibers were weaved into elongated fiber bundles (consisting ofapproximate 30 fibers) with a diameter of around 100 μm andlength up to 1 m (Figure 6A and Figure S7A). Then, 1 g of theresulting silk fiber bundles was immersed in a 20 mLmultiwalled carbon nanotube (MWCNT)−isopropyl alco-hol−HFIP solution stabilized by poly(vinyl alcohol) (PVA)(1:3:17 w/v/v) and sealed for incubation at 60 °C for 2 daysto generate conductive RBSFs. In these conductive RBSFs, theMWCNTs bonded with single B. mori silk fibers, and differentfibers partially adhered to each other due to the dissolved silkfibroin by the HFIP (Figure S7).Benefiting from these distinct hierarchical structures, the

conductive RBSFs were sensitive to elastic deformation. Whenthe conductive RBSFs were stretched, the single fibers in thefiber bundles slipped; thereby the corresponding resistance wasreduced. When the fibers were unloaded, they returned to theiroriginal configuration, and the resistance returned to itsoriginal value. For example, loading−unloading cycles (Figure

6B) showed that neither plastic deformation or loss of strengthoccurred in the conductive RBSFs at a set strain of 7.5%, andno significant hysteresis loop was found, indicating that theconductive RBSFs exhibited good shape recovery propertiesafter the first cycle. More remarkably, the variation inconductive resistance was synchronized with the tensile strain(Figure 6C and Movie 1). Therefore, such RBSFs can bedirectly utilized to monitor the deformation of a material. As aprototype (Figure 6D), two conductive RBSFs were weavedinto a grid substrate, where the first (fiber (i) and the secondconductive fiber (fiber (ii) were located in the middle and edgeof the grid, respectively (Figure 6E). Further, sphere stress wasapplied and released circularly on the grid. As shown in Figure6F and Movie 2, the changes in resistance of the two fibersfollowed the cyclic “press-and-release” process, and fiber ishowed more substantial changes than fiber ii, owing to itslarger external deformation.In addition, these conductive RBSFs were also sensitive to

changes in temperature due to thermal expansion andcontraction stress in the MWCNT-PVA coating layers. Whenthe temperature increased, the expansion of PVA on singlefiber surfaces led to increased contact area between fibers inthe conductive RBSFs. As a result, the resistance of theconductive RBSFs was reduced. In contrast, when thetemperature was reduced, the PVA shrank to its original size,so the contact area between the fibers decreased accordingly,and thus led to an increase in the corresponding resistance. Anin situ experiment was designed to record this temperatureresponse (Figure 7A). A conductive RBSF was first fixed bytwo tensile clamps, which was then connected by a resistancedetector. In this system, a tensile device recorded the force

Figure 6. Conductive RBSF-based sensor for monitoring mechanical stimuli. (A) Polarized light microscopy image of B. mori silk bundlecoated with MWCNTs produced by yarn-spinning technique after the tensile fracture. At the fracture position (indicted by white dashedframe), the silk fibers were spread out while the near adjacent area was still bundled together, presenting similar fracture behaviors with A.yamamai silk fibers. (B) Cyclic load−unload stress−strain curve of conductive RBSFs. (C) Relationship between strain and electricresistance of conductive RBSFs during cyclic load-unload tensile measurement. (D) Schematic of the method to measure the electricresistance response for spherical stress. (E) Photograph of the RBSF grid. Two black fibers are conductive RBSFs. The other white fibers areB. mori silk fiber bundles. (F) The electric resistance changes of these two fibers with the change of stress. These two fibers followed thecyclic “press and release” process.

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generated by a change in temperature, while the resistancedetector collected the resistance changes synchronously.During the testing, a movable heat source was close to (butnot touching) the conductive RBSF, and temperature changesof the fiber were monitored in real time by a thermal imager.As shown in Figure 7B, the stress of the conductive RBSFincreased immediately when a heat source was close to thefiber and then decrease gradually. More importantly, the stressof the fiber was changed circularly and repeatably with changesof temperature (Figure 7C). Meanwhile, the resistance of theconductive RBSF also changed circularly and repeatably inresponse to temperature changes (Figure 7D). These stressand temperature sensitive RBSFs may find applications inwearable sensors or as medical implants, considering thebiocompatible nature of the composites.

CONCLUSIONS

A. yamamai silk was chosen for study in order to understandthe structure−property relationships of natural silks, because ofits superior advantages in terms of mechanical extensibility thatare superior to B. mori silk. A multiscale structural comparisonof A. yamamai and B. mori silks confirmed that the distinctivemicrofibrillar architectures and hierarchical designs of A.yamamai silk dominate its outstanding extensibility andtoughness. Inspired by this fiber construction strategy usedby the A. yamamai silk moth, RBSFs were generatedexperimentally with highly organized hierarchical structures.These RBSFs had extensibility (23 ± 4%) and modulus (14 ±

3 GPa) that compared to natural A. yamamai silk and werefurther functionalized to conductive RBSFs by introducingcarbon nanotube during the spinning processing. Benefitingfrom the microfibrillar construction and weak microfibrillarinteractions, these conductive RBSFs were highly sensitive toforce and temperature stimuli and responded as sensors tomonitor these changes. These features suggest utility in smarttextiles. More importantly, the synergistic integration ofmultiscale characterization and biomimetic preparation offersan innovative route to the rational design of functionalmaterials using an optimized “structure-property” relationship.

METHODSPreparation of Degummed A. yamamai Silk Fibers. Raw A.

yamamai silkworm cocoon silk fibers were degummed by boiling intwo 30 min changes of 0.5% (w/w) NaHCO3 solution. Thedegummed fibers were then washed with distilled water and obtainedby drying at room temperature.

Preparation of A. yamamai Silk Nanofibrils. A. yamamai silkfibers (1 g) were suspended in a beaker containing 100 mL of DIwater, and then a 30 min chemical oxidation of silk was initiated byadding the desired amount of sodium hypochlorite (NaClO) solution(15 mmol of NaClO per gram of protein). Further, the oxidized A.yamamai silk pulp was dialyzed (Pierce, molecular weight cutoff =10 000) against DI water for 3 days, followed by ultrasonic treatment(QSonica500, USA) at 150 μm amplitude and 20 kHz frequency withan interval of 30 min. A. yamamai silk nanofibrils/water dispersionwas harvested by centrifugation at 8000 rpm for 5 min.

Preparation of Conductive RBSFs. First, B. mori silkwormcocoon silk fibers were degummed by boiling in two 30 min changes

Figure 7. Conductive RBSF-based sensor for monitoring thermal stimuli: (A) experimental setup for recording the thermal and electricconductivity of conductive RBSFs; (B) time-force load curve of the conductive RBSFs with the change of temperature; (C,D) force andresistance of the conductive RBSFs with change of temperature. The force and resistance of the conductive RBSF repeatably response tochanges in temperature (three cycles).

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of 0.5% (w/w) NaHCO3 solution. The degummed silk fibers werewashed with distilled water and allowed to air-dry at roomtemperature. Then, the degummed single B. mori silk fibers werewoven into elongated fiber bundles (consisting of approximately 30fibers) with a diameter of 100 μm and length up to 1 m. To preparethe MWCNT/isopropyl alcohol/HFIP solution, 3 mL of isopropylalcohol dispersed MWCNT solution stabilized by PVA (containing≈200 mg MWCNT, Chengdu Organic Chemicals Co. Ltd., China)was mixed with 17 mL of HFIP in a 20 mL sealed glass bottle withsufficient stirring. Finally, 1 g of elongated B. mori silk fiber bundleswere immersed in MWCNT/isopropyl alcohol/HFIP solution. TheMWCNT-coated silk fibers were obtained after incubating in airtightcontainers as mixtures at 60 °C for 3 days. The residual solvent wasremoved thoroughly by drying the fibers at room temperature for 1day. All of these steps were conducted in a chemical hood with thenecessary precautions since HFIP is a toxic solvent.Polarized S-FTIR Microspectroscopy of Single A. yamamai

Silk Fibers. The experiments were performed at BL01B in theShanghai Synchrotron Radiation Facility (SSRF) with the Nicolet6700 Fourier transform infrared spectrometer, infrared microscopy,and imaging systems. A 15 × 15 μm2 square aperture was selected tocollect the S-FTIR microspectra of the single A. yamamai silk fiber.This aperture size was smaller than the width of a single fiber and thusavoids the diffraction and the scattering of the infrared light. To studythe dichroism of specific absorption bands, a KRS-5 IR polarizer wasinserted in the infrared beam. S-FTIR microspectra were collected inthe mid-infrared range of 800−3800 cm−1 at a resolution of 4 cm−1

with 256 coadded scans. During the measurement, the backgroundwas collected each time before all FTIR spectra of single silk fiberswere collected. Deconvolution of amide III band was carried out usingPeakFit 4.12. The number of peaks and their positions were obtainedfrom the second derivative spectra and fixed during the subsequentdeconvolution process. The orientation of individual moieties can beobtained from the angular dependence of the absorbance A(ν) atwavenumber ν which corresponds to a vibration of the moleculargroup under investigation. In the general case, the angulardependence of the absorbance can be determined using eq 1.

ν Ω = − { Ω − Ω

+ Ω − Ω }

ν

ν

−

−

A( , ) log 10 cos ( )

10 sin ( )10

Amax( ) 20

Amin( ) 20 (1)

Where A(ν, Ω) is the peak intensity of a certain band, Ω is thepolarization angle, Ω0 is the angle at maximum absorption, and Amaxand Amin are the maximum and minimum absorbance, respectively.The molecular order parameter (Smol) of the corresponding secondarystructural component was calculated as eq 2.

ν νν ν

=−+

SA A

A A( ) ( )

( ) 2 ( )mol max min

max min (2)

Mechanical Testing of Natural and Regenerated Silk Fibers.The degummed A. yamamai silk fibers were cut into 40 mm segmentsfor tensile tests. For tensile testing, the 40 mm segments weremounted on a hard-cardboard frame with a base length of 20 mm andfixed with cyanoacrylate. After the cyanoacrylate was dried overnight,the frame was mounted in the testing machine (Instron 5966machine, Instron, Norwood, USA) and the side support of the framewas cut away so that the force was transmitted through the fibers.Meanwhile, the initial length of the fiber was measured with a caliperat zero load point (the point in which the fibers are tight, but no forceexerted on it). To understand the crack propagation mechanism of Ayamamai silk fibers, nylon-66 and RBSFs were compared. A fiber wascut into two segments. One segment was further notched by a smallincision that operated with the assistance of a microscope, while theother one without a notch was used for the control. All of the tensilemeasurements were carried out at 25 °C and 50% RH with a tensilespeed of 2 mm/min. To calibrate the cross-sectional area of the fibers,the fibers were fixed with an epoxy resin, and after drying overnight,the samples were sectioned into three segments in liquid nitrogen.The cross-section area (CSA) of the fibers was then measured by

SEM observation, and the CSA was estimated by ImageJ software(NIH). The CSA of the notched fibers was the effective area sectionswhich have not been cut off after the incision. The average area ofthree segments was used as the CSA of adjacent RSFs and then usedfor stress calculations.

The Mechanical and Thermal Response of the ConductiveRBSFs. The ends of the twisted conductive RBSFs were clamped bytwo pieces of copper sheets and then fixed by tensile devices. Beforecyclic tests, the copper sheets were connected with a digitalmultimeter (CEM, DT-9989). The initial length of the fiber wasmeasured with a caliper at zero load point. The mechanical tests werecarried out using an Instron 5966 machine (Instron, Norwood, USA)in cycle load−unload mode at 25 °C and 50% RH with a tensile speedof 2 mm/min. To test the deformation-electric resistance relationshipof the conductive RBSFs, two conductive RBSFs and 13 weaved B.mori silk fiber bundles were weaved into a grid substrate. These twoconductive RBSFs were located in the middle and edge of the grid,respectively. All fibers were fixed with cyanoacrylate. The ends of theconductive RBSFs were clamped by two pieces of copper sheets andfurther connected with a digital multimeter. During the tests, a 100mL round-bottom flask, which applied sphere stress on the grid, wasused to press the grid intermittently. The resistance values wererecorded by a Bluetooth device with a time resolution of 1 s. Todetect the thermal response of the sample, a movable copper heaterwas first moved close to (but not touching) the conductive RBSF forheating and then was removed for cooling. These processes can beiterative. The temperature changes of the fiber were monitored in realtime by a thermal imager, and the changes in fiber tension wererecorded by using an Instron 5966 machine (Instron, Norwood,USA). The variation of resistance was collected using a digitalmultimeter (CEM, DT-9989). The resistance and temperature valueswere extracted from each frame with a time resolution of ≈0.3 s.

Characterization. The surface and cross sections of all the fiberswere observed by SEM (JEOL JSM-7800F) at an acceleration voltageof 5 kV and polarizing optical microscope (Olympus BX51-P, Japan).To prevent electrical charging, all specimens were coated with a 5 nm-thick gold layer before observation. For atomic force microscopy(AFM) measurements, A. yamamai silk nanofibril aqueous dispersionwas diluted to ∼0.01% (w/v) with DI water. The resultant solutionwas added dropwise onto a mica substrate for 120 s, followed bypurging with nitrogen gas. The topologic structures of nanofibril werecharacterized by Dimension ICON AFM fast scanning system(Bruker, Germany) with tapping mode. An aluminum reflectivecoated silicon cantilever with a tip radius of 2 nm was used (k = 0.4N/m).

ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsnano.8b02430.

Stess-strain curves; spectra; images as described in thetext (PDF)Movie 1: Cyclic Load-Unload Tensile Measurement ofConductive RBSF (Six Times Fast Forward) (AVI)Movie 2: The Electric Resistance Response for SphericalStress (AVI)

AUTHOR INFORMATIONCorresponding Authors*E-mail: David.Kaplan@Tufts.edu.*E-mail: mbuehler@mit.edu.*E-mail: lingshj@shanghaitech.edu.cn.*E-mail: fanyimin@njfu.edu.cn.ORCIDYimin Fan: 0000-0003-2764-1310Markus J. Buehler: 0000-0002-4173-9659

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6975

David L. Kaplan: 0000-0002-9245-7774Author Contributions#W.Z. and C.Y. contributed equally to this work.

NotesThe authors declare no competing financial interest.

ACKNOWLEDGMENTS

Y.F. acknowledges the National Key R&D Program of China(2016YFD0600803) and the Priority Academic ProgramDevelopment of Jiangsu Higher Educational Institutions(PAPD). S.L. acknowledges the starting grant of ShanghaiTechUniversity. M.J.B. acknowledges NIH Grant U01 HS 4976 andOffice of Naval Reasearch Grant N00014-16-1-2333. We thankthe staff from BL01B beamline of National Center for ProteinScience Shanghai (NSPSS) at Shanghai Synchrotron RadiationFacility, for assistance during data collection.

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