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Structural Protein-based Flexible Whispering Gallery Mode Resonators Huzeyfe Yilmaz, Abdon Pena-Francesch, Robert Shreiner, Huihun Jung, Zaneta Belay, Melik C Demirel, Sahin Kaya Ozdemir, and Lan Yang ACS Photonics, Just Accepted Manuscript • DOI: 10.1021/acsphotonics.7b00310 • Publication Date (Web): 07 Jun 2017 Downloaded from http://pubs.acs.org on June 18, 2017
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Structural ProteinProtein-Based Whispering Gallery Mode Resonators Huzeyfe Yılmaz†, Abdon Pena-Francesch‡, Robert Shreiner‡, Huihun Jung‡, Zaneta Belay‖, Melik C. Demirel*‡§, Şahin Kaya Özdemir*‖, Lan Yang†‖ †Institute
of Materials Science and Engineering, ‖Department of Electrical and Systems Engineering, Washington University, St. Louis, MO 63130, USA. ‡Department of Engineering Science and Mechanics, §Materials Research Institute and Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA. KEYWORDS: Structural proteins, protein-based photonics, optical switch, protein-based whispering gallery mode resonators, thermo-refractivity, flexible photonics ABSTRACT: Nature provides a set of solutions for photonic structures that are finely tuned, organically diverse and optically efficient. Exquisite knowledge of structure-property relationships in proteins aids in the design of materials with desired properties for building devices with novel functionalities, which are difficult to achieve or previously unattainable. Here we report whispering-gallery-mode (WGM) microresonators fabricated entirely from semi-crystalline structural proteins (i.e., squid ring teeth, SRT, from Loligo vulgaris and its recombinant) with quality factors as high as 105. We first demonstrate versatility of protein-based devices via facile doping, engaging secondary structures. Then we investigate thermorefractivity and find that it increases with beta-sheet crystallinity which can be altered by methanol exposure and is higher in the selected recombinant SRT protein than its native counterpart. We present a set of photonic devices fabricated from SRT proteins such as add-drop filters and fibers. Protein based microresonators demonstrated in this work are highly flexible and robust where quality factors and spectral position of resonances are unaffected from mechanical strain. We find that the thermo-optic coefficients of SRT proteins are nearly hundred times larger than silica and more than ten times larger than polydimethylsiloxane. Finally, we demonstrate an optical switch utilizing the surprisingly large thermo-refractivity of SRT proteins. Achieving 41 dB isolation at an input power of 1.44 µW, all-protein optical switch is ten times more energy efficient than a conventional (silica) thermo-optic switch.
Protein-based devices provide an alternative to synthetic polymers and glasses granting comparable or better performances while presenting diverse functionalities and clean processing capabilities.1 Recent bio-inspired photonic platforms made from proteinaceous materials lay the groundwork for many functional device applications such as electroluminescence in peptide nucleic acids,2 multiphoton absorption in amyloid fibers,3 sensing with protein single fibers,4 stimulus responsive protein WGM lasers,5 WGM emitting protein microcavity assemblies6 along with silk waveguides, optical fibers and inverse opals.7-9 The ability to manipulate and control light flow and light– matter interactions using whispering-gallery-mode (WGM) resonators has created significant interest in various fields of science, including but not limited to biosensing10-12 and detection,13-16 cavity-QED,17 optomechanics,18 and paritytime symmetric photonics.19 WGM resonators are currently manufactured using standard lithography techniques with conventional materials such as silica,20 silicon,21 and silicon nitride.22 However, recent developments in optical technologies have revealed the strong need for developing soft, biocompatible, and biodegradable photonic devices
and photonic structures with novel functionalities that cannot be attained with current optical materials.23 Towards this aim, all-polymer24 and polymer- coated silica WGM resonators,25,26 as well as silica WGM resonators encapsulated in low- index polymers27 have been fabricated using conventional and commercially available polymers, such as polydimethylsiloxane (PDMS) and polystyrene (PS) to address the need for flexible structures. Nature, on the other hand, has optimized proteins through millions of years of evolution to provide diverse materials with complex structures.28,29 Thus studying structural proteins at the molecular level will help us in our endeavor for efficiently designing diverse materials, which are finely tunable, flexible, biodegradable, and have physical properties and functionalities different or superior to those currently present in conventional materials.30 This will also enable the production of functional devices with less chemical processing and energy consumption, addressing growing environmental concerns. In this article, we report high quality factor WGM microresonators fabricated solely from semi-crystalline structural proteins and demonstrate their use as add-drop filters and optical switches. We fabricated
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Figure 1. All-protein whispering-gallery-mode (WGM) microresonators. (a) Scanning electron microscope (SEM) image of a WGM resonator fabricated from SRT (Scale bar 20 μm). (b) Transmission spectrum of a WGM resonance around 672 nm. (c) Confocal fluorescence obtained from silica microresonators that are spin-coated entirely (top) and partially (bottom) with SRT and then immersed in thioflavin-T (ThT) dye. (d) Green fluorescence emission from an all-SRT chip, doped with ThT dye, under UV-blue light and confocal fluorescence image of a single microtoroid on this chip before and after urea treatment (bottom). (e) Fluorescence intensities of ThT doped SRT microtoroid before (top) and after (bottom) urea treatment. solutions, a plasticizer (butanediol) was used to reduce the glass transition temperature and temporarily soften the material. Also, the fabrication of the silica master was tailored to endure demolding step of fabrication (see Supporting Information). Light from a tunable laser was evanescently coupled in and out of the fabricated resonators through a tapered optical fiber to measure their transmission spectra and characterize resonances. Structural proteins are characterized by long-range ordered molecular secondary structures (e.g., β-sheets, coiled coils, or triple helices) that arise due to highly repetitive primary amino acid sequences within the proteins. These features promote the formation of structural hierarchy via self-assembly. SRT is a semi-crystalline structural protein, which are flexible, biodegradable, and thermally and structurally stable. Semi-crystalline morphology of these proteins, which emanates from their β-sheet secondary structures, is ideal for tunability of their physical
microresonators using native and recombinant SRT (Figure 1a) to demonstrate the possibility of building flexible all-protein functional photonic devices. Although, proteinbased three dimensional WGM resonators have been demonstrated before,5,6 the resonators provided here are superior in performance, allow studies of relations between the nanostructure (β-sheet content) and fundamental properties (thermal refraction). Therefore, a fabrication method that preserves the nanostructure unlike e-beam lithography4 or photo-crosslinking5 was chosen. Allprotein microtoroid WGM resonators were fabricated using a molding and solution casting technique (Figure S1). From initial silica templates fabricated as previously described,20 we prepared PDMS negative molds for solution casting of the proteins. The microtoroid structures were then fabricated by filling the respective protein solutions into the corresponding PDMS molds. To avoid deformation of SRT WGM resonators during demolding of the protein
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Figure 2. Thermal response of WGM resonators fabricated entirely from SRT proteins. Resonance wavelengths shift to shorter wavelengths as the temperature is increased for all resonators fabricated from (a) native or (b) recombinant SRT proteins. Methanol-treated WGM resonators prepared from native (a) or recombinant (b) SRT proteins have stronger thermal response than the corresponding untreated resonators. Among the resonators fabricated from native SRT proteins, methanol-treated resonators prepared from aged solution of native SRT proteins have the largest thermal response. Note that both the methanol treatment and the shearing increase the number of β-sheets. onators, immersed them into ThT dye and rinsed them multiple times with deionized water to test selective binding of ThT to SRT. First, confocal fluorescence microscope images revealed that for the fully SRT-coated resonator (Figure 1c top), the green fluorescence circled the boundary of the resonator uniformly, whereas for the partially SRT coated resonator (Figure 1c bottom) the fluorescence appeared only along half of the boundary and there was no fluorescence from the uncoated silica portion of the resonator. These confirm that the emission is from the ThT dyes bound to the SRT protein but not from the residual ThT dyes on the surface. Second, while the all-SRT chips exhibited green fluorescence (Figure 1d top), which confirms the large red-shift in the emission from ThT due its binding to β-sheets of SRT, there was no green fluorescence from all-PDMS chip doped with ThT, implying the absence of secondary structures in PDMS. These results confirm the selective binding of ThT to β-sheets in SRT: The dye does not bind to silica or PDMS protein due to the absence of secondary structures in these materials. Since β-sheets are formed via hydrogen bonds, disruption of the H-bonds with urea should eradicate the fluorescence of the ThT dye. After treating the SRT chips with urea (2 mM for 20 minutes), we observed a significant decrease in the fluorescence from the chip and individual resonators (Figure 1d and Figure 1e). We also note that the WGM structure deformed due to loss of rigidity in the resonators upon disruption of β-sheets with urea. We experimentally determined thermo-optic coefficients of SRT proteins by monitoring the resonance wavelength of an all-SRT resonator as the temperature of the all-protein chip substrate was varied. Then we kept this resonator in methanol for 4 hours- a process known to increase the number of β-sheets in semi-crystalline structural proteins such as silk and
properties. SRT is composed of several proteins varying in molecular weight (e.g., 10-55 kDa).31 Although optical properties do not vary with polydispersity in molecular weight due to linear dielectric response or statistically driven size, they may change in special cases due to cooperative non-linear response or thermal effects. Therefore, a recombinant SRT (Rec) protein with unique molecular weight (i.e., 18 kDa) is also selected to create a monodisperse material.32 A combination of RNA-sequencing, protein mass spectroscopy, and bioinformatics tools (i.e., transcriptome assembly) was performed to produce 18 kDa recombinant SRT protein. Rec, and SRT proteins were analyzed using X- ray diffraction (XRD), which showed that SRT protein had lower crystallinity compared to Rec protein at room temperature (Figure S3a-c). Rec, and SRT proteins were also are composed of β-sheets and α-turns (Figure S3d-f). Figures 1a and 1b present an example proteinbased WGM resonator (Figure 1a) and its resonance transmission spectrum in the 660 nm band (Figure 1b). The quality factors of these proteinaceous WGM resonators ranged from 104 to 105 (Figure S4). We probed the nano-crystalline regions in these structural proteins, delineated by anti-parallel β-sheets, using fluorescent microscopy. We selected a fluorescent dye, thioflavin-T (ThT), which binds specifically to the β-sheets, but does not bind to amorphous polypeptides.33, 34 When ThT dye binds to β-sheets, it undergoes a characteristic red shift of its excitation/emission spectrum that may be selectively excited with blue light at 450 nm, resulting in a green fluorescence signal with maximum at 482 and extending to 570 nm. We prepared all-SRT chips containing SRT microresonators (Figure 1a and Figure 1d), silica microresonators fully (Figure 1c top) or partially (Figure 1c bottom) coated with SRT, and a PDMS chip with microres-
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Table 1. Thermo-optic and thermal expansion coefficients of silica, polydimethylsiloxane and native and recombinant SRT proteins. Material
Thermo-optic Coefficient (/ )10 !
Thermal Expansion Coefficient (1/)(/ )10 " !
Resonance Wavelength Shift (1/)(/ )10 !
0.119 -1.0
0.055 30.46
0.124 1.646
-6.82
-9.5
-5.43
-8.76
-9.5
-6.70
-8.33
-9.5
-6.42
-10.31
-9.5
-7.72
No methanol treatment
-8.42
-9.5
-6.48
Methanol-treated
-10.45
-9.5
-7.81
Silica Polydimethylsiloxane Native SRT Unaged solution No methanol treatment Methanol-treated Aged solution No methanol treatment Methanol-treated Recombinant SRT
SRT35 and measured the change in its resonance wavelength as a function of temperature once more. For all resonators, we observed blue shift in resonance wavelength, λ (i.e., the WGM resonance shifted to a shorter wavelength) as the temperature T was increased (Figure 2a and Figure 2b), i.e., (, )/ < 0. It is clearly seen that methanoltreated samples (i.e., samples with higher number of βsheets) experience a larger resonance shift than the untreated samples (i.e., (1/)∆/∆ is larger for methanoltreated samples). Since the number of β-sheets crystals in SRT proteins can also increase due to shearing in the solution, we also fabricated microresonators from aged native SRT proteins to compare their thermal response with the resonators fabricated from fresh (i.e., none or minimal shear) samples. We observed that resonators prepared from aged SRT proteins experienced larger resonance shifts (Figure 2a). This is consistent with the methanol treated SRT protein microtoroids since this treatment is also known to increase the number of β-sheets and crystallinity of SRT proteins. When the resonators prepared from aged SRT proteins were further treated with methanol, the resonance shifts they experienced were even larger. These results suggest a relation between the crystallinity (i.e., number of β-sheets) of the samples and their thermal response, implying that one can tune thermal properties of photonic devices prepared from semi-crystalline proteins such as SRT by controlling the number of β-sheets. Using a thermomechanical analyzer, we measured linear thermal expansion coefficients (1/) / prepared from native and recombinant SRT proteins as −95 × 10-6 ± 7 × 10-6 K-1. Moreover, our ellipsometry measurements revealed the refractive index n of these samples to be 1.523 at 1420 nm. Using the measured parameters (1/)(, )/ , (1/) / and n in the equation of WGM resonance 1 (, ) 1 1 = + (1) for a resonator of radius r, we calculated thermo-optic coefficient dn/dT for the prepared samples (Table 1). For
comparison purposes, we performed the same analysis with WGM resonators fabricated from silica and PDMS. As seen in Table 1, contrary to silica which has positive thermo-optic coefficient and positive thermal expansion coefficient, SRT proteins have negative thermo-optic and negative thermal expansion. Thermal refraction can be characterized by the following equation36
= + (2)
which can be rewritten in terms of coefficient of thermal expansion, , and density, , as = − + (3)
The thermo refractivity is then due to changes in the density of the material ( /) and/or changes in its molecular polarizability (/ ). The first term on the right-hand side of equation 3 is density dependent and represents the change in thermo refractivity due to thermal expansion, α. Lorentz-Lorenz equation gives a relation for the first term ( + 2)( − 1) = (4) 6 Given a positive density and a negative coefficient of thermal expansion (SRT proteins) the, first term on the righthand side of equation 3 cannot be negative. This means that the density independent thermo refractivity is extraordinarily large and negative for SRT proteins. One explanation for this phenomenon is the water content of SRT proteins. When heated, water leaving SRT proteins can cause significant reduction in the refractivity of the material. Large thermo-refractivities are seen advantageous for low power thermo-optic switches,37 otherwise, composites with opposite sign thermo-refractivities are employed to attain athermal behaviors in photonics.25
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Figure 3. Device examples of semi-crystalline proteins. (a-b) An all-SRT microtoroid resonator with two coupling taperedfiber waveguides forming an add-drop filter. Transmission spectrum at 667 nm band shows a resonant dip in the transmission port and a resonance peak in the drop port (scale bars 20 μm). (c) Light transmission through a fiber waveguide prepared from recombinant SRT by electrospinning. Coupling of laser light into the protein fiber was done using a silica tapered fiber (scale bar 50 μm). (d) Flexibility of the substrate (scale bar 1 cm) and of the WGM resonators fabricated from SRT (scale bar 1 mm). Inset presents the results of dynamic mechanical analysis (DMA) measurements used to quantify the bending of native and recombinant SRT films. (e) A WGM resonance measurement while the substrate is under strain. (f) Wavelength and quality factor measurements of flexible substrate as a function of strain show stable resonance in the protein resonator. The calculated thermo-optic coefficients given in Table 1 suggest a relation between crystallinity (i.e., number of βsheets) and the thermo-optic coefficient of SRT proteins, because (i) processes such as aging (shearing) and methanol treatment which result in higher crystallinity leads to more negative thermo-optic coefficients, and (ii) recombinant SRT which has higher crystallinity has also more negative thermo-optic coefficient compared to the native SRT. By coupling proteinaceous WGM resonators to two separate fiber-taper waveguides, we designed add-drop filters (Figure 3a) that are frequently integrated in optical communication architectures (such as optical filters, multiplexers and routers), as well as used as schemes for high performance optical sensing devices. As seen in Figure 3a, the add-drop filter routed the light from the input waveguide to the drop port in the second waveguide when the wavelength of the light coincided with the resonance wavelength of the SRT resonator. Non-resonant light passed through the input waveguide to the transmission port. The dip in the transmission port and the peak in the drop port seen in Figure 3b shows that the SRT add-drop filter performed its function with an add-drop efficiency of 51%, which may be further increased by more careful setting of the waveguide-resonator coupling and by decreasing scattering losses. We also produced proteinaceous waveguides (Figure 3c) using electro-spinning (e-spin) technique (Figure S5). Engaging a single protein fiber to
silica fiber taper via van der Waals attractions, we achieved evanescent coupling of light from the silica fiber taper to the protein fiber, clearly demonstrating the waveguiding capability of proteinaceous fibers. The flexible nature of these protein-based WGM resonators is shown in Figure 3d, where the soft base of the WGM microresonators is bent in a bowed shape. This characteristic enables significant bending on a macroscopic scale (see inset). We performed three-point bending dynamic mechanical analysis (DMA) for the recombinant and native SRT films to quantitatively describe the flexibility of the fabricated photonic microstructures (Figure 3d-inset). The loaddeflection curve shows similar bending modulus (~1 GPa) for all films. Figure 3e shows bending of the protein substrate while a resonance measurement is made. Unlike flexible ring resonators,38 flexible and on-chip toroidal WGM resonators are unresponsive to mechanical strains. The resonance wavelength of the SRT microtoroid did not change under mechanical strains as large as 4.5%. Also, the quality factor was unaffected during bending of the substrates (Figure 3f). This robustness makes flexible, protein based WGM resonators an attractive platform to be employed in future applications such as biologically integrated sensing. Finally, to utilize the strong thermo-optic response of structural proteins, we constructed an all-optical on/off switch using protein based WGM structure (Figure 4a)
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Figure 4. All-optical switch based on thermal shift of WGMs in an all-protein microtoroid resonator. (a) Initially the signal light in the 980 nm band is coupled to the resonator such that its transmission is zero and the control light in the 1450 nm band is set off-resonant so that its completely transmitted (left). When the control light is brought to on-resonance, cavity temperature increases and the WGM in the 980 nm band is also shifted bringing the signal to off-resonant. As a result, the transmission of signal increases to one whereas that of the control decreases to zero (right). (b) Normalized transmission of the signal through the waveguide-coupled all-SRT microresonator when the control field of power 1.44 μW was modulated by a square wave bringing it on- and off-resonance. (c-d) Ratio of normalized transmission for the signal field at various input (c) and circulating (d) powers of the control field. The minimum power of the control field required to observe switching was 16.43 μW for a silica microtoroid and 1.44 μW for an all-SRT microtoroid. where the transmission of a signal field through a waveguide-coupled all-SRT microresonator was switched between "on" (i.e., unit transmission) and "off" (i.e., zero transmission) states by a control field via the thermal response of the SRT protein. We first identified two resonance modes: one at 974.8 nm and the other at 1451.7 nm to serve as the resonance modes for the signal and control fields, respectively. The signal field provided by a tunable laser with emission in the 980 nm was set on-resonance with the mode at 974.8 nm, and the waveguide-resonator coupling for this mode was set at the critical coupling so that the signal had zero transmission in the absence of the control field (Figure 4a, left). The wavelength of the control field provided by a tunable laser in the 1450 nm band was first set detuned from the resonance at 1451.7 nm such that normalized transmission of the control laser was close to unity, and the signal was kept at its zero-transmission state (Figure 4a, top). When the control laser was modulated by a square wave and its wavelength was switched between its detuned state (i.e., close to unity transmission, Figure 4a, top) to on-resonance state (i.e., close to zero transmission, Figure 4a, bottom), we observed that the transmission of the signal field switched between its “off”
(Figure 4a, top) and "on" (Figure 4a, bottom) states, respectively. This can be understood as follows: When the control field is on-resonance, the field built up inside the cavity is absorbed by the protein material and transformed into heat, which in turn shifts the resonance modes through the strong thermo-optic and thermal expansion responses of the SRT material. As a result, the signal field becomes off- resonant and its transmission increases to a value close to unity. When the control field is adjusted to be off-resonant, the cavity cools down and the resonances return back to their initial positions. As a result, the signal field becomes on-resonant again and its transmission drops to near-zero value. Figure 4b presents the experimental results depicting all-optical switching with 41 dB isolation at a control field power of 1.44 μW (i.e., circulating power 0.129 mW) using an all-SRT microresonator. Repeating this experiment at different input powers of the control field, we obtained the switching ratio versus control power for all-SRT and all-silica microresonators (Figure 4c and 4d). It is clear that an all-SRT switch has an extremely low power budget compared to an all-silica switch. The best isolation we achieved with the all-silica switch was 25 dB with a control power of 16.43 μW, correspond-
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ing to a circulating power of 219.76 mW (Figure 4c and 4d). Comparing the required pump powers to achieve switching with a desired isolation, we find that the allprotein resonator is fourteen times more energy efficient (Figure 4d). Since the quality factors of the SRT and silica resonators are between 104 to 106, the circulating power inside the silica microtoroid is thousand times more than that of the SRT microtoroid. This striking performance is both due to the strong thermo-optic coefficients and negative coefficient of thermal expansion of SRT proteins. Our results show that protein-based photonic devices can be used for low power consumption applications. Although our previous work39 on SRT based WGM resonators aided in optical characterization of SRT proteins, this study provides the first energy efficient protein based switch. In summary, proteinaceous WGM microcavities demonstrated here provide a platform to manipulate photons within microscale volumes of structural proteins at high power efficiency. As illustrated, protein-based materials could provide the next generation of adaptive photonic structures whose optical, mechanical and thermal properties can be engineered at the molecular level for energy efficient applications in photonics. This approach will also expedite the design, fabrication and synthesis of ecofriendly, recyclable, flexible materials and devices.
kit). PDMS and curing agent were mixed in a 10:1 ratio, stirred and degassed under vacuum for 30 minutes. The mix was poured on the silicon master and it was cured at room temperature for 24 hours. Due to mechanical processing requirements (e.g., viscosity in solvent depends on protein molecular size and solubility), the shape and size of WGM resonators varied. Unaged, aged and recombinant SRT microresonators had respectively major diameters of 54 μm, 72 μm and 80 μm and a minor diameter of 9 μm, 9 μm and 10 μm which were used in quality factors and thermo-optic coefficient measurements. For ThT dye experiments, we used larger SRT microresonators (i.e., 100 μm). Protein casting: SRT solution (either recombinant or native protein) was prepared by dissolving 50 mg/mL of protein in hexafluoro-2-propanol (HFIP). The solution was sonicated for 1 hour and vacuum-filtered in a 4-8 µm mesh size filter. 80 µL of SRT solution were poured into a PDMS toroid mold in successive 20 µL additions 1 minute apart. After the last addition, the HFIP was evaporated at room temperature in the fume hood for 5 minutes and the moldSRT system was immersed in butanediol (plasticizer) at 80°C (above Tg) for 30 minutes. The thermoplastic SRT film was peeled off with tweezers and excess butanediol was removed by rinsing with ethanol. Characterization of WGM resonances: Taper-fibers fabricated by heating and pulling single mode fibers were used to couple light from a tunable laser into and out of the microtoroid resonators. The coupling distance between the taper and the resonators was controlled by a nanopositioning system. Four tunable lasers with emission in the wavelength bands of 670 nm, 780 nm, 980 nm and 1450 nm were used to characterize the resonators in different bands of the spectrum. Their wavelengths were linearly scanned around resonances of the resonators. The realtime transmission spectra were obtained by a photodetector connected to an oscilloscope. The scanning speeds and powers of the lasers were optimized to eliminate thermally-induced (due to heat build-up in High-Q resonators) line-width broadening and narrowing effects. Typical operating conditions for scanning speed and laser power were 10-20 nm/s and 15 μW, respectively. Mechanical characterization: Protein films were prepared by casting 50 mg/mL SRT/HFIP solution on PDMS molds, resulting in 40x8x0.03 mm rectangular films (length, width, thickness). The samples were analyzed in a dynamic mechanical analysis instrument (TA 800Q DMA) with a three-point bending clamp of 20 mm length between supports. Strain ramp experiments were performed at room temperature at a rate of 250 µm/min with a preload of 0.02 N. The flexural modulus was calculated as described in ASTM D790 – 10. Fluorescence imaging: Thioflavin-T (Sigma) aqueous solution is prepared at 0.125 μM concentration. The absorbance and emission spectra are measured using a fluorescence microscope (Zeiss LSM 5 PASCAL system coupled to a Zeiss Axiovert 200M microscope) at 458 nm wavelength.
METHODS Native squid ring teeth (SRT): European common squid (Loligo vulgaris) were caught from the coast of Tarragona (Spain). The squid ring teeth (SRT) were removed from the tentacles, immediately soaked in deionized water and ethanol mixture (70:30 ratio v/v) and vacuum dried in a desiccator. Expression and characterization of recombinant squid ring teeth (SRT) proteins: The SRT protein family is comprised of SRTs of different size that exhibit different physical properties. Heterologous expression of the smallest (~18 kDa) SRT protein extracted from Loligo vulgaris (LvSRT) was performed using the protocols described earlier.32 Briefly, the full-length sequence was cloned into Novagen’s pET14b vector system and transformed into E. coli strain BL21(DE3). Recombinant SRT expression was achieved with a purity of ~90% and an estimated yield of ~50 mg/L. The yield increases approximately ten-fold (i.e., ~0.5 g/L) in auto induction media. The size of the protein was confirmed via an SDS-page gel (Fig. 2d). WGM resonator fabrication: Details on the Si master preparation and etching for micro-WGM fabrication were reported earlier.20 Briefly, the microtoroids were fabricated from a 2 μm thick oxide layer on a silicon wafer. First, series of circular pads of 80 μm in diameter were created through a combination of standard photolithography technique and buffered HF etching. These circular pads serve as etch mask for isotropic etching of silicon in XeF2 gas chamber, leaving under-cut silica disks supported by silicon pillar. The microdisks were then selectively reflowed using a 30W carbon dioxide (CO2) laser to form a toroidal shape. A negative mold was made from polydimethylsiloxane (PDMS, Dow Corning Sylgard® 184 silicone elastomer
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SRT fibers: Fibers were obtained through an electrospinning process that utilized a conventional horizontal setup from Dr. Seong Kim’s laboratory at the Pennsylvania State University. The schematic design consisted of three components (Fig. S6): a syringe for supplying the SRT solution, a collecting platform for accumulating the array of fibers, and a voltage source for generating the necessary electric field. A standard procedure began with the preparation of an approximately 50 mg/mL solution of SRT in hexafluoro2-propanol (HFIP). Once fully dissolved, 0.5 mL of the solution was drawn into the 1 cm diameter plastic syringe, whose 22-gauge bevel tip needle was previously cut perpendicularly to its cylindrical axis, straightening the eventual orientation of the fiber jet. Placing the syringe securely in the pumping device (PHD 2000 Programmable, Harvard Apparatus, Holliston, MA) contained in the ventilation hood, the collecting platform was then configured. Supported by a ring stand and clamps, the collecting platform, constructed from cut pieces of aluminum held 0.8 cm apart by glass slides and epoxy, was positioned such that its center was aligned with the syringe needle, 6.5 cm away from the tip. With the setup complete, the syringe needle was attached to the 10 kV source (HV Power Supply, Gamma High Voltage Research, Ormond Beach, FL), and the aluminum plates of the collecting platform were grounded. After establishing the voltage difference, the pumping device was activated with an infusion rate of 250 μL/min. Following the discharge of approximately 0.25 mL of the solution, the pumping process was terminated and the voltage source detached. To ensure that the HFIP evaporated completely, five minutes were allowed for drying, and the collecting platform was then removed and inspected for fibers between its plates.
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ACKNOWLEDGMENT MCD, AP, HJ and RS were supported by the Army Research Office under grant No. W911NF-16-1-0019, and Materials Research Institute of the Pennsylvania State University. LY, ŞKÖ, and HY were supported by the Army Research Office under grant No. W911NF-12-1-0026 and the National Science Foundation under grant No 1264997. We thank Dr. Seong Kim for providing electro-spinning set up.
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ASSOCIATED CONTENT Supporting Information Proteinaceous WGM resonator fabrication, XRD and FTIR characterization of recombinant and native SRT proteins, transmission spectra and quality factors, and electrospinning procedure for SRT protein fibers. “This material is available free of charge via the Internet at http://pubs.acs.org.”
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected]. * E-mail:
[email protected].
Author Contributions M.C.D. and S.K.O conceived the idea and designed the experiments, H.Y. and A.P-F. performed the experiments and analyzed the results. M.C.D., Ş.K.Ö and H.Y. wrote the manuscript with the help of the other authors. M.C.D. and Ş.K.Ö supervised the project.
Notes The authors declare no competing financial interest.
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