Understanding Secondary Structures of Silk Materials via Micro- and

Review Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3161−3183

pubs.acs.org/journal/abseba

Understanding Secondary Structures of Silk Materials via Micro- and Nano-Infrared Spectroscopies Jiajia Zhong,†,⊥ Yawen Liu,‡,⊥ Jing Ren,‡ Yuzhao Tang,† Zeming Qi,§ Xiaojie Zhou,† Xin Chen,∥ Zhengzhong Shao,∥ Min Chen,*,# David L. Kaplan,*,∇ and Shengjie Ling*,‡

Downloaded via UNIV OF SOUTHERN INDIANA on July 21, 2019 at 17:14:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

National Facility for Protein Science in Shanghai, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China ‡ School of Physical Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, China # Shanghai Synchrotron Radiation Facility, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China ∥ State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai 200433, China § National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230029, China ∇ Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States S Supporting Information *

ABSTRACT: The secondary structures (also termed conformations) of silk fibroin (SF) in animal silk fibers and regenerated SF materials are critical in determining mechanical performance and function of the materials. In order to understand the structure−mechanics−function relationships of silk materials, a variety of advanced infrared spectroscopic techniques, such as micro-infrared spectroscopies (micro-IR spectroscopies for short), synchrotron micro-IR spectroscopy, and nano-infrared spectroscopies (nano-IR spectroscopies for short), have been used to determine the conformations of SF in silk materials. These IR spectroscopic methods provide a useful toolkit to understand conformations and conformational transitions of SF in various silk materials with spatial resolution from the nano-scale to the micro-scale. In this Review, we first summarize progress in understanding the structure and structure−mechanics relationships of silk materials. We then discuss the state-of-the-art micro- and nano-IR spectroscopic techniques used for silk materials characterization. We also provide a systematic discussion of the strategies to collect high-quality spectra and the methods to analyze these spectra. Finally, we demonstrate the challenges and directions for future exploration of silk-based materials with IR spectroscopies. KEYWORDS: infrared spectroscopy, silk, secondary structure, synchrotron, nano-IR spectroscopy

1. INTRODUCTION

addition, animal silks, as abundant biopolymers, in nature have been of interest in a variety of task-specific fields due to their biocompatibility and biodegradability, as well as their tunable optical and mechanical properties.6,7 Especially, silk fibroins (SFs) derived from domestic and wild silkworm cocoons and genetically engineered spidroins have shown promising applications in biomedicine,8−11 optics,12,13 electronics,14−18 and environmental engineering.19−22 Modulation of SF conformations is also the basis to control the mechanical

Animal silk, one of the most attractive and unique fibers in nature, is usually described as semicrystalline polymer fibers that consist of antiparallel β-sheet nanocrystals and an amorphous matrix (includes random coil and/or helix).1,2 These structures in silks are termed as secondary structures or conformations. The secondary structures of silks are constructed from amino acid sequences (primary structure), which are composed of highly repeated alternating hydrophilic and hydrophobic domains with conservative N- and C-termini.3 Hydrophobic and hydrophilic domains along the protein chains tend to form β-sheet and random coil/helical structures, respectively.4 These organizations at the secondary structural level play a central role in determining the mechanical performance of silk fibers.5 In © 2019 American Chemical Society

Received: March 2, 2019 Accepted: June 12, 2019 Published: June 12, 2019 3161

DOI: 10.1021/acsbiomaterials.9b00305 ACS Biomater. Sci. Eng. 2019, 5, 3161−3183

Review

ACS Biomaterials Science & Engineering Scheme 1. Respective Time-Scale and Length-Scale Domains of Applicability of IR Spectroscopic Techniquesa

a

The schematic of micro-IR spectroscopy (bottom right) is reproduced with permission from ref 64. Copyright 2014 Springer Nature.

properties of the SF materials, as well as the degradation of the materials in vivo.23 To understand the structure−mechanics relationships of natural silk fibers and to rationally design SF materials, a variety of the state-of-the-art infrared spectroscopic techniques, such as micro-infrared spectroscopies (micro-IR spectroscopies for short) and nano-infrared spectroscopies (nano-IR spectroscopies for short), have been widely used to monitor the conformations and conformational transitions (assembly processes) of silk materials (both natural silks and engineered SF materials). These IR techniques provide unique and powerful toolkits to disclose the conformation of the whole silk material system with spatial resolution from the nano-scale to macroscales (Scheme 1). The structural models of animal silk fibers were substantially refined by using these characterization methods. This Review provides a critical summary of the spatial exploration of silk materials using micro- and nano-IR spectroscopies. Of note, all of the techniques mentioned in this Review are based on Fourier transform infrared (FTIR) spectroscopy, which are abbreviated as IR techniques. The FTIR spectra introduced in this Review are also abbreviated as IR spectra for short. We first summarize recent progress in understanding the conformations and conformation−mechanics relationships of silks. We then describe the principles of micro- and nano-IR spectroscopies and discuss their use in silk material characterization. Further, we provide a systematic discussion of the strategies used to collect high-quality spectra and the methods to analyze these spectra. The use and misuse of IR spectroscopies in silk materials characterization are also highlighted. Finally, we demonstrate the challenges and

directions for future exploration of silk materials with microand nano-IR spectroscopies.

2. SECONDARY STRUCTURES OF ANIMAL SILKS 2.1. Spider Dragline Silks. The amino acid sequences (also refer to primary structures) of spider silks are complex. This complexity stems from both the large molecular size (∼350 kDa) and the diversity of the sequence with the specie source of silk fibers. There are more than 30 000 known species of spiders that produce silks.24 And one spider often can produce seven different kinds of silks.24 Different silks usually have different amino acid sequences, and most remain uncharacterized. In this Review, we only discuss the secondary structures of Nephila spider dragline silks (abbreviate to Nephila silks), which are the most well-studied spider silk sources. The amino acid sequences of Nephila silks have highly repetitive core sequences alternating between hydrophilic and hydrophobic segments flanked by highly conserved shorter terminal domains (N- and C-termini). In the hydrophobic motifs, Nephila silks consist of (A)n (n = 4− 6) (Figure 1),25 which are also well known as the domains that form crystals. For the hydrophilic motifs, they are formed with glycine- and tyrosine-rich sequences. During natural spinning, the hydrophilic domains maintain random coil and/or helical structures, while the hydrophobic domains transition into βsheet structures via extrusion and shear flow. β-Sheet nanocrystals serve as physical cross-linkers to connect the amorphous chains to form a network of structures.26 The lattice constants of the orthogonal unit cell of β-sheet are (1.03, 0.944, 0.695) nm for Nephila silks.27 The minimum crystal dimensions of Nephila silks are reported to be 2 nm × 5 nm × 7 nm from wide-angle Xray diffraction (XRD).28 In another report, by using the Debye− 3162


Review

ACS Biomaterials Science & Engineering

Moreover, low-voltage transmission electron microscopy revealed that the distribution of the major axis length of βsheets was 13.0 ± 11.9 nm while the minor axis distribution was 5.7 ± 2.3 nm.38 Therefore, the crystal sizes of both spider silks and B. mori silks show a large distribution of sizes depending on the researcher, and there is no consensus.41 These amorphous and nanocrystal components are further organized into silk nanofibrils with a diameter of ∼20−200 nm. A single silk nanofibril has a bead-like structure connected by nonhomogeneous nanoglobules. At the macro-scale, B. mori silks are also core−shell fibers with a SF core (the major component) and a sericin shell.29−31 2.3. Antheraea Silkworm Cocoon Silks. Antheraea is a moth genus belonging to the family Saturniidae.42 Several species of this genus, such as A. pernyi and A. yamamai, are caterpillars which produce silks commonly called “tussar silk”.42 Antheraea silkworm cocoon silks (abbreviate to Antheraea silks)) have similar amino acid sequences to the partial sequence of the Nephila silks but with longer poly(A) repeat regions. Specifically, the repetitive regions of Antheraea SFs consist of (A)n (n = 11−13) alternating with glycine-rich regions with motifs of GGYG, GSGA, and GGAG (Figure 1). The longer poly(A) length in the Antheraea silk contributes to higher crystallinity compared with Nephila silks, but less than that of B. mori silks. The crystallinity tendency reflects B. mori silks > Antheraea silks > Nephila silks based on the content of β-sheet forming domains in these three silks (Table 1). Of note, as

Figure 1. Secondary structures (conformations) and typical amino acid domains in animal silks of B. mori, Antheraea, and Nephila.

Scherrer formula,29 the crystal sizes were assessed to be 5.3 nm × 4.7 nm × 6.0 nm. These amorphous and nanocrystal components are further organized into intensively stacked and highly oriented nanofibrils. At the fiber scale, silk fibers can be considered as core−shell fibers with a SF core (the major component) and a skin layer that composed of lipid, glycol and protein.29−31 2.2. Bombyx mori Silkworm Cocoon Silks. Bombyx mori silkworm cocoon silks (abbreviated to B. mori silks) consist of a heavy chain (Fib-H) with 5,263 amino acids (391.6 kDa), a light chain (Fib-L) with 262 amino acids (27.7 kDa), and glycoprotein P25 (30 kDa).32−34 The heavy chain, light chain, and P25 have been reported to assemble to a high molecular weight elementary unit,35 where the heavy chain and light chain are joined into a single branched polymer, via a covalent disulfide linkage, while only physical interactions occur between P25 and the branched polymer of heavy and light chains. The complete sequence of the heavy chain contains a highly repetitive and glycine-rich core and two structurally nonrepetitive ends (N- and C-termini).36 The repetitive core is usually divided into four typical domains: GAGAGS, tyrosinecontaining segments (Y), GAAS, and non-repetitive segments. Among these, the recurring GAGAGS segment is the building block for β-sheet nanocrystallites, while the tyrosine-containing segments cannot fit into the crystalline unit cell of the aliphatic GAGAGS segments, and may form another β-sheet crystallite that has different cell parameters.37 The lattice constants of the orthogonal unit cell of β-sheets are (0.938, 0.949, 0.698) nm.27 GAAS is thought to form a tetrapeptide β-turn, which is used to disrupt the crystalline domains of (GX)n (X is a non-glycine residue) repeats.38 The non-repetitive segments between the repeat domains are hydrophilic and may form ring structures (twisted omega shape) with the role to reverse the forward direction of the protein chain by 180° to promote the formation of antiparallel β-sheet, a structure is associated with the nanocrystal of silk proteins (Figure 1).37 X-ray diffraction has been widely used to evaluate the size of silk protein crystallites. Using this method, the size of the βsheets has been detected with a value of 21 nm × 6 nm × 2 nm.39 The crystallite size was also assessed to be 2.6 nm (hydrogen bond direction) × 3.2 nm (sheet stacking direction) × 11.5 nm (chain direction) in another report but also using XRD.40

Table 1. Comparison of β-Sheet Contents of Domestic Silkworm Silk, Wild Silkworm Silk, and Spider Dragline Silks Acquired by Different Methods β-sheet content (%) B. mori silks

wild silkworm silks

spider dragline silks

IR spectroscopy

28a (ref 109)

20−23b (refs 109, 155) 40c (ref 15)

17 ± 4e (ref 109) 36f (ref 156)

Raman spectroscopy

50a (ref 157)

45d (ref 157)

36−37e,f (ref 157)

13

60.5−62a (refs 158−161

41−50b,d (refs 155, 161, 162)

34f (ref 163) 46.5g (ref 161)

X-ray diffraction

49−56a (refs 161, 164)i 37.1a (ref 165)j 50a (ref 38)k 53.8a (ref 166)l

32−33.2b,d (ref 161)i 25.2d (ref 165)j 25.6 (ref 166)l

10−15f,h (refs 28, 167k 31.3g (ref 161)i

ordered fraction35(43)

77a

57b

29−31f

C CP-MAS NMR

a

B. mori. bA. pernyi. cA. yamamai. dS. c. ricini. eN. edulis. fN. clavipes. A. assamensis. hN. inaurata. iSynchrotron X-ray diffraction. jWideangle X-ray diffraction. kWide-angle X-ray scattering. lX-ray fiber diffraction. g

presented in Table 1, the large distribution of crystal content is obtained even though the same kind of silk and the same characterization method were used; the values obtained from XRD of B. mori silk for an example. This considerable disagreement can be attributed by both uncertainty inherent in the method and the differences between the specimens 3163


Review

ACS Biomaterials Science & Engineering Table 2. Mechanical Properties of Animal Silk Fibers, Synthetic Fibers, and Biological Materials density (g cm−3) Araneus dragline silk Araneus viscid silk Nephila clavipes dragline silk B. mori silk A. yamamai silk A. pernyi silk A. mylitta silk A. assamensis silk P. ricini silk

1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.307 1.308

modulus (GPa)

toughness (MJ m−3)

refs

10 0.5 11−13 7 9 12 8 8.5 3.6

160 150 110 70 113 65 79 95 71

168 168 169 5 15 15 170 170 170

18 2.7 850 17 15 540 26 10 56 1.3 0.8

5 130 0.001 0.9−1.55 13.8 0.0085 2.75 4.2 5.3 300 190−210

80 50 100 64 55 78 30 18 − 25 6

5 168 168 171 171 171 171 171 172 5 5

Biological Materials 120 12 60−100 4−9 10 2 150 3 190 200 50 1000 160 3 22−65 0

1.2 6−20 0.5−3 0.04 0.0011 0.002 0.5 100 20 13−21.5

6 5−9 0.5−0.8 15a 2 4 60 − 4 (3−4) × 10−3 a

5 173 173 174 5 168 168 174 175 175

strength (MPa)

strain (%)

Animal Silk Fibers 1100 27 500 270 880−970 0.17−0.18 600 19 650 31 460 23 513 26 564 26.4 400 27.5 Synthetic Fibers

nylon fiber Kevlar 49 fiber silicone rubber polypropylene (UIstron) PET polyurethane PVA PE (Courlene X3) PLA carbon fiber high-tensile steel

tendon collagen wood (longitudinal) wood (transverse) skin elastin resilin wool, 100% RH cellulose fiber bone coral

1.14 1.44 0.98 0.89−0.91 1.29−1.4 1.12−1.24 1.19−1.31 0.95−0.96 1.2−1.3 1.5−2 7.82

0.6−0.8 0.6−0.8 1.3

1.3 1.5 1.8−2.08 0.89−1.5

950 3600 50 585 610 370 210 320 350 4000 1650

a

Unit is kJ/m2.

measured. At the higher scale, as with B. mori silks, Antheraea silks consists of two SF brins that are covered by a sericin coating. The SF brins also present a highly oriented nanofibril structure with diameter roughly ∼20−500 nm.

highest tenacity (resistance to break) with a value of 508 MPa was achieved by wet-spinning of native-sized recombinant spider silk proteins produced in metabolically engineered Escherichia coli.44 The highest modulus with a value of 110 gpd was achieved by wet-spinning of soluble recombinant silk produced in mammalian cells.45 The first artificial silk with toughness comparable to native spider silk has been reported most recently by using recombinant Araneus diadematus spidroins.46,47 These differences in mechanical properties are directly related to the differences in the conformations of the different kinds of silks. Several structure-mechanics models of animal silks have been established to correlate the mechanical properties with conformations of silks. 3.1. Two-Phase Cross-Linking Network Model. A twophase cross-linking network model (Figure 2a) was first established to evaluate the roles of β-sheets and amorphous regions in the determination of the mechanical properties of Nephila silks.48 In this model, the nanocrystals with high modulus function as cross-linkers to connect amorphous chains of the proteins to form a network structure. Moreover, a third phase with an intermediate modulus was proposed to exist at the interface between the nanocrystals and the amorphous regions.

3. CONFORMATION−MECHANICS MODELS OF ANIMAL SILKS Different silks typically feature different mechanical properties (Table 2). For instance, Nephila silks have a remarkable mechanical performance with a unique combination of strength, modulus, and toughness with values of ∼1−2 GPa, ∼10 GPa, and ∼350 MJ m−3, respectively.5 They are toughest materials in both natural and engineered polymer material fields.5 Although the mechanical properties of B. mori silks are inferior to the Nephila silks, the breaking stress and toughness of silk fibers directly reeled from B. mori silkworms were similar to that of benchmark of Nephila silks, further such silks pulled from the animal at different rates were mechanically superior to Nephila silks.26 Further, the mechanical properties (e.g., Young’s modulus and toughness) of Antheraea silks were between those of Nephila silks and directly reeled B. mori silks.43 A series of recombinant silk fibers have also shown mechanical performance comparable with natural silks. Specifically, the 3164


Review


Figure 2. Secondary structure−mechanical models of animal silks. (a) Two-phase cross-linking network model by Termonia.48 Adapted with permission from ref 48. Copyright 1994 American Chemical Society. (b) Mawell model by Krasnov et al.51 Adapted with permission from ref 51. Copyright 2008 American Physical Society. (c) Hierarchical chain model by Zhou et al.54 Adapted with permission from ref 54. Copyright 2005 American Physical Society. (d) String of beads model by Porter et al.55 Reproduced with permission from ref 55. Copyright 2009 John Wiley and Sons. (e) Nanoconfinement model by Buehler et al.1 Reproduced with permission from ref 1. Copyright 2010 Springer Nature.

curves of B. mori silks, whether this model can be extended to other animal silks has not yet been clarified. A hierarchical chain model was further proposed to explain the nonlinear force− extension response of spider capture silks (Figure 2c).54 The simulation results agreed well with the experimental observations, but the application of the model to Nephila silks or other silkworm silks was not discussed. 3.3. String of Beads and Order-and-Disorder Model. Although the SF chains in animal filaments are highly oriented, they do not differ significantly in modulus parallel to the fiber direction and perpendicular to the fiber direction. Based on this phenomenon, a string of beads model was proposed in which specific fragments of silk protein chains were folded to form nanobeads, the scale of which determined the strength of the silk (Figure 2d).55 The mean field method was also employed to simplify the complex SF structure into a crystal formation (alanine-rich for Nephila silks and Antheraea silks or GAGAGS for B. mori silks) and amorphous formation (glycine-rich) chain, and the secondary structure of Nephila silks was represented by an order-and-disorder fraction. The order fraction and disorder fraction identified with a crystal of the alanine-rich motifs and amorphous regions of the glycine-rich motifs in the silk proteins.55 This simplified approach explains the molecular scale structural origins of macroscopic (thermo-)mechanical properties in silks and enables the prediction of changes in

The mechanical properties calculated from this model agreed well with the experimental tensile results. However, the modulus of the β-sheet in this model was proposed to be as high as 160 GPa, which is the theoretical value that can be achieved when the protein chains in the crystallites are fully extended. However, the modulus obtained from X-ray diffraction (XRD) experiments or molecular dynamics (MD) simulations are much lower than this value.49−51 Based on the fact that the nanocrystals are highly oriented along the fiber axis, the two-phase cross-linking network model was improved and the crystalline phase was divided into two components: a highly oriented and tightly packed crystalline phase and a poorly oriented and non-closepacked crystalline phase.52 On the basis of nuclear magnetic resonance (NMR) characterization, this model was further refined by involving staggered and oriented 31-helix in β-sheet and amorphous region networks.53 3.2. Viscoelastic Model. As a biopolymer fiber, the tensile behavior of animal silks, B. mori silks for instance, can be simulated by a classical three-parameter Maxwell model (Figure 2b).51 β-Sheets in silk fibers can be modeled as springs and connected in series with a secondary spring that represents the elasticity of the amorphous regions. These springs are further connected in parallel with a viscoelastic Maxwell model, which represents the amorphous regions of the silk fibers. Although this phenomenological model fits well based on the tensile 3165


Review

ACS Biomaterials Science & Engineering properties induced by environmental conditions.56 The simulation results obtained from the order-and-disorder fraction models agrees with the results from experimental mechanical and dynamic mechanical analyses. This order-and-disorder model also gives a numerical relationship between the ordered fraction in the model and the super-contraction rate of some silks in water.57 3.4. Nanoconfinement Model. A series of MD models was developed to evaluate the effects of the size of β-sheet nanocrystals on mechanical properties (strength and modulus, for example) of Nephila silks (Figure 2e).1 MD simulations disclosed that the confined β-sheet sizes were crucial to achieving higher stiffness, strength, and toughness than larger nanocrystals. Specifically, when the β-sheet nanocrystal size was larger than a critical scale (4 nm in length for Nephila silks1), the deformation mode of the nanocrystal changed from a favorable shear mode into an unfavorable bending mode. In the bending mode, the competing hydrogen bonds were in varying extents of compression and tension across the β-strands, which leads to crack-like flaws. When there were open cracks in large nanocrystals, water molecules enter the crystal regions that are under tension and split the crystal, leading to early catastrophic failure through brittle fracture. In contrast, smaller sheardominated crystals feature self-healing ability unless complete rupture occurs. Hydrogen bonds can reform during stick−slip shear motions, significantly increasing the total dissipated energy and protecting hydrogen bonds from adverse exposure to surrounding water.

Figure 3. Micro-IR spectroscopy. Reproduced with permission from ref 64. Copyright 2014 Springer Nature.

producing an IR spot and specifying the area of the sample to be measured. The objective can be further divided into visible objective, IR objective, attenuated total reflectance, and grazing angle objective. The visible objective in this system is to find the sample or the interesting sample area which has the same function as a conventional visible objective. The IR objective is to focus the IR light to the sample and collect the reflected or partially scattered light to the detector in the reflection mode. The condenser is only used in the transmission mode to focus the divergent light. Both the IR objective and the condenser are Schwarzchild reflecting elements. The detector is another important element in the IR microscopy. Liquid nitrogencooled mercury−cadmium−telluride (MCT) detector is the most advanced detector and has been used widely in micro-IR spectroscopy system.65 The MCT detector is about 15 times more sensitive than the deuterated triglycine sulfate (DTGS) detector.66 However, the DTGS detector has a larger useful wavelength range than that of MCT detector. The wavelength range of the DTGS detector with a KBr window and PE window can reach to 12 500−350 cm−1 and 700−10 cm−1, respectively, while the largest wavelength range of the MCT detector can only reach to 11 700−400 cm−1.66 Focal plane array (FPA) detectors have also been employed in the micro-IR spectroscopy.67 Some MCT FPA detectors contain 64 × 64 pixels, and each pixel can be used as a separate IR light source to collect the spectrum of the sample. Therefore, 4096 IR spectra can be collected at the same time.68 With this imaging technology, the fidelity of the images is determined by the number of pixels on the FPA detector, and only a few seconds is required for one IR image acquisition. Transmission mode and transflection mode (also known as reflection−absorption) are two of the most common experimental methods in micro-IR spectroscopy (Figure 4a). The

4. SPATIAL-RESOLVED IR SPECTROSCOPIES IR spectroscopies involve the interaction of IR radiation with materials, including a range of techniques (IR spectroscopy, micro-IR spectroscopies, synchrotron micro-IR spectroscopies, and nano-IR spectroscopies) with different test modes (absorption, emission, and reflection). As with other vibrational spectroscopic techniques, such as Raman spectroscopy and CD spectroscopy, IR spectroscopy is sensitive to the molecular vibrations of chemicals.58−60 Samples for IR measurements can be solid, liquid, or gas. The method or technique of IR spectroscopy is conducted with an instrument called an IR spectrometer (or spectrophotometer) to produce an IR spectrum. According to the characteristic IR absorption band, characteristic functional groups and conformations (for protein samples) can be identified.61,62 The vibrational frequencies of one given group depend on the bond strength and are also sensitive to the surrounding group environment.63 IR spectroscopies can be further divided into near-, mid-, and far-IR spectroscopy according to the IR absorption wavenumbers (or wavelength).58 The higher-energy near-IR, ∼14 000−4000 cm−1 (0.8−2.5 μm in wavelength), is able to excite overtone or harmonic vibrations. The mid-IR, ∼4000− 400 cm−1 (2.5−25 μm) is the region of fundamental vibrations and this region is associated with rotational−vibrational structure. The far-IR, ∼400−10 cm−1 (25−1000 μm), lying adjacent to the microwave region, has low energy and may be used for rotational spectroscopy. 4.1. Micro-IR Spectroscopy. Micro-IR spectroscopy, a technique that couples microscopy with a Fourier transform IR spectrometer, has been developed to measure the micro-sized samples (Figure 3).64 A micro-IR spectroscopy system mainly consists of an aperture, objective, condenser, glass eyepiece and camera, detector, compensation ring, sample stage, and window material for microscopy. The aperture is responsible for

Figure 4. Three experimental methods in micro-IR spectroscopy: (a) transmission mode and transflection mode, and (b) attenuated total reflection (ATR) mode. 3166


Review

3167

F F F

From ref 69. Abbreviations: D, diamond; C, carbon graphite; F, fragile (knoop < 200); O, opaque, dark to visible light (only reflected light is visible); T, toxic (if cell death >10 % total cells). Entries shown in bold indicate unsuitable substrate parameters for high-quality FTIR imaging of cells.

a

1 600 − 36 2±2 107 ± 37 O 0.0005 720 − 13 6±4 164 ± 44 F 0.45 720 − 30 3±1 157 ± 39 O 0.5 750 − 31 4±2 108 ± 34 F 1 100 − 7 1±1 65 ± 24 F 2 900 − 3 21 ± 11 7±5 F/T 2 1100 − 3 15 ± 14 14 ± 11 T 2 750 − 15 23 ± 8 9±8 F/T 2 600 − 17 19 ± 6 11 ± 9 F/T 2 200 + 7 2 490 + 5 2 520 + 2 680 + 5

thickness (mm) IR low-frequency cutoff (cm−1) hygroscopic energy loss (%) biotoxicity (% dead cells) adhesion (cells/mm2) remark

BaF2 CaF2 ZnS ZnSe CsI KBr KCl NaCl

Table 3. Main Characteristic of the IR-Transparent Substrates Tested for the 18-h Cell Culturea

D

Si-FI

Si-O

Si3N4

Ge

C

transmission mode is suitable for samples with a smooth surface and thin thickness (∼5−10 μm). Thin films can be directly used for measurements; however, supporting substrates are required in most cases. The most commonly used substrate (or window) materials are barium fluoride (BaF2), calcium fluoride (CaF2), potassium bromide (KBr), and diamond. These materials are transparent or partly transparent in the mid-IR region (Table 3).69 In the transflection mode, the incident light passes through the film and is then reflected from the substrate, then the light is collected by the IR objective. This method is usually suitable for characterization of the surface of thin films. The most commonly used substrates materials are highly reflective materials, such as gold-coated materials or low-E glasses. However, in practice, the spectra collected from transmission and transflection mode differ because the real part of the complex dielectric function affects the reflectance and transmittance of light. At normal incidence only the imaginary part of the complex dielectric function causes absorption, while at tilted incidence the real part of the dielectric function gains influence. As a result, the reflectivity spectrum in the transflection mode usually causes drift of the baseline, and this spectral deformation can be corrected by the Kramers−Kronig transform.70,71 A variety of accessories, such as micro-attenuated total reflection (micro-ATR), IR polarizer, microfluidic chips, in situ tensile and temperature control devices, have also been widely used in micro-IR spectroscopy. In the approach of micro-ATR, the IR light penetrates to the sample for only a few micrometers through the internal reflection element (Figure 4b).72 This characteristic permits the micro-ATR to measure the aqueous samples (e.g., protein crystallization,73 and live cells74) and image samples with a rough surface or polymer materials with high IR absorbance.75 IR polarizers are widely used to measure IR dichroism of functional groups and crystals. For instance, the orientation of β-sheet along the animal silk fiber axis has been investigated by this method.76,77 Microfluidic chips which are suitable for IR measurements have also been developed in life science and material fields, especially for IR imaging of living cells and chemical reactions.78 In situ temperature control devices and tension devices are also used in polymer science to investigate structural responses to external stimuli, such as temperature, force and strain.79,80 To date, these accessories have rarely been used to characterize silk materials, but they would be very useful in further understanding the structure− property relationships of silk materials. Another advantage of micro-IR spectroscopy is the efficiency in high-pressure characterization. For high-pressure IR measurements, a diamond anvil cell, an ultra-hard and highly IR transparent material, is used to pressurize the samples and to transmit the IR light. The higher the pressure, the smaller aperture size required, in line with the characteristics of the synchronous light source.82,83,88 4.2. Synchrotron Micro-IR Spectroscopy. Synchrotron light sources provide ultrahigh-brightness electromagnetic radiation, the energy of the synchrotron radiation can range from the ultraviolet to X-ray regions.81 This is a powerful method to investigate the structure of materials. In terms of IR region, synchrotron can produce a wider spectral range, extending to IR and longer wavelengths (terahertz region, e.g., up to 2 mm for the Diamond Light Source).84 The synchrotron IR light can be generated by bending magnet and edge radiation from the storage ring.85 In synchrotron micro-IR spectroscopy, the facilities are similar to conventional micro-IR spectroscopy, while the internal light source of micro-IR spectroscopy is

0.5 100 − 17 4±2 121 ± 48 O



Review


Figure 5. IR imaging with a multi-beam synchrotron IR source. (a) Schematic of the beamline optical paths. M1−M4 are mirror sets. (b) Full 128 × 128 pixel focal plane array image with 12 overlapping beams illuminating an area of ∼50 μm × 50 μm. Scale bar, 40 μm. (c) Visible-light photograph of the 12 beams projected on a screen in the beam path (dashed box in panel a). Scale bar, ∼1.5 cm. (d) Long-exposure photograph showing the combination of the 12 individual beams into the beam bundle by mirrors M3 and M4. Scale bar, ∼20 cm.89,154 Reproduced with permission from ref 89. Copyright 2011 Springer Nature.

Figure 6. Basic principles of (a, left) AFM-IR and (b, right) s-SNOM. The schematic is adapted from Anasys Instruments Inc.

replaced by synchrotron IR light.81 The brightness of the synchrotron light is 100−1000 times higher than the blackbody radiation.86 Benefiting from this unique advantage, the spatial resolution of synchrotron micro-IR spectroscopy is able to reach the diffraction limit. Accordingly, this is a powerful technique to characterize micrometer-sized samples or sample areas (∼5−20 μm for proteins, decided by the diffraction limit of the specific wavenumbers). Compared with other IR spectroscopic techniques, synchrotron micro-IR spectra have higher signal-to-noise ratios for micrometer-sized samples and thus can be available to investigate biomaterials in the several micrometer scales.87 Recently, the FPA detector has been coupled with synchrotron micro-IR spectroscopy (Figure 5).64,89 In this system, a wide aperture of synchrotron IR light was split into multiple beams and then refocused to match the entrance aperture of the IR microscopy.90,91 Due to the high brightness of synchrotron IR light, each pixel in the FPA detector is ∼1000 times higher energy than conventional light. Therefore, FPA-based synchrotron micro-IR spectroscopy can image a micron-sized sample with diffraction-limited resolution and high signal-to-noise ratio in only a few minutes.69 The dynamics of bioreactions and micrometer-sized in situ biomineralization92,93 have been studied by using the FPA detector-based synchrotron microIR spectroscopy.94 However, the problem of IR heating to the sample under synchrotron illumination cannot be ignored. Mid-

IR photons are too low in energy to break bonds directly or to cause ionization; however, IR heating can lead to a temperature increase and a wavenumber shift of the biological samples.95,96 4.3. Nano-IR Spectroscopy. Due to the diffraction limit of the IR light, micro-IR spectroscopy is unable to reach the nanometer-scale even when coupled with synchrotron light source. Therefore, two nano-IR spectroscopies, i.e., atomic force microscopy (AFM)-IR spectroscopy and scattering scanning near-field optical microscopy (s-SNOM) techniques, were developed to overcome the spatial resolution limits of microIR spectroscopy, making the characterization of the nano-sized materials possible. Most recently, synchrotron light sources have been involved in nano-IR spectroscopy. The resultant synchrotron nano-IR spectroscopy provided a brilliant and continuous broadband IR source.97,98 AFM-IR spectroscopy directly detects light absorbed by the sample through the AFM probe tip. The absorbed IR light causes the temperature to rise and in turn results in the thermal expansion of the sample. The thermal expansion then has a short duration of force to the AFM cantilever, and in turn, causes the cantilever to oscillate (Figure 6a). The degree of thermal expansion mainly depends on the sample absorption coefficient. Accordingly, IR absorption spectra can be directly obtained by measuring the amplitude of the cantilever oscillation that is related to the IR source wavelength. This technique is preferred for measuring soft materials with high thermal expansion. With 3168


Review

ACS Biomaterials Science & Engineering the development of laser sources and AFM tips, much more sensitive AFM-IR spectroscopy has been achieved. Resonanceenhanced AFM-IR adopts metal-coated probe tips and quantum cascade laser (QCL) with higher repetition and can provide continuous wave oscillation of the cantilever with a main advantage in measuring small and thin materials.99−101 The detailed AFM-IR principles and applications have been mentioned in several critical reviews.102,103 The s-SNOM technology is based on the near-field optical technology, replacing the traditional aperture with scattering points. An illuminated particle will form an enhanced light field around it, and this near field will be changed by the sample nearby.104 The interaction of the near fields between the sample and the scattering point lead to the change of far field which contains the optical information on the sample. An AFM probe tip is used as a scattering point, and the resolution of the sSNOM is determined by the radius of the AFM probe tip. Therefore, s-SNOM detects IR light scattered by nanometerscale regions under the AFM tip. The scattered field contains rich information which depends on the complex optical properties of the AFM tip, sample, and substrate (Figure 6b). Careful modeling of the tip and sample interactions may be needed to interpret the data results.101 According to this basic working principle, s-SNOM technology is suitable for a variety of materials, especially for hard materials with high light scattering ability.105

coil to β-sheet, which is also a common strategy to produce water-insoluble silk materials. The film-casting method, therefore, is generally applied to investigate the effects of solvents and environmental stimuli on the structure of silk materials rather than to directly study the structure of silk protein within silk fibers. Water molecules have strong IR absorption at 3000−3750 cm−1 (antisymmetric and symmetric stretching vibration of H2O) and 1600−1700 cm−1 (bending vibration of H2O) (Table 4). In most cases, water vapor in the optical path is even more

5. EXPERIMENTAL STRATEGIES TO COLLECT HIGH-QUALITY IR SPECTRA OF SILK MATERIALS 5.1. Transmission Mode. The transmission mode does not require a separate accessory. The sample is directly placed in the IR beam and as the IR beam passes through the sample the transmitted energy is measured and a spectrum generated. However, before transmission measurements, the analyst must often prepare the sample into a pellet or film format. One routine approach is to finely grind the SF powder with a purified salt (usually potassium bromide) with the aim of eliminating scattering effects from large crystals. This powder mixture is pressed into a translucent pellet through which the IR light can pass. In order to avoid pressure-induced conformational transformations of SF, this method generally needs to be carried out at room temperature with pressure lower than 500 MPa. The second approach is to use a microtome to cut a thin film (∼5−20 μm) from a solid SF sample. This is an effective way to analyze the internal structure of 3D samples because the integrity of the structures is preserved. A final and the most used method for SF characterization is “film casting”. In this method, the animal silk is first dissolved into an aqueous solution. A drop of this solution is then cast onto a polyethylene (PE) plate or onto the surface of a CaF2 or BaF2 cell and allowed to dry in air. The key to this method is to ensure that the film is not too thick to hinder light passage. The thickness of the SF film for transmission mode characterization is ∼5−20 μm. For near-IR measurements, thicker specimens are allowed since the combination and overtone vibrations absorb much more weakly than that of vibrations in mid-IR region. It is worth noting that β-sheets in silk fibers are destroyed during the dissolution processing, thus the SF films produced from filmcasting processing appear as random coil structures when the films are formed at room temperature. In these cases, different solvents (acetone and alcohols) and environmental inputs (such as heating and water vapor-annealing) are often used to induce the conformational transition of silk proteins from the random

problematic than liquid water, as the combination of rotational and vibrational bands produces multiple absorbance peaks between 1200−2000 and 3200−4000 cm−1.106 In particular, the absorption of water at 1650 cm−1 overlaps with the absorption of amide I band of proteins, so it is necessary to eliminate water and atmospheric water vapor in the surrounding conditions. In addition, atmospheric CO2 in the optical path also affects the quality of the resultant spectra, especially, peaks around 2330 cm−1. The changes in CO2 concentration can occur quite easily, for instance, due to the spectroscopist breathing while loading a sample. Nitrogen purging and evacuation to the entire light path of the instrument are often required during measurement. Table 4 lists the absorption peaks and absorption coefficients of water in the mid-IR region as well as the optimum path length for obtaining the best signal-to-noise ratio.107 Besides the measurement conditions, the morphologies of the samples are another key factor that can affect the quality of the spectrum. Nephila silks and B. mori silks, with their diameters ∼2−50 μm, is equivalent to IR wavelength. Therefore, the IR light will scatter when passing through the fibers. As a result, the collected IR spectrum (usually from micro-IR spectroscopy) contains both structural information on the sample as well as signals caused by IR scattering. The IR scattering, in this case, can be solved by the Mie solution (Figure 7), which describes the scattering of an electromagnetic plane wave by a homogeneous sphere.108 Mie scattering causes the spectrum to produce a baseline of sinusoidal oscillations, resulting in the distortion of the peak position and the intensity. To address this problem, paraffin oil can be used to wet the sample to decrease scattering due to the cylindrical geometry of the fibers. Although paraffin oil has absorption at 2800−2950 and 1450 cm−1, these two bands do not overlap with the amide I and amide III bands of SFs. Another strategy is to press the silk fibers (or other irregular silk materials) into a flat shape to remove IR scattering. However, some in situ experiments, such as in situ tensile approaches, would be affected by using this approach.

Table 4. Vibrational Spectroscopic Data of H2O and D2O vibrational mode

ϖ0/cm−1

ε0/M−1 cm−1

A10 μm/a.u.

lopt/μm

H2O s, H2O as H2O b H2O b+η D2O s, D2O as D2O b D2O b+η

3404.0 1643.5 2127.5 2504.0 1209.4 1555.0

99.9 ± 0.8 21.8 ± 0.3 3.50 ± 0.1 71.5 ± 0.4 17.4 ± 0.2 1.90 ± 0.05

5.53 3.6 0.194 3.94 0.962 0.105

0.8 3.6 22.4 1.1 4.5 41.2

Data from ref 107. Abbreviations: ϖ0, peak frequency, expressed in wavenumber; ε0, molar absorption coefficient; A10 μm, absorbance for a 10 μm path length; lopt, optimal path length, at which the S/N ratio is maximized; s, symmetric stretching mode; as, antisymmetric stretching mode; b, bending mode; b+η combination bending and libration mode.

a

3169


Review


cause different degrees of scattering, resulting in slight changes in the peak position.111 However, by contrast, the limited range of the evanescent wave can also be used beneficially, such as, for surface measurements. For example, the ATR element may provide a surface to which protein from solution specimens can adhere, thereby producing a spectrum that is not representative of the bulk. Moreover, the ATR mode allows the freely accessible surface of the ATR device simultaneous sample preparation or manipulation and in situ measurements.112,113 5.3. Solution Samples. To monitor the conformation transition of SF or to detect in situ reactions, solution samples are required. However, as mentioned earlier, water molecules have strong IR absorption in the mid-IR region. Especially, absorption intensities at 1650 and 3200−3600 cm−1 will be saturated when the thickness of the water layer is larger than 15 and 2 μm, respectively. Accordingly, for SF aqueous solutionbased samples, 8−10 μm is the maximum thickness, and when a higher quality spectrum is required, the thickness of the sample preferably is reduced to 3−4 μm.116,117 Time-resolved IR spectroscopy has been used to deduct the water absorption of the sample. These time-resolved spectra were collected from the same position of the sample; thus, the first spectrum can be used as a reference spectrum for deducting the water absorption by difference spectrum analysis.116,117 A more general method is to use deuterated water (D2O) instead of water (H2O) as a solvent. This method has been widely used in time-resolved experiments.116,117 The absorption of D2O is mainly at 1200 and 2500 cm−1, which correspond to the bending vibration as well as antisymmetric and symmetric stretching vibration of D2O (Table 4). Therefore, the amide I band is not affected by D2O and can be applied to analyze the conformations of SF. However, of note, compared to the amide I band of protein/H2O system, the peak assignments of amide I band in proteins/D2O system are shifted to the lower wavenumbers due to the H/D exchange. For instance, the αhelix shifts from 1654 to 1652 cm−1, the β-sheet shifts from 1633 to 1630 cm−1, while a random coil shifts from 1654 to 1645 cm−1.59,60

Figure 7. Mie correction of IR spectrum. (a) Optical image of a PC-3 cells and an IR spectrum of the smaller cell. (b) Corrected spectrum using new RMieS-EMSC algorithm. Reproduced with permission from ref 108. Copyright 2010 The Royal Society of Chemistry.

Synchrotron micro-IR spectroscopy, which combines the ultrahigh brightness of synchrotron source (usually 100−1000 times brighter than the conventional globar source) with the powerful magnification of the microscope, allows the collection of highquality spectra of single animal silk fibers with an aperture size ∼10 × 10 μm.109 5.2. ATR Mode. The sampling path length of ATR mode is independent of the sample thickness, so there is no specific need to control the thickness of the sample.110 Theoretically, ATR mode is suitable for any liquid and solid silk materials.114,115 For liquid samples, dropping a shallow amount of the material over the surface of the crystal is sufficient. For solid samples, they are pressed into directly contact with the ATR crystal, with firm clamping between sample and crystal required because the evanescent wave (i.e., the electric field associated with the IR photons) can be improved with more intimate contact. The pressing of the sample also helps to remove trapped air, which can affect the wave and distort the spectra. The signal-to-noise ratio obtained depends on the number of reflections but also on the total length of the optical light path which dampens the intensity. Therefore, a general claim that more reflections give better sensitivity cannot be made. However, the ATR mode has significant limitations. First, the penetration depth of the evanescent wave is limited; thus, ATR mode can only detect structural information on the surface layer of the sample (with a depth of 1−2 μm) and the signal-to-noise ratio of the resultant spectrum compared to transmission mode is poor.110 Additionally, a more significant issue with ATR mode measurement is that the use of ATR crystal with the different refractive index will

6. METHODS FOR IR SPECTRA ANALYSIS After collecting the IR spectra, some pre-treatment, such as baseline correction and spectra smoothing, are usually required, especially for the spectra not collected in optimized conditions. Calculation of the second derivative spectrum is a useful approach to identify the peak positions in the original spectrum and to allow more specified identity of small and nearby absorption peaks which are not resolved in the original spectrum. The minimum value of the second derivative directly corresponds to the peak positions of the original spectrum. Moreover, the second derivative spectrum can remove baseline errors. However, compared with the original spectrum, the second derivative spectrum causes a significant loss in signal-tonoise ratio. The signal is reduced approximately an order of magnitude while the noise is magnified by a factor of 6.5, provided that no smoothing is applied. Therefore, the quality demands of the IR spectroscopic data are increased over the requirements of conventional absorption spectrum analysis techniques.118 In addition to identifying the peak positions, peak-fitting of amide I or amide III is usually conducted to evaluate the content of conformations of the proteins. As an initial step, the numbers and positions of peaks used for peak-fitting are defined from the results of second derivatives or deconvolution spectra. The 3170


Review

ACS Biomaterials Science & Engineering Table 5. FTIR Band Assignments of Silk Materialsa wavenumber (cm−1)

potential energy distribution

conformation

transition moment orientation

refs

B. mori OH s and OH sc amide A and amide II

5160 4870 4860 4615 4580 4530 4430 4360 4320 4250 4170 4090 4050 4010 1700 1655 1627 1540 1530 1268 1233 553 428 387 371 335 260 125

CH s and CH sc CH s and CH sc CH s and CH sc CH s and CH sc CH s and CH r CH s and CH skeletal CH s and CH r CH s and CH skeletal amide I, CO amide I, CO amide I, CO amide II, out-of-phase CN s, and NH ib amide II, out-of-phase CN s, and NH ib amide III, in-phase CN s, and NH ib amide III, in-phase CN s, and NH ib CO opb, CαCN def, CO ib Cβ b, NCαC def, Cβ b, NH opb CO ib, NH opb, CNCα def, NCαC def Cβ b, NCαC def, CαCN def Cβ b CαCβ tw, CNCα def, H···O s H···O s

1670 1660 1624 1545 1525 1455 1445 1405 1373 1332 1306 1265 1242 1222 1168 1105 1054 965 925 892 658 620 526 448 373 330 245 118

amide I, CO s amide I, CO s amide I, CO s amide II, out-of-phase CN s, and NH ib amide II, out-of-phase CN s, and NH ib CH3 ab, CH2 b CH3 ab, CH2 b Hα b, CH2 w CH3 sb, Hα b CH3 sb, Hα b Hα b amide III, in-phase CN s, and NH ib amide III, in-phase CN s, and NH ib amide III, in-phase CN s, and NH ib Hα b CH3 r, CαCβ s CαCβ s, CH3 r, Hα b C−N s, CH3 r CH3 r, CN s CαC s, CN s CN tw, NH opb, CO opb CO ipb, CαC s Cβ b, CO ipb, CαC s Cβ b, NCαC def Cβ b, NCαC def, CαCN def NCαC def CαCβ tw, NCαC def NCαC def, CNCα def NCCα def

amide B and amide II amide A and amide III

α-helix and random coil β-sheet α-helix β-sheet

β-turn random coil and/or α-helix β-sheet random coil and/or α-helix β-sheet β-sheet random coil and/or α-helix β-sheet

∥ ⊥ ∥

∥

α-helical β-sheet

Antheraea β-turn associated with β-sheet random coil and/or α-helical β-sheet random coil and/or α-helical β-sheet

β-sheet α-helix α-helix α-helix random coil β-sheet

∥

∥ ⊥ ∥ ∥ ⊥ ∥ ⊥

∥ ∥ ∥

α-helix β-sheet β-sheet α-helix β-sheet α-helix β-sheet α-helix β-sheet

3171

⊥ ∥ ⊥

176, 122, 123 122, 176, 123 122 122 122, 122, 122, 122 122 123 109 109 109 109 109 109 109 131 131 131 134 131 131 131

177 176−181 176−181 177

181 176, 181 176, 181

109 109 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 77, 182 134 133 134 133 134 133 133 133


Review

ACS Biomaterials Science & Engineering Table 5. continued wavenumber (cm−1) 1700−1690 1655−1660 1630−1620 1265 1235 1224

potential energy distribution amide amide amide amide amide amide

I, CO I, CO I, CO III, in-phase CN s, and NH ib III, in-phase CN s, and NH ib III, in-phase CN s, and NH ib

conformation Nephila Spider β-turn random coil and/or α-helical β-sheets α-helix random coil β-sheet

transition moment orientation ∥ ⊥ ⊥ ⊥

refs 109, 109 109, 109 109, 109,

133 133 133 133

a

Abbreviations: s, stretching; sc, scissoring; b, bending; ab, antisymmetric bending; sb, symmetric bending; w, wagging; ib, in-plane bending; opb, out-plane bending; tw, twisting; r, rocking; def, deformation; ∥, IR light parallel to the fibers; ⊥, IR light perpendicular to the fibers.

Figure 8. FTIR (solid line) and second derivative (dashed line) spectra of regenerated silk protein membranes: (a) B. mori SF membrane as cast, (b) B. mori SF membrane treated with 70% ethanol aqueous solution, (c) A. pernyi SF membrane as cast, (d) A. pernyi SF membrane treated with 70% ethanol aqueous solution, (e) Nephila spidroin membrane as cast, and (f) Nephila spidroin membrane treated with 0.3 mol/L KCl aqueous solution. Reproduced with permission from ref 109. Copyright 2011 American Chemical Society.

7. PEAK ASSIGNMENTS OF ANIMAL SILKS IN THE IR REGION

estimates of the width, height, and shape of identified peaks are then used as input parameters in an iterative least-squares routine that attempts to reproduce the experimentally obtained band profile by varying these parameters. The integrated area under each peak after the final iteration is calculated as a percentage of the total band area, and this value can be taken to be the content of the particular conformation in measured protein.119

7.1. Near-IR Region. Table 5 summarizes the assignments of SF from near-IR to far-IR regions. Near-IR absorption is based on molecular overtone and combination vibrations. For protein characterization, the method is sensitive to the secondary structure and hydration state of the peptide chains. The absorption peaks of water and SF are relatively independent in near-IR regions, near-IR spectroscopy thereby provides a 3172


Review


It is noticeable that the IR peak positions are not only impacted by individual factors but by multiple factors, such as Hbonding with residual solvents as well as chain conformation and crystal morphology. Hence, the peak positions between liquidand solid-state silk proteins often differ. The attribution of specific factors to position and intensity of a peak need to be evaluate carefully to avoid the influence of other factors. In addition, for liquid state proteins, temperature can also be a factor to affect peak position through the strength of H-bonding interactions and physical properties of the solvent.128 In addition, animal silks (Lepidopteran silks, for example) number in the thousands and display a vast diversity of structures and properties. Assignment of the main bands present in animal silks between 1800 and 779 cm−1 provides an effective approach to identify and screen different silk feedstocks and fibers from a number of spider and silkworm species. The multivariate analysis of IR spectra collected from different species allowed the grouping of these silks into distinct hierarchies with a classification that agrees with phylogenetic data and taxonomy.129 7.3. Far-IR Spectra. Far-IR spectroscopy has been utilized for the characterization of peptides and polyamides since the 1960s.130 Compared to middle IR frequencies, the far IR region is more sensitive to vibrational modes from hydrogen bonds and peptide skeletons.130 However, far-IR spectroscopy has been overlooked for protein characterization. This is because most proteins, such as myoglobin, β-lactoglobulin, serum albumin, lysozyme, hemoglobin, chymotrypsinogen, subtilisin, and ribonuclease, only have weak and broad absorptions in the farIR region. By contrast, animal silks exhibit several sharp absorption peaks in this region. The far IR spectra of B. mori SF (from 200 to 700 cm−1) has also been reported as early as the 1960s, but the peak assignments in this region have not been identified in detail.131 Because of the highly repeated GAGAGS and (A)n motifs in B. mori silk and Nephila/Antheraea silk proteins, the vibrational modes of animal silk proteins in the farIR region can be assigned according to the vibrational analysis of poly(L-alanylglycine) and poly(L-alanine). In addition, the conformation sensitivity of the animal silk proteins in 100− 500 cm−1 regions can also be assigned by comparing the variation of the far-IR spectra before and after alcohol treatment.132 Figure 9a shows the far-IR spectra of as-cast and ethanoltreated B. mori SF132, where several sharp bands appeared, including 553 cm−1 (CO opb), 428 cm−1 (Cβ bend and NCαC deformation),133 335 cm−1 (Cβ bend 2), 387 cm−1 (CO ipb), 371 cm−1, and 260 cm−1 (CαCβ torsion and CNCα deformation).133 Among these peaks, 371 cm−1 is assigned to the α-helix conformation, whereas the peaks at 428 and 260 cm−1 are only present in the ethanol-treated films and are assigned to the βsheet.131 Figure 9b presents the far-IR spectra of Antheraea SF films, compared with the far-IR spectra of B. mori SF, more absorption peaks exist in the far-IR region.132 For instance, the peaks at 658, 526, and 373 cm−1 are strong and sharp in the untreated films (curve a) but almost disappear after ethanol treatment and thus are assigned to the α-helix conformation according to the assignments of the far-IR spectra of poly(Lalanine).134 Two peaks at 448 and 245 cm−1 appear, and a peak at 620 cm−1 that becomes sharp after ethanol treatment is assigned to the β-sheet conformation.135,136 Interestingly, B. mori SF and Antheraea SF have distinct absorption in far-IR frequencies; thus, far-IR spectroscopy may provide a useful tool to study the silk protein blends.

possibility to investigate the interaction between water molecules and SF.120 General assignments of polypeptides have been conducted.121,122 The 4000−5000 cm−1 is mainly the combination vibrations of C−H and N−H groups, and the frequencies larger than 5500 cm−1 is an overtone of different amide bands. The assignments confirmed near-IR spectroscopy of B. mori SF by comparing the near-IR spectra of the film before and after ethanol treatments.123 In the near-IR region, the peak at 4530 cm−1 is assigned to β-sheet conformation. The peak at 4870 cm−1 is attributed to the combination vibration of amide A and amide II, which are directly related to the abundance of the hydrogen bonds (i.e., the total number) of SF. Accordingly, from the position shift of 4870 cm−1, the amount of β-sheet in the test system also can be inferred, because β-sheet has a much higher intensity of hydrogen bonds than the random coil conformation. 7.2. Middle-IR Region. The mid-IR is the most useful region for evaluation of conformation of silks.124−127 Several amide bands, including amide A, amide B, amide I, amide II, and amide III bands exist in this region. Figure 8 presents typical IR spectra of films prepared from Nephila silks, B. mori silks, and Antheraea silks, respectively.109 Among these amide bands, amide I, amide II and amide III bands of these three silks are the most investigated and their peak assignments generally agree. The amide I band mainly stems from CO stretching (80%) with minor contributions from N−H in-plane bending; the amide II band is mainly due to the out-of-phase combination of the C−N stretching and N−H in-plane bending, while the amide III band is less clear but has been assigned by Barth et al. to the in-phase combination of the C−N stretching and N−H in-plane bending.59,60 In addition to amide bands, there are several helical conformational sensitive bands, such as 1332, 1306, and 1105 cm−1, that are detected in Nephila silks and Antheraea silks.109 Amide I, amide II, and amide III vibrations are also directly associated with the secondary structure of the protein backbones. IR spectra of films before and after alcohol or salt solution treatments are displayed in Figure 8. Alcohol or salt treatment are useful to induce the conformation transition of SF from the random coil and/or helix to β-sheet.23 Three kinds of animal silks have almost the same absorbance in amide I bands, that the main absorption occurs at 1655−1660 cm−1 in the three untreated silk protein films assigned to the random coil/helix structure. The main peak at 1620−1630 cm−1 in three silk proteins treated with alcohol/salt treatment is attributed to βsheet conformation, and a shoulder peak appeared at 1690− 1700 cm−1 are attributed to the β-turns of the hairpin-folded antiparallel β-sheet structure.109 In contrast, these three silk proteins have different peak assignments in the amide III region.124 For B. mori SF, the peak of 1233 cm−1 appears in the as-cast film and is assigned to the random coil/helical conformation, while the peak at 1266 cm−1 after the alcohol treatment is assigned to the β-sheet conformation. For Antheraea SF, 1270 and 1235−1241 cm−1 exist in the as-cast film and are assigned to α-helix and random coil, respectively, while a new peak appears at 1221 cm−1 belonging to the β-sheet conformation. The amide III band of Nephila silks has very similar assignments with Antheraea SF due to their similar amino acid sequences, where 1265, 1235, and 1224 cm−1 are attributed to the α-helix, random coil, and β-sheet structure, respectively. The detailed peak assignments of animal silks reported in literature are summarized in the Supporting Information. 3173


Review


Figure 10. Synchrotron micro-IR spectra for single B. mori, A. pernyi, and N. edulis silk fibers. Reproduced with permission from ref 109. Copyright 2011 American Chemical Society.

spectra for single B. mori, Antheraea, and Nephila silk fibers.109 All three spectra show good resolution in the amide I, II and III regions, despite the baseline distortions at 1700 cm−1 caused by the interaction of the real and imaginary parts of the dielectric function. In this condition, a correction with Kramers−Kronig relation is necessary. The deconvolution of the amide III bands provides the evaluation of β-sheet content of each animal silks. In addition, IR dichroism of polarized synchrotron micro-IR spectroscopy offers the possibility to disclose the orientation of single silk fibers.77 Further, when the macroscopic orientation of the fiber or fiber bundle is known, orientation and order of particular structural elements, such as conformation and molecular order parameter, within a fiber can be determined.135,138−141 As shown in Figure 11, the β-sheet peaks at 965 cm−1 (C−N stretching) and 1222 cm−1 (in-phase C−N stretching and N−H bending) showed dichroism parallel to the fiber long axis. Other β-sheet peaks at 925, 1054, and 1373 cm−1 presented perpendicular dichroism. Moreover, the peaks at 1405 and 1168 cm−1 showed parallel dichroism, but these peaks were not sensitive to the conformation change. Quantitatively, the orientation of certain moieties can be obtained from the angular dependence of the absorbance A(ν) at wavenumber ν which corresponds to a vibration of the molecular group under assessment. The angular dependence of the absorbance can be determined using the following function:135,138,139

Figure 9. Far-IR spectra of silk fibrion films: (a) as-cast (i) and ethanoltreated (ii) B. mori SF film; (b) as-cast (i) and ethanol-treated (ii) A. pernyi SF film. Reproduced with permission from ref132. Copyright 2018 The Royal Society of Chemistry.

8. SPATIAL EXPLORATION OF SILK MATERIALS WITH MICRO- AND NANO-IR TECHNOLOGIES 8.1. Single Silk Fibers. Conventional IR spectroscopy has been applied to monitor the structural evolution of silk fibers during stretching or super-contraction, but these measurements are conducted with silk fiber bundles rather than single fibers. Accordingly, these measurements are not ideal for insights into structure−mechanical property relationships of natural silks. For example, the stress−strain curves of spider dragline silk and wild Antheraea silkworm cocoon silks can be divided into three stages: the elastic region, the platform region, and the stressreinforced region. However, if the in situ IR characterization was conducted by using a silk fiber bundle, the structural changes of silk proteins at the stress-reinforced region cannot be monitored since the fibers in a bundle do not fracture at same strain. In addition, for silkworm cocoon silks, the variability of the fibers in structures and morphologies are intrinsic,137 so single -fiber-based characterization is better that fiber-bundle-based measurement when the focus is on the structure of the fiber. However, for the spider dragline silks, if we only pay attention to their structural characteristics instead of in situ and online experiments, fiber bundle-based measurements may also be appropriate to their structural uniformity. ATR IR spectroscopy with a custom-built sample holder has been used to analyze single silk fibers. The focused IR beam with a diameter of ∼750 μm is still larger than the diameter of single silk fiber. Synchrotron micro-IR spectroscopy is a rational technique to monitor single silk fibers qualitatively and quantitatively.109 Figure 10 presents synchrotron micro-IR

A(v , Ω) = −log10{10−A max (v) cos2(Ω − Ω 0) + 10−A min(v) cos2(Ω − Ω 0)}

(1)

where A(ν, Ω) is the peak intensity of a certain band, Ω is the polarization angle, Ω0 is the angle at maximum absorption, and Amax and Amin are the maximum and minimum absorbance, respectively. The molecular order parameter (Smol) of the corresponding secondary structure in silk fiber was calculated as follows:140 S mol

A max (v) − A min (v) A max (v) + 2A min (v)

(2)

8.2. SF Composites. As with other polymer blends, the phase behavior of SF blends is crucial for the determination of 3174


Review


Figure 11. IR dichroism of single Antheraea silk fibers. (a) S-FTIR microspectra of single A. pernyi silk fibers with different polarization angle from 0° to 90°. (b) Polar plot of the absorbance of the characteristic peaks in S-FTIR microspectra in the 1500−800 cm−1 region from single A. pernyi silk fibers. (c) Polar plot of the relative intensity of different component from the deconvolution of the amide III band in S-FTIR microspectra of single A. pernyi silk fibers. Reproduced with permission from ref 77. Copyright 2013 American Chemical Society.

Figure 12. IR image and data analysis of silk fibroin and poly(ethylene oxide) blend. The peak height or peak areas of the amide I bands can be used as characteristic signals of SF for imaging integration. IR spectrum in each pixel can be extracted from FTIR image, and deconvolution of amide I bands can provide the β-sheet content. The false color map can be reconstructed by calculating the β-sheet content in SF-rich and PEO-rich regiosn. Reproduced with permission from ref 142. Copyright 2014 The Royal Society of Chemistry.

the mechanical, optical and thermal properties of the materials. However, the conventional methods for phase behavior characterization, such as AFM, scanning electron microscopy, differential scanning calorimetry, and dynamic thermomechanical analysis, are unable to directly provide chemical structure information on these blends. In this respect, IR imaging is a

useful tool to provide both spectral and spatial information, thereby enabling spatial chemical visualization of the sample. In order to image the spatial distribution of specific component in blends, the characteristic band (a non-overlapping band or a band with much higher intensity than that of other components) of this component needs to be identified for further imaging integration. When B. mori SF and poly(ethylene 3175


Review


Figure 13. Univariate and multivariate IR imaging of SF/SPI blend film. (a) Univariate amide II image of two pieces of pristine SF and SPI film, the joint between two films are marked with a white dotted line. (b) PCA and CA imaging of the SF/SPI blend film. The mean spectra of the red and blue domains from the CA imaging are similar to the single spectra extracted from the pristine SF and SPI film. Reproduced with permission from ref 144. Copyright 2014 The Royal Society of Chemistry.

further provide β-sheet content in B. mori SF-rich (

Understanding Secondary Structures of Silk Materials via Micro- and

Recommend Documents