Aqueous multiphoton lithography with multifunctional silk-centred bio-resists

Silk and silk fibroin, the biomaterial from nature, nowadays are being widely utilized in many cutting-edge micro/nanodevices/systems via advanced micro/nanofabrication techniques. Herein, for the first time to our knowledge, we report aqueous multiphoton lithography of diversiform-regenerated-silk-fibroin-centric inks using noncontact and maskless femtosecond laser direct writing (FsLDW). Initially, silk fibroin was FsLDW-crosslinked into arbitrary two/three-dimensional micro/nanostructures with good elastic properties merely using proper photosensitizers. More interestingly, silk/metal composite micro/nanodevices with multidimension-controllable metal content can be FsLDW-customized through laser-induced simultaneous fibroin oxidation/crosslinking and metal photoreduction using the simplest silk/Ag+ or silk/[AuCl4]− aqueous resists. Noticeably, during FsLDW, fibroin functions as biological reductant and matrix, while metal ions act as the oxidant. A FsLDW-fabricated prototyping silk/Ag microelectrode exhibited 104-Ω−1 m−1-scale adjustable electric conductivity. This work not only provides a powerful development to silk micro/nanoprocessing techniques but also creates a novel way to fabricate multifunctional metal/biomacromolecule complex micro/nanodevices for applications such as micro/nanoscale mechanical and electrical bioengineering and biosystems.


Supplementary Methods
Some experimental details of silk-centered FsLDW. The fabricated structures were directly left and supported on FsLDW substrates (glass or ITO slices as needed) after water-rinsing. Before using, SF-centered inks were kept under 4 °C and in the dark for preventing self-gelling of SF and photo-accelerated reduction of metal ions. For pre-exposure, SF/AgNO 3 or SF/[AuCl 4 ]inks were sealed in polystyrene-based centrifugal tubes, and exposed under an incandescent light bulb (40 W) for appropriate time. During FsLDW, two details needed to be paid attention: i) proper encapsulation of FsLDW-fabrication area for restraining ink evaporation; ii) avoiding long-time exposure of high-intensity illumination (a fiber optic light is used for illumination and observation during FsLDW in our experiment). After FsLDW, the SF-centered micro/nano-structures were easily developed via water-rinsing and obtained on substrates. As-formed SF-centered micro/nano-structures/devices can keep their stable appearance during long-time storage in proper environments, for example, in dry air or pure water in our work.
where G was electric conductance (G=current/voltage), L was the length and S was the cross-section area of a silk/Ag micro-wire. Figure 4). In Figure 2e of main text and Supplementary Figure 4c, the indentation loading curves were presented for a FsLDW-fabricated all-SF-based micro-square (20×20×4 μm 3 ) under dry condition in air (red curve, about 25% relative humidity, 25 °C) and equilibrated in water (blue curve), respectively. As the indicative of the stiffness, the curve slope of contact parts (vertical displacement of Z-piezo > 0) was obviously smaller for the RSF based micro-square immersed in water (red curve) than that dried in air (blue curve). This might be caused by that the RSF based micro-square was significantly equilibrium-swollen and therefore softened in water. Along with continuous AFM indentation, RSF-based biopolymers swollen in water might be compacted, and  Figure 8). Relatively high Ag + concentration (150-mg•mL -1 AgNO 3 in 1.5-wt% RSF aqueous solution) was applied for applicable electric conductibility (see Methods). As displayed in Figure 3h of main text, current-voltage curves of the silk/Ag composite microwire in air (25-% relative humidity, 23 °C) were tested, resulting electrical resistances of about 118 Ω (intact ITO film and unconnected ITO electrodes as controls). By AFM characterization, 3D topography of the silk/Ag microwire was measured (Supplementary Figure 8), by which the cross sectional area was estimated to be about 10 μm 2 (length, 100 μm; width, 7 μm; thickness, 2 μm).

Silk/Ag composite microdevices for bio-electrics/electronics (Supplementary
According to Ohm's law, the electric conductivity of the silk/Ag microwire was calculated to be about 8.47×10 4 Ω -1 •m -1 , which was about 3 order of magnitude smaller than that of bulk silver (6.25×10 7 Ω -1 •m -1 ).

Cell viability (%) = ([A]test-[A] blank) / ([A]control-[A]blank) ×100%
(2) Besides, we also experimentally tested the toxicity of silk-centered aqueous inks as recommended (Supplementary Figure 15). As mentioned by the reviewer, we observed that live cells would die in a short time (several minutes, confirmed by cell morphology) in inks containing high-concentration Ag + or AgNPs. However, for silk/MB, silk/[AuCl 4 ]or silk/AuNPs inks, biocompatibility was acceptable. As FsLDW-fabrication of micro/nanostructures here usually took a short time from a few seconds to tens of minutes, we investigated cell viability after 1-hour incubation in silk-centered inks (see Supplementary Figure 15  First, for silk/MB ink, ~75-mW•μm -2 (measured before the objective lens) Fs-laser average power intensity is applied as the optimal FsLDW parameter. When the focused laser beam passed through the silk/MB ink, about 6.3-mW•μm -2 laser power would be deposited (experimentally measured value; equivalent to ~ 66 J•cm -3 per pulse; focal spot is an approximate ellipsoid with ~1800-nm major axis and ~700-nm minor axes). Accordingly, the focal temperature might be increased by ~ 73 °C (from ~ 22-°C room temperature to ~ 95 °C , without obvious boiling/cavitation) as the enough long Fs-laser irradiation (Movie 6 and Supplementary Figure 19). Besides, the smallest line width is ~ 350-400 nm when using Fs-laser intensity of ~ 75 mW•μm -2 in our work (see Figure 2(a) in the manuscript). It is precisely consistent with the span of 1000-μs isothermal profile at ~50-°C increase of temperature (namely, ~ 70-°C local temperature that could cause SF β-conformation transition [3]) in radial direction of laser focus [1]. This coincidence implies that the probable temperature distribution in laser focal spot should be approximate to the calculation.
In addition, obvious SF curing started at about 50-mW•μm -2 laser power intensity (measured before the objective lens). Accordingly, ~ 4.  [4,5]. To achieve an optimal balance between energy absorption and heat diffusion, and therefore a maximum temperature increase，the optimum size of AuNPs should be around 40-50 nm [4]. For an ideal condition, a steady ~30-°C temperature increase will be resulted in water proportionally along with Bubbles started to form as the Fs-laser intensity increased to over ~ 5 mW•μm -2 , (probably about 100 °C ; also without obvious damage to fabricated microstructures; see Movie 10 and Supplementary Figure 22b). Therefore, since focal temprature probably increased ~ 78 °C for 5-mW•μm -2 laser, 3.5-mW•μm -2 optimal power intensity should proportionally result into a 55-°C increase (about 77-°C focal temperature) [4,5]. Figure   23-29). We tested attenuated-total-reflection Fourier transform infrared (ATR-FTIR) absorption spectra of FsLDW-fabricated all-silk, silk/Ag, and silk/Au micro-patterns (see Supplementary Figure 18). First, the 1050-1150 cm -1 absorption strongly demonstrated formation of C-O-C bonds (probable result of Tyr oxidization and polymerization [8]), suggesting the oxidation crosslinking of RSF during FsLDW. As we mentioned, it has been widely accepted and applied to various proteins' FsLDW that enhanced production of singlet oxygen via two-photon MB-photosensitization ( 1 O 2 , or other oxidizing species by other photosensitizers) promoted protein crosslinking under high-intensity irradiation of femtosecond laser pulses [9,10].

Details on probable mechanisms of silk-centered FsLDW (Supplementary
Meanwhile, series of existing researches have also experimentally demonstrated that light-irradiated nano-metals did enhance localized electromagnetic field [11,12,13,14], photo-thermal effects (also proved in our experiments described above), and photo-production of 1 O 2 (or other oxidizing species) [15]. Therefore, we believe that the photo-oxidation crosslinking in our RSF system was probably also "catalyzed" by MB or nano-metals. Meanwhile, the amide-I absorption peak at 1635 cm -1 (shifted from 1651 cm -1 of amorphous SF) proved β-sheets in SF molecules likely formed by photo-heating during FsLDW [16]. Thus, these two simultaneous effects (oxidation-crosslinking and β-folding) illustrate why micro/nano-gels could be formed during FsLDW.
More detailed discussions are in the following.
In silk/MB ink, focal temperature could be around 70-95 °C during FsLDW with optimized processing parameters. Therefore, photoheating caninduce β-crystallization and cure SF from ink. However, "any thermal denaturation of biomolecules will thus always be mixed with free-electron-induced chemical effects, and the latter will probably dominate" [1]. Another evidence in our work is that FsLDW-fabricated SF For the silk/Ag + and silk/[AuCl 4 ]inks, optimized FsLDW parameters (weaker than those for silk/MB ink) induced focal temperatures probably around ~80-100 °C as discussed above. Similarly, photothermal effect played an important part in micro/nano-curing SF. As for other probable photochemical processes, we investigated the UV single-photon process (e.g. ~ 400 nm) in inks, which is a reasonable and frequently used parallel of infrared multiphoton process (e.g. 800 nm).
405-nm laser light (~ 6.2 mW•mm -2 ) was used to irradiate fresh silk/AgNO 3 ink (4-wt% SF and 5-mg•mL -1 AgNO 3 ). Under UV exposure, the irradiated area of ink immediately became brown (Supplementary Figure 25) and it went darker and more feculent (Supplementary Figure 26), as the increase of exposure time.
After standing in dark for hours, more sediment deposited in longer UV-exposed silk/AgNO 3 inks (Supplementary Figure 27). Considering that diameters of AgNPs were about 6-10 nm (Supplementary Figure 28), the higher turbidity was probably related to crosslinks and SF micro/nano-gels formed in inks [18]. On the other hand, the ATR-FTIR absorption peak shifted step by step from 1651 cm -1 to 1628 cm -1 formation (a clue of Tyr redox polymerization) [8]. Therefore, both two effects (oxidation-crosslinking/polymerization and β-sheet formation) involved in and enabled SF micro/nano-curing during FsDLW.
Accordingly, the photoreduction of Ag + under 800-nm Fs-laser irradiation might also exist, which was supposed to be induced by a MPA process [11][12][13][14]. Furthermore, metal nanoparticles were reported to greatly help to reduce more metal [11][12][13] and to MPA-polymerize surrounding pre-polymers or monomers [13,14] via enhanced plasmonic field. Especially, the nanomaterials of noble metal, such as Ag, Au, Pt, were well demonstrated and applied as photosensitizers to MPA-generate 1 O 2 (or other oxidizing species) for photodynamic therapy and photopolymerization [15].
Actually, specific crosslinking mechanisms of various protein FsLDW (including SF) are very complex with plenty of reaction pathway and many probable catalysis mechanism. This issue still needs more in-depth investigations by chemists and we will also try to do more research in this domain.

Discussion on photoluminescence of FsLDW-fabricated RSF microstructures.
Actually, SF itself originally has the ability of photoluminescence, for instance, a 305-nm-stimulated fluorescent peak at ~ 340 nm [18]. The enhanced fluorescence and even new peaks emerged in 400~470-nm region (305-nm excitation).
Correspondingly, 405-nm-excited FsLDW-fabricated micro-silks exhibited enhanced fluorescence in 500-nm region in our work. It suggested that new photoproducts were formed during UV processing [18]. For example, phenylalanine and tyrosine crosslinked via photooxidation in the irradiated SF solution [19]; photooxidation of methionine could change SF fluorescence [20]; radicals formed in SF chain might interact with oxygen to form peroxy radicals [21]; carbonyl groups formed as a result of the photooxidation of SF to α-keto-acids at glycine and alanine residues [22]. So, these new photoproducts and crosslinks might bring new chromophores to change absorption and fluorescence spectra of SF. Importantly, di-tyrosine cross-links were known to emit around 400-nm fluorescence under 305-nm excitation (accordingly, 500-nm fluorescence under 405-nm excitation), which was considered to be related to fluorescent changes of UV-processed RSF (as well as SF microstructures here multiphoton fabricated and irradiated by 800-nm Fs laser; see Figure 5b) [18].
Namely, the changed fluorescence is also a clue indicating probable Tyr-involved covalent crosslinking of SF during FsLDW.