Precise Protein Photolithography (P3): High Performance Biopatterning Using Silk Fibroin Light Chain as the Resist

Precise patterning of biomaterials has widespread applications, including drug release, degradable implants, tissue engineering, and regenerative medicine. Patterning of protein‐based microstructures using UV‐photolithography has been demonstrated using protein as the resist material. The Achilles heel of existing protein‐based biophotoresists is the inevitable wide molecular weight distribution during the protein extraction/regeneration process, hindering their practical uses in the semiconductor industry where reliability and repeatability are paramount. A wafer‐scale high resolution patterning of bio‐microstructures using well‐defined silk fibroin light chain as the resist material is presented showing unprecedent performances. The lithographic and etching performance of silk fibroin light chain resists are evaluated systematically and the underlying mechanisms are thoroughly discussed. The micropatterned silk structures are tested as cellular substrates for the successful spatial guidance of fetal neural stems cells seeded on the patterned substrates. The enhanced patterning resolution, the improved etch resistance, and the inherent biocompatibility of such protein‐based photoresist provide new opportunities in fabricating large scale biocompatible functional microstructures.

centrifuged three times. Finally, lyophilization for 48 hours yielded a pure white powder.

Solubility of silk fibroin protein
Solubility was initially investigated to facilitate analysis of the fabrication process. Native silk fibroin protein displays limited solubility when extracted from silk fiber, owing to its secondary structural content. [3] But, with the increase of degumming time, the silk fibroin proteins display enhanced solubility ( Figure S1) due to the shorter protein chain length. In addition, the silk fibroin protein preliminary dissolution in salt solution, a process which 'activates' the fibroin. [4] Alcohols are known to follow a trend of protein solubilization: HFIP > trifluoroethanol (TFE) > isopropanol > ethanol > methanol, where the fluorinated alcohols HFIP and TFE are specifically known to enhance solubility thorough stabilization of the αhelical conformation. [5] Furthermore, the solubility of silk fibroin protein in non-aqueous solvents DMSO, N,N-dimethylacetamide (DMAc), and N,N-dimethylformamide (DMF) is negligible without the use of salts to disrupt hydrogen bonding. With the chemical conjugation described, the solubility characteristics of the silk fibroin protein varied significantly since the added groups present hydrophobic alkene and carbonyl groups at modified amino acids which were occupied via the conjugation reaction. Among the above organic solvents, HFIP was the only solvent to provide solubility and was thus used as a carrier for characterization as well as deposition of photoresists. Thus, HFIP was used as the primary carrier of the photoresist for incorporation into later photolithography steps.

Self-assembly of silk fibroin protein
Silk fibroin protein consists of several polypeptides, thus contributing to a variability in molecular weights ranging from a few tens to a few hundreds of kDa [6] . The protein is characterized by the presence of strongly polar side groups, such as hydroxyl, carboxyl, and amino groups. Silk fibroin protein molecules, thus, follow a diffusion-limited aggregation process (DLA) on surfaces to form nano-and macro-scale architectures with remarkable morphologies and mechanical properties. On drying, silk fibroin protein spontaneously forms branched patterns ( Figure S2) that are qualitatively and quantitatively similar to the fractal structures formed by a DLA [7] that has been observed in the assembly of a range of materials including colloids, polymer thin films [8] , peptides [9,10] , and proteins [11] .
To prove that the formation of wrinkled patterns was attributed to the physical phenomenon but not the secondary structure change, the s-SNOM was employed to measure the absorbance and reflection spectrum of the silk film. According to the properties of s-SNOM, if the chromatic difference at 1,631 cm −1 (characteristic peak of the secondary structure corresponding to the beta sheet) exists along with the height on the surface, the form of wrinkled patterns is resulted from the secondary structure change. Figure S2(c) indicates the AFM image, and the s-SNOM images of both absorbance and reflection spectrum on the surface of UV-silk30 pattern. No contrast difference in the absorbance and reflection images at 1,635 cm −1 was observed, which was attributed to the secondary structure at different regions on the surface were basically the same, and only physical phenomenon existed in the self-assembly procedure depending on properties such as particle size, dispersity and charge.
The underlying substrate is an important factor that may direct the nature of self-assembly of silk fibroin protein. We initially used silicon as the surface in which the surface was treated by IPA solvent. Due to the more hydrophobicity of silicon with the IPA treatment, the solutions tended to dry unevenly, resulting in aggregated clusters and the formation of a film in many cases ( Figure S2). We therefore used the substrate without the IPA treatment in the experiments. The relevant hydrophilic and atomically flat nature of the substrate permitted us to spread the solution and to dry it with smaller concentration gradients, yielding highresolution AFM images. In addition, the lyophilization process did not change the behavior of silk fibroin protein.

Measurements of Resolution
The resolution characterization has been carried out using a Siemens Stars method which is widely used in imaging quality test of optical instruments, printers and displays (https://en.wikipedia.org/wiki/Siemens_star). The method provides information about the resolution at different positions in the image. It is stable and leads to reliable results especially in the case of sharpening and compression algorithms. Due to the self-assembly phenomenon of silk proteins on the silicon substrate, when the pattern dimension decreases to a certain size, the pattern cannot reflect the precise shape of mask. In this case, by detecting the lines in the center of the Siemens Star, we observed swallow-tailed phenomenon of silk resists (the area between two red circles in Figure S3) which was attributed to the self-assembly properties of silk proteins. By measuring and calculating the size of outer circle, we can obtain the resolution of the patterns. The surface roughness has been characterized using Atomic Force Microscopy.  and L-Fibroin from reference [12] .

Fourier transform infrared spectroscopy (FTIR)
Fourier transform infrared spectroscopy (FTIR) was employed on unmodified silk fibroin proteins film to confirm the presence of the methacrylate moiety. Cast films (5.0 mg) were analyzed in attenuated total reflectance (ATR) mode using a Ge ATR crystal, and data was collected between 4000 -1000 cm −1 , for 32 scans at a resolution of 1 cm −1 . Here, comparison of spectra to that of the IEM, demonstrates functionalization of IEM to different silk fibroin protein under 10, 30, 60, 90 minutes degumming time.
Native silk fibroin under 10, 30, 60, 90 minutes degumming time shows intrinsic Amide I/II/III signals (Figure S6 (a)), which are simultaneously observed for the silk fibroin protein photoresist. The IEM reagent (Figure S6 (b)) shows stretching vibrations at 1720 cm -1 , representing carbonyl ester C=O, and 1640 cm -1 , assigned to vinyl C=C (which confers photoreactivity), and an additional intense peak is present at 1160 cm -1 , resulting from CH 3 rocking vibrations [13] . The silk fibroin protein photoresist under 30 minutes degumming time also shows peaks representing an ester carbonyl, terminal C=C stretching vibrations as broad shoulder peaks of Amide I, and CH 3 rocking vibrations, all provided by the methacrylate group. The absence of this isocyanate stretching vibration from the product indicates the absence of residual isocyanate. The clear observation of characteristic IEM groups in the product provides evidence to support the successful grafting of IEM onto the silk fibroin protein. In addition, no obvious enhancement of methacrylate group peak with an increase of IEM ratio during the fabrication of silk fibroin photoresists (Figure S6 (b)). The secondary structural content of the polypeptide backbone remains relatively unchanged with the methacrylate bioconjugation reaction [14] .

s-SNOM
s-SNOM set-up employing a tunable single line IR quantum cascade laser (1,450 to 1,750 cm -1 ) for tip illumination (Figure S7). During instrument operation, the light backscattered from the tip is collected and analyzed with a Michelson interferometer operating in pseudoheterodyne mode. The laser was attenuated to ∼10 mW such that the detector yields a nominal signal of 1.5 V. The AFM was operated in tapping mode with 65 nm tapping. Gold-coated AFM tips with about 250 kHz resonance (Tap300G-B-G, budgetsensors.com) were used to optimize the IR near-field signal. The IR signal was detected simultaneously with AFM signals. The IR signal used for analysis in this work was measured by a pseudo-heterodyne technique and a lock-in amplifier. Such amplifier was set at the second and third harmonics of the tapping frequency which provides both reflection and absorption that are (mostly) free of background. Both amplitude and phase information is collected. The near-field interaction leads to a phase spectrum that resembles a familiar molecular absorbance band, while the near-field amplitude spectrum acquires a dispersive line shape similar to a far-field reflectivity spectrum [15] .

Young's Modulus measurement
The Young's Modulus is measured by the Force-Distance Spectroscopy module of a commercial AFM (NT-MDT, Russia). The AFM records the applied Z-movement of the cantilever and the real deflection of the tip. The Young's modulus can then be calculated (given a simple model of the AFM tip). For all the measurement, a single ARROW-NCPT tip (NanoWorld, Switzerland) is used and the tip radius is assumed to be 25 nm according to the specification. Figure S8. Microscope images of the patterns of Silk fibroin protein using the contact photolithography procedure. All scale bars in the images are 200 m. Figure S9. Microscope images of the patterns of L-fibroin protein using the contact photolithography procedure. All scale bars in the images are 200 m.