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

Abstract

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.

Keywords: biopatterning; protein photolithography; silk fibroin light chain.

Figure 1

Synthesis of the UV‐reactive silk l‐fibroin (UV–LC) and the result of photolithography using UV–LC as a negative resist. a) B. mori cocoons are degummed for 60 min to obtain b) silk fibroin, and c) the l‐fibroin is then separated from the silk fibroin using formic acid; d) photoactive l‐fibroin (UV–LC precursor) is obtained by conjugating IEM to the l‐fibroin; e) by adding the photoinitiator (Irgacure 2959), the UV–LC resist can be synthesized; f) photolithography using UV–LC resist; g) optical images of the fabricated patterns (linewidth: 5 µm, zoom‐in image) shows that UV–LC has better lithographic performance than UV–Silk30. Scale bar: 50 µm. h) Dark‐field stereomicroscopic photograph of double immunofluorescence staining with Nestin (green fluorescence) and nuclear staining (blue DAPI staining) of fetal neural stems cells that were guided to be cultured on a micropatterned UV–LC resist (dash line) on a silicon substrate. Scale bar: 100 µm.

Figure 2

Characterization and analysis of patterns fabricated by protein photolithography using different types of silk‐based materials (e.g., UV–Silk30, UV–SilkHTP, and UV–LC). a) Schematic comparison between structures of UV–LC and UV–Silk (including both UV–Silk30 and UV–SilkHTP) precursor, where UV–Silk30 has longer protein chains than UV–HTP. UV–LC contains only l‐fibroin; b) morphological characterization (using an optical microscope and AFM, scale bar: 200 µm) of micropatterns fabricated by protein photolithography using UV–Silk30, UV–SilkHTP, and UV–LC. It shows that the UV–LC can achieve better resolution and surface smoothness than UV–Silk30 and UV–SilkHTP; c,d) quantitative analysis of resolution and surface roughness of micropatterns fabricated using various UV–Silk and UV–LC. The result is consistent with the observations from optical and AFM images.

Figure 3

Structural characterization of the UV–Silk and UV–LC using FTIR and s‐SNOM. a) Schematic of ATR–FTIR setup, where the sample is illuminated from the back of the ATR crystal; b) FTIR spectrum of IEM, silk fibroin protein, UV–Silk, and UV–LC. The peaks vanish in the UV–Silk and UV–LC, indicating the binding of IEM on silk fibroin and l‐fibroin; c) schematic of the s‐SNOM system. An infrared laser is focused onto the AFM tip, and the scattered signal is collected by the detector; d,e) IR nanoimaging and absorbance (acquired by s‐SNOM measurement performed at 1635 cm⁻¹) of UV–Silk30, UV–Silk90, UV–SilkHTP, and UV–LC with various exposure time. The disappearance of the absorbance with increasing exposure time indicates the increasing crosslinking degree of IEM until about 90 s, after which time all the available IEM active conjugated acrylate group sites are crosslinked.

Figure 4

Etching rate measurements and schematic structures of various UV–Silk and UV–LC. a) Etching rate measurement of the UV–Silk30 and UV–LC with increasing exposure time. The etching rate of UV–LC decreases faster than the UV–Silk30 with increasing exposure time but reaches a constant rate that is higher than UV–Silk30; b) etching rate comparison between various UV–Silk (including both UV–Silk and methanol treated UV–Silk) and UV–LC at two exposure times (20 and 120 s). All the samples with 20 s exposure times have larger etching rate than the samples with 120 s exposure times. UV–Silk has an increasing etching rate with increasing degumming time because the mechanical strength is better with longer chain length. UV–LC has slightly less etching rate because its highly defined molecular structure help form better crystalline structure; c) Young's modulus of UV–Silk and UV–LC and ratio of Young's modulus before and after methanol treatment. It shows the similar trend with the data of etching speed, where the largest Young's modulus value corresponds to slower etching rate. It also shows no obvious change on Young's modulus by treating with methanol, suggesting no formation of beta sheet structures in UV–Silk and UV–LC resists induced by the methanol treatment; d) schematic structure and the corresponding etching rate of the photocrosslinked UV–Silk and UV–LC with 20 s exposure time and UV–Silk30 exposed for 120 s. For UV–Silk30, the etching rate decreases with longer exposure time because of the increased crosslinking degree. With the same exposure time, the etching rate increases with increasing degumming time because of the shorter chain length, and thus less mechanical strength. With 20 s exposure (partially crosslinking) UV–LC has less etching speed because its highly defined molecular structure helps it form better IEM‐induced crystalline structure.

Figure 5

a) Bioactivity evaluation of HRP‐doped silk resist and HRP enzyme after UV exposure. The ELISA test shows that enzyme activities are negatively affected during UV exposure (as shown in the photos on the right) and silk resists can help to stabilize the bioactivities to some extent during UV exposure; b) portion of the designed photomask. c–f) Double immunofluorescence staining with Nestin (green fluorescence) and nuclear staining (blue DAPI staining) of fetal neural stem cells cultured on UC–LC substrates showing the spatial guidance of cell seeding. Scale bar: 100 µm.