Multiphoton lithography with protein photoresists.

Recently, 2D/3D direct laser writing has attracted increased attention due to its broad applications ranging from biomedical engineering to aerospace. 3D nanolithography of water-soluble protein-based scaffolds have been envisioned to provide a variety of tunable properties. In this paper, we present a functional protein-based photoresist with tunable mechanical properties that is suitable for multiphoton lithography (MPL). Through the use of methacrylated streptavidin or methacrylated bovine serum albumin in combination with polyethylene glycol diacrylate or methacrylated hyaluronic acid as crosslinkers and a vitamin-based photoinitiator, we were able to write two- and three-dimensional structures as small as 200 nm/600 nm lateral/axial features, respectively. We also demonstrated that Young's modulus can be tuned by the photoresist composition, and we were able to achieve values as low as 40 kPa. Furthermore, we showed that Young's modulus can be recovered after drying and rehydration (i.e. shelf time determination). The retained biological functionality of the streptavidin scaffolds was demonstrated using fluorescently labelled biotins. Using single-molecule fluorescence microscopy, we estimated the density of streptavidin in the written features (1.8 ± 0.2 × 105 streptavidins per 1.00 ± 0.05 µm³ of feature volume). Finally, we showed applicability of our 2D scaffold as a support for a fluorescence absorbance immuno-assay (FLISA), and as a delivery platform of extracellular vesicles to HeLa cells.

Biofunctionalization of Sub-Diffractionally Patterned Polymer Structures by Photobleaching.

Stimulated emission depletion (STED) nanolithography allows nanofabrication below the diffraction limit. Recently, it was applied to nanoanchors for protein fixation down to the single molecule level. We now combined STED nanolithography with laser-assisted protein adsorption by photobleaching (LAPAP) for optical and selective attachment of proteins to subdiffractional structures. In turn, STED was used for imaging of fluorescently tagged streptavidin to reveal protein binding to STED-lithographically patterned acrylate structures via LAPAP. Protein localization down to 56 nm spots was achieved using all-optical methods at visible wavelengths.

Selective Cell Adhesion and Biosensing Applications of Bio-Active Block Copolymers Prepared by CuAAC/Thiol-ene Double Click Reactions.

N-Acetyl-l-cysteine (NAC)-capped poly(methyl methacrylate)-b-polycaprolactone block copolymer (PMMA-b-PCL-NAC) was prepared using the previously described one-pot photoinduced sequential CuAAC/thiol-ene double click procedure. PMMA-b-PCL-NAC had previously shown good applicability as a matrix for cell adhesion of cells from the Vero cell line (African green monkey kidney epithelial). Here, in this work, PMMA-b-PCL-NAC served as an excellent immobilization matrix for biomolecule conjugation. Covalent binding of RGD (R: arginine, G: glycine, and D: aspartic acid) peptide sequence onto the PMMA-b-PCL-NAC-coated surface was performed via EDC chemistry. RGD-modified PMMA-b-PCL-NAC (PMMA-b-PCL-NAC-RGD) as a non-toxic cell proliferation platform was used for selective "integrin αvß3-mediated cell adhesion and biosensing studies. Both optical and electrochemical techniques were used to monitor the adhesion differences between "integrin αvß3" receptor positive and negative cell lines on to the designed biofunctional surfaces.

Simultaneous Photoinduced ATRP and CuAAC Reactions for the Synthesis of Block Copolymers.

Atom transfer radical polymerization (ATRP) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions, both utilizing copper(I) (Cu(I)) complexes, make a tremendous progress in synthetic polymer chemistry. Independently or in combination with other polymerization processes, they give access to the synthesis of polymers with well-defined structures, desired molecular architectures, and a wide variety of functionalities. Here, a novel in situ photoinduced formation of block copolymers is described by simultaneous ATRP and CuAAC processes. This approach relies on the direct reduction of initially charged copper(II) complexes to Cu(I) complexes to trigger both ATRP and CuAAC reactions coinciding under UV light at ambient temperature in one pot. Its synthetic utility is demonstrated on a model block copolymerization process by photoinduced ATRP of methyl methacrylate (MMA) using an initiator possessing acetylene functionality and concomitant click reaction between thus formed α-acetylene-poly(methyl methacrylate) (Ac-PMMA) and independently prepared azide functional polystyrene (PS-N3 ). Successful formation of PS-b-PMMA block copolymer is confirmed by FT-IR and 1 H NMR spectral analysis and gel permeation chromatography (GPC) measurements.