Pico- and femtosecond laser-induced crosslinking of protein microstructures: evaluation of processability and bioactivity.

This study reports the pico- and femtosecond laser-induced photocrosslinking of protein microstructures. The capabilities of a picosecond Nd:YAG laser to promote multiphoton excited crosslinking of proteins were evaluated by fabricating 2D and 3D microstructures of avidin, bovine serum albumin (BSA) and biotinylated bovine serum albumin (bBSA). The multiphoton absorption-induced photocrosslinking of proteins was demonstrated here for the first time with a non-toxic biomolecule flavin mononucleotide (FMN) as the photosensitizer. Sub-micrometer and micrometer scale structures were fabricated from several different compositions of protein and photosensitizer by varying the average laser power and scanning speed in order to determine the optimal process parameters for efficient photocrosslinking. In addition, the retention of ligand-binding ability of the crosslinked protein structures was shown by fluorescence imaging of immobilized biotin or streptavidin conjugated fluorescence labels. The surface topography and the resolution of the protein patterns fabricated with the Nd:YAG laser were compared to the results obtained with a femtosecond Ti:Sapphire laser. Quite similar grain characteristics and comparable feature sizes were achieved with both laser sources, which demonstrates the utility of the low-cost Nd:YAG microlaser for direct laser writing of protein microstructures.

Direct laser writing of 3D scaffolds for neural tissue engineering applications.

This study reports on the production of high-resolution 3D structures of polylactide-based materials via multi-photon polymerization and explores their use as neural tissue engineering scaffolds. To achieve this, a liquid polylactide resin was synthesized in house and rendered photocurable via attaching methacrylate groups to the hydroxyl end groups of the small molecular weight prepolymer. This resin cures easily under UV irradiation, using a mercury lamp, and under femtosecond IR irradiation. The results showed that the photocurable polylactide (PLA) resin can be readily structured via direct laser write (DLW) with a femtosecond Ti:sapphire laser and submicrometer structures can be produced. The maximum resolution achieved is 800 nm. Neuroblastoma cells were grown on thin films of the cured PLA material, and cell viability and proliferation assays revealed good biocompatibility of the material. Additionally, PC12 and NG108-15 neuroblastoma growth on bespoke scaffolds was studied in more detail to assess potential applications for neuronal implants of this material.

Quantification of the activity of biomolecules in microarrays obtained by direct laser transfer.

The direct-writing technique laser-induced forward transfer has been employed for the micro-array printing of liquid solutions of the enzyme horseradish peroxidase and the protein Titin on nitrocellulose solid surfaces. The effect of two UV laser pulse lengths, femtosecond and nanosecond has been studied in relation with maintaining the activity of the transferred biomolecules. The quantification of the active biomolecules after transfer has been carried out using Bradford assay, quantitative colorimetric enzymatic assay and fluorescence techniques. Spectrophotometric measurements of the HRP and the Titin activity as well as chromatogenic and fluorescence assay studies have revealed a connection between the properties of the deposited, biologically active biomolecules, the experimental conditions and the target composition. The bioassays have shown that up to 78% of the biomolecules remained active after femtosecond laser transfer, while this value reduced to 54% after nanosecond laser transfer. The addition of glycerol in a percentage up to 70% in the solution to be transferred has contributed to the stabilization of the micro-array patterns and the increase of their resolution.

Fabrication of three-dimensional structures by three-photon polymerization.

We report the fabrication of three-dimensional structures of submicrometer resolution by three-photon polymerization. This resolution has been achieved by polymerizing ORMOCER, a UV photocurable organic-inorganic hybrid material, with an ultrafast laser irradiation at 1028 nm. To our knowledge, this is the first demonstration of three-photon polymerization, a process that may allow the fabrication of components of very high resolution.

Microfabrication by use of a spatial light modulator in the ultraviolet: experimental results.

We report the development of a new microstereophotolithography technique for creation of three-dimensional microcomponents by use of a planar, layer-by-layer process of exposure, in which a spatial light modulator is used as a dynamic lithographic mask. The system operates in the UV to take advantage of the wide supply of commercially available photopolymers designed for conventional stereolithography. With this novel procedure it is possible to build components with feature sizes as small as a few micrometers. The experimental setup is briefly described, and the first microcomponent fabricated by this system is shown.

UV Microstereolithography System that uses Spatial Light Modulator Technology.

A new stereophotolithography technique utilizing a spatial light modulator (SLM) to create three-dimensional components with a planar, layer-by-layer process of exposure is described. With this procedure it is possible to build components with dimensions in the range of 50 mum-50 mm and feature sizes as small as 5 mum with a resolution of 1 mum. A polysilicon thin-film twisted nematic SVGA SLM is used as the dynamic photolithographic mask. The system consists of eight elements: a UV laser light source, an optical shutter, beam-conditioning optics, a SLM, a multielement reduction lens system, a high-resolution translation stage, a control system, and a computer-aided-design system. Each of these system components is briefly described. In addition, the optical characteristics of commercially available UV curable resins are investigated with nondegenerate four-wave mixing. Holographic gratings were written at a wavelength of 351.1 nm and read at 632.8 nm to compare the reactivity, curing speed, shrinkage, and resolution of the resins. These experiments were carried out to prove the suitability of these photopolymerization systems for microstereolithography.