Towards understanding the mechanism of 3D printing using protein: Femtosecond laser direct writing of microstructures made from homopeptides.

Femtosecond laser direct write (fs-LDW) is a promising technology for three-dimensional (3D) printing due to its high resolution, flexibility, and versatility. A protein solution can be used as a precursor to fabricate 3D proteinaceous microstructures that retain the protein's native function. The large diversity of protein molecules with different native functions allows diverse applications of this technology. However, our limited understanding of the mechanism of the printing process restricts the design and generation of 3D microstructures for biomedical applications. Therefore, we used eight commercially available homopeptides as precursors for fs-LDW of 3D structures. Our experimental results show that tyrosine, histidine, glutamic acid, and lysine contribute more to the fabrication process than do proline, threonine, phenylalanine, and alanine. In particular, we show that tyrosine is highly beneficial in the fabrication process. The beneficial effect of the charged amino acids glutamic acid and lysine suggests that the printing mechanism involves ions in addition to the previously proposed radical mechanism. Our results further suggest that the uneven electron density over larger amino acid molecules is key in aiding fs-LDW. The findings presented here will help generate more desired 3D proteinaceous microstructures by modifying protein precursors with beneficial amino acids. STATEMENT OF SIGNIFICANCE: Femtosecond laser direct write (fs-LDW) offers a three-dimensional (3D) printing capability for creating well-defined micro-and nanostructures. Applying this technology to proteins enables the manufacture of complex biomimetic 3D micro-and nanoarchitectures with retention of their original protein functions. To our knowledge, homopeptides themselves have never been used as precursor for fs-LDW so far. Our study gains several new insights into the 3D printing mechanism of pure protein for the first time. We believe that the experimental evidence presented greatly benefits the community of 3D printing of protein in particular and the biomaterial science community in general. With the gained insight, we aspire to expand the possibilities of biomaterial and biomedical applications of this technique.

High Immobilization Efficiency of Basic Protein within Heparin-Immobilized Calcium Phosphate Nanoparticles.

Previously, we achieved one-pot fabrication of heparin-immobilized calcium phosphate (CaP) nanoparticles with high dispersibility by a precipitation process in a highly supersaturated reaction solution. In this study, we revealed that the heparin-immobilized CaP nanoparticles have a greater co-immobilizing capacity for basic proteins than for acidic proteins. In this process, heparin acted as not only a particle-dispersing agent but also as an immobilizing agent for basic proteins; it remarkably (approximately three-fold) improved the immobilization efficiency of cytochrome C (a model basic protein) within the CaP nanoparticles. The content of cytochrome C immobilized within the nanoparticles was increased with an increase in cytochrome C concentration in the reaction solution and by aging the nanoparticles. The obtained nanoparticles were dispersed well in water owing to their large negative zeta potentials derived from heparin, irrespective of the content of cytochrome C. Similar results were obtained also for another basic protein, lysozyme, but not for an acidic protein, albumin; the immobilization efficiency of albumin within the nanoparticles was decreased by heparin. These findings provide new insights into the co-immobilization strategy of proteins within heparin-immobilized CaP nanoparticles and will be useful in the design and fabrication of nanocarriers for protein delivery applications.

Biological modification of tooth surface by laser-based apatite coating techniques.

BACKGROUND: Development of new clinical regenerative procedures is needed for the reconstruction of the connective tissue attachment lost to periodontal disease. Apatite coating on the affected root surfaces could improve root surface biocompatibility and promote the reestablishment of connective tissue attachment. HIGHLIGHT: We developed two novel techniques that use laser light for coating the tooth surface with apatite. In the laser-assisted biomimetic (LAB) process, a tooth substrate was placed in a supersaturated calcium phosphate solution and irradiated for 30 min with low-energy pulsed laser light. Due to the laser-assisted pseudo-biomineralization, a submicron-thick apatite film was created on the laser-irradiated tooth surface. Furthermore, we created a fluoride-incorporated apatite film on the tooth surface using the LAB process and demonstrated its antibacterial activity against Streptococcus mutans. In the laser-induced forward transfer with optical stamp (LIFTOP) process, a thin apatite film loaded with the cell-adhesion protein, fibronectin, was prepared beforehand as a raw material on the optical stamp (carbon- and polydimethylsiloxane-coated support) by a conventional biomimetic process. After irradiation with a single laser pulse, the film (microchip) was transferred onto a tooth substrate via laser ablation of the carbon sacrificial layer. The LIFTOP process requires only a short processing time and has a minimal heat effect on the film; thus, the film exhibits cell adhesion activity even after the LIFTOP process. CONCLUSION: The LAB and LIFTOP processes have the potential as novel tools for tooth surface modification in the treatment of periodontal disease.

Laser-Induced Transfer of Noble Metal Nanodots with Femtosecond Laser-Interference Processing.

Noble metal nanodots have been applied to plasmonic devices, catalysts, and highly sensitive detection in bioinstruments. We have been studying the fabrications of them through a laser-induced dot transfer (LIDT) technique, a type of laser-induced forward transfer (LIFT), in which nanodots several hundred nm in diameter are produced via a solid-liquid-solid (SLS) mechanism. In the previous study, an interference laser processing technique was applied to LIDT, and aligned Au nanodots were successfully deposited onto an acceptor substrate in a single shot of femtosecond laser irradiation. In the present experiment, Pt thin film was applied to this technique, and the deposited nanodots were measured by scanning electron microscopy (SEM) and compared with the Au nanodots. A typical nanodot had a roundness fr=0.98 and circularity fcirc=0.90. Compared to the previous experiment using Au thin film, the size distribution was more diffuse, and it was difficult to see the periodic alignment of the nanodots in the parameter range of this experiment. This method is promising as a method for producing large quantities of Pt particles with diameters of several hundred nm.

Local Melting of Gold Thin Films by Femtosecond Laser-Interference Processing to Generate Nanoparticles on a Source Target.

Shape- and size-controlled metallic nanoparticles are very important due to their wide applicability. Such particles have been fabricated by chemosynthesis, chemical-vapor deposition, and laser processing. Pulsed-laser deposition and laser-induced dot transfer use ejections of molten layers and solid-liquid-solid processes to fabricate nanoparticles with a radius of some tens to hundreds of nm. In these processes, the nanoparticles are collected on an acceptor substrate. In the present experiment, we used laser-interference processing of gold thin films, which deposited nanoparticles directly on the source thin film with a yield ratio. A typical nanoparticle had roundness fr=0.99 and circularity fcirc=0.869, and the radius was controllable between 69 and 188 nm. The smallest radius was 82 nm on average, and the smallest standard deviation was 3 nm. The simplicity, high yield, and ideal features of the nanoparticles produced by this method will broaden the range of applications of nanoparticles in fields such as plasmonics.

Fabrication of microarrays on fused silica plates using the laser-induced backside wet etching method.

A novel approach in the fabrication of microarrays of dye and protein on fused silica plates using the laser-induced backside wet etching (LIBWE) technique is described. The surface of fused silica plates was initially precoated using trimethoxysilane self-assembled monolayers (SAMs) and then etched using the LIBWE method to obtain the desired microstructures on the plate surface. Using this technique, the SAMs on the nonirradiated areas were able to survive the LIBWE process and were used as templates for the subsequent deposition of dye molecules or proteins via chemical bonding or physical adsorption. In the case of fused silica plates precoated with fluorinated SAMs, the LIBWE method is used to remove the SAMs to expose the etched silica surfaces, on which a thin layer of pyranine molecules can be site-selectively deposited using an aqueous solution of pyranine. In another application, an ethanol solution of rhodamine 6G was preferentially deposited onto the nonirradiated areas. In yet another application, bovine serum albumin was preferentially deposited onto the laser-irradiated areas; in this case, the fused silica plates were precoated with poly(ethylene oxide) SAMs. Interestingly, when an aqueous suspension of polystyrene (PS) microbeads was cast onto the fused silica precoated with the fluorinated SAMs, hexagonally close-packed PS microbeads were deposited into the etched cavities. Depositions of the dye, protein, and microbeads were confirmed by visualization using a fluorescence microscope and scanning electron microscope.

Generation and photoreactions of 2,4,6-trinitreno-1,3,5-triazine, a septet trinitrene.

We have studied the matrix photolysis of 2,4,6-triazido-1,3,5-triazine (cyanuric triazide, 1). Stepwise generation of the corresponding mononitrene, dinitrene, and trinitrene was observed by matrix IR and electron paramagnetic resonance (EPR) spectroscopy. The generated species were identified by comparison of their matrix IR spectra with density functional theory (DFT) computational results. The generation of 2,4,6-trinitreno-1,3,5-triazine with a septet ground state was confirmed for the first time by matrix EPR spectroscopy. The trinitrene readily decomposed into three NCN molecules upon further photoirradiation. This process was also confirmed by matrix EPR spectroscopy.

Site-selective dye deposition on microstructures of fused silica fabricated using the LIBWE method.

Using laser-induced backside wet etching (LIBWE) technique, microstructures were fabricated onto the surface of fused silica plates, which were pre-coated with self-assembled monolayers (SAMs). Dye molecules and proteins were alternately deposited onto the laser-irradiated or non-irradiated areas by either chemical bonding or physical adsorption.