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.

Attomolar Sensing Based on Liquid Interface-Assisted Surface-Enhanced Raman Scattering in Microfluidic Chip by Femtosecond Laser Processing.

Surface-enhanced Raman scattering (SERS) is a multidisciplinary trace analysis technique based on plasmonic effects. The development of SERS microfluidic chips has been exploited extensively in recent times impacting on applications in diverse fields. However, despite much progress, the excitation of label-free molecules is extremely challenging when analyte concentrations are lower than 1 nM because of the blinking SERS effect. In this paper, a novel analytical strategy which can achieve detection limits at an attomolar level is proposed. This performance improvement is due to the use of a glass microfluidic chip that features an analyte air-solution interface which forms on the SERS substrate in the microfluidic channel, whereby the analyte molecules aggregate locally at the interface during the measurement, hence the term liquid interface-assisted SERS (LI-SERS). The microfluidic chips are fabricated using hybrid femtosecond (fs) laser processing consisting of fs laser-assisted chemical etching, selective metallization, and metal surface nanostructuring. The novel LI-SERS technique can achieve an analytical enhancement factor of 1.5 × 1014, providing a detection limit below 10-17 M (<10 aM). The mechanism for the extraordinary enhancement afforded by LI-SERS is attributed to Marangoni convection induced by the photothermal effect.

Three-Dimensional Printing of Pure Proteinaceous Microstructures by Femtosecond Laser Multiphoton Cross-Linking.

Laser direct write (LDW) is a promising three-dimensional (3D) printing technology for creating proteinaceous microstructures in which the proteins retain their original function, enabling the manufacture of complex biomimetic 3D microenvironments and versatile enhancement of medical microdevices. A photoactivator has commonly been used to date in the laser direct write of proteins to enhance the cross-linking process. However, incomplete conversion results in photoactivator molecules remaining trapped inside the protein microstructure, causing their gradual leaching and subsequent undesirable effect on biological applications. Here, we demonstrate the 3D fabrication of microstructures made of pure serum albumin protein using photoactivator-free fabrication, confirmed by Raman data. For the first time, acid-catalyzed hydrolysis of the created structures provides evidence that chemical cross-links are induced by exposure to femtosecond laser irradiation. The diversity of the biomaterial protein available for the precursors for LDW offers capability of the fabrication of complex biomimetic 3D microenvironments and biochip applications.

Mechanical Characterization of Microengineered Epithelial Cysts by Using Atomic Force Microscopy.

Most organs contain interconnected tubular tissues that are one-cell-thick, polarized epithelial monolayers enclosing a fluid-filled lumen. Such tissue organization plays crucial roles in developmental and normal physiology, and the proper functioning of these tissues depends on their regulation by complex biochemical perturbations and equally important, but poorly understood, mechanical perturbations. In this study, by combining micropatterning techniques and atomic force microscopy, we developed a simple in vitro experimental platform for characterizing the mechanical properties of the MDCK II cyst, the simplest model of lumen-enclosing epithelial monolayers. By using this platform, we estimated the elasticity of the cyst monolayer and showed that the presence of a luminal space influences cyst mechanics substantially, which could be attributed to polarization and tissue-level coordination. More interestingly, the results from force-relaxation experiments showed that the cysts also displayed tissue-level poroelastic characteristics that differed slightly from those of single cells. Our study provides the first quantitative findings, to our knowledge, on the tissue-level mechanics of well-polarized epithelial cysts and offers new insights into the interplay between cyst mechanics and cyst physiology. Moreover, our simple platform is a potentially useful tool for enhancing the current understanding of cyst mechanics in health and disease.

Multi-Component Microscaffold With 3D Spatially Defined Proteinaceous Environment.

In this paper, we present a multicomponent microenvironment consisting of proteinaceous networks with submicron-sized features optionally embedded into a photoresist microscaffold. By two-photon direct laser writing, free-standing 3D proteinaceous microstructures were fabricated for cell culture application, demonstrated with NIH/3T3 fibroblast cells. A Young's modulus of megapascal-order contributes to the challenge of structural sustainability of the proteinaceous microstructures for experiments as well as sequential fabrication steps. We propose to embed proteinaceous networks into a mechanically robust photoresist microscaffold. We investigate the limits of this 3D microfabrication of embedded proteinaceous networks and demonstrate the embedment of two different proteinaceous networks within one microscaffold. Performing cell culture of PC12 cells, we observe cell adhesion and cell motility on embedded proteinaceous networks of collagen type-IV mixed with bovine serum albumin into a photoresist microscaffold. The ability to structure proteinaceous elements for 3D spatial control of microenvironment might be a key feature in cell culture to decouple environmental cues to control cellular behavior.