Review of fabrication of three-dimensional proteinaceous micro- and nano-structures by femtosecond laser cross-linking


Lasers enable creation of architected 3D protein structures | We are living in a three-dimensional (3D) world where most of objects have 3D structures. Although mass production has revolutionized industry including automotive, electronic, chemical manufacturing to change consumption chains, most of conventional fabrication techniques rely on the planar process. Meanwhile, the 3D process would enhance functionality and integration degree of devices. Thus, techniques quickly fabricating customized 3D structures are increasingly more important not only to produce value-added goods but also to meet individual requirement. To this end, 3D printing as rapid prototyping which enables manufacturers to quickly fabricate architected 3D structures with individual design has been recently attracting much attention. Depending on the material properties and sizes of the 3D printed objects, different 3D printing techniques are available. 
    For true freedom in 3D creation, the printing process needs to be possible to be executed at any positions in the material. The unique features of femtosecond (fs) lasers with extremely high peak intensity can have access to this requirement. Specifically, fs laser light whose wavelength has no absorption by the material transmits the material, but due to nonlinear multiphoton absorption processes affects the material at the laser focus only where the photon intensity is adjusted to be high enough to fulfill the multiphoton absorption condition. Then, scanning of the focused fs laser beam utilizing this feature allows not only to directly create 3D structures with arbitrary shapes, but also to realize feature sizes below the diffractive limit in the submicron-and nanometer-regimes.
    In the recent decades, this concept has been applied rigorously to various polymers where polymerization is controlled by absorption properties, additive photoactivators and precursor concentration. More recently, due to application of photodynamic therapy in cancer treatment, this technique was transferred to cross-link protein for 3D printing.
Prof. Sugioka’s team investigates laser processing of various materials. Amongst others, the team aims to understand the protein cross-linking mechanism and to identify diverse applications for the 3D fabrication in biochips, microfluidic devices and cell culture. In this on-going effort, a comprehensive review summarizes recent developments, applications and current challenges.

Diversity of protein functions | The most appealing factor of protein as a 3D printing material is that protein function can be retained after 3D fabrication to be utilized for many applications as unanimously reported so far. Because protein is an element material in all kinds of organisms, including human beings, there is a vast diversity available from native proteins with functions such as DNA manipulation, stimuli response and molecular transport. Regarding the study of disease, the search of relevant drugs and the culture of cells, absence or presence of certain protein functions are equally important. Moreover, chemical engineering allows to generate synthetic proteins with artificially designed functions, which expectedly expand a field rapidly in the near future. Due to the diversity of protein functionality, protein as 3D printing precursor material promises a great potential for applications in broad fields. The potential is yet unexplored because details of mechanisms and fabrication parameters are still under investigation. First applications however confirm that an interesting protein function or structure design can open a new possibility.

Great potential for diverse applications | Of course, protein is important for cellular environment as protein largely constitutes the extra cellular matrix of cells or mediates cell-cell communication. 3D printed protein providing a biomimetic microenvironment for cells can contribute to tissue engineering, cell culture and organ-on-a-chip devices for drug screening. The challenge are to create the microenvironment for target cells with sufficient complexity and mechanical properties. Integration of such microenvironment into microfluidic devices yields also other approaches beyond cell culture environments. Simple protein structures of antibody can be used to capture what binds to the antibody. Enzymatic protein in a microfluidic device can be utilized as reactor in a confined micrometer-sized chamber. The on-going challenges here are to identify useful proteins and integrating multiple proteins into a single device to achieve multi-purpose devices. In addition to biological functions of protein, secondary function of pH-dependent deformability that some proteins possess gives an opportunity to fabricate some dynamic devices. In this case, pH-change induces swelling and shrinkage of the hydrogel-like proteinaceous microstructures, which relates to movement when designed appropriately. Here, the environmentally friendly nature of protein is also important factor for practical use. This concept can be applied to actuate well-designed structures such as pore sizes in a grid and to create soft, even bendable, micro-optical devices.
    As the outlook of 3D printing of protein, diversity of protein functions offers infinite possibilities for wide range of applications. Hybrid 3D structures of protein with other materials such as polymer will further expand the possibilities. Thus, although 3D fabrication of proteinaceous micro- and nano-structures and its applications are still in its infancy, a bright and broad future is highly expected.

Figure: Illustration of connective protein structure by interaction of protein molecules (light blue) with photoactivated molecules to form a cross-linked protein molecules (dark blue) only at a focal volume of the fs laser. The inset SEM image is reprinted from Serien and Takeuchi, Appl. Phys. Lett. 107, 013702, with the permission of AIP Publishing, copyright 2015.
(scale bar: 1 µm)



Serien D, Sugioka K. Fabrication of three-dimensional proteinaceous micro- and nano-structures by femtosecond laser cross-linking. Opto-Electronic Advances 1, 180008 (2018).
DOI: 10.29026/oea.2018.180008