Femtosecond Laser Direct Write Integration of Multi-Protein Patterns and 3D Microstructures into 3D Glass Microfluidic Devices

Abstract

Microfluidic devices and biochips offer miniaturized laboratories for the separation, reaction, and analysis of biochemical materials with high sensitivity and low reagent consumption. The integration of functional or biomimetic elements further functionalizes microfluidic devices for more complex biological studies. The recently proposed ship-in-a-bottle integration based on laser direct writing allows the construction of microcomponents made of photosensitive polymer inside closed microfluidic structures. Here, we expand this technology to integrate proteinaceous two-dimensional (2D) and three-dimensional (3D) microstructures with the aid of photo-induced cross-linking into glass microchannels. The concept is demonstrated with bovine serum albumin and enhanced green fluorescent protein, each mixed with photoinitiator (Sodium 4-[2-(4-Morpholino) benzoyl-2-dimethylamino] butylbenzenesulfonate). Unlike the polymer integration, fabrication over the entire channel cross-section is challenging. Two proteins are integrated into the same channel to demonstrate multi-protein patterning. Using 50% w/w glycerol solvent instead of 100% water achieves almost the same fabrication resolution for in-channel fabrication as on-surface fabrication due to the improved refractive index matching, enabling the fabrication of 3D microstructures. A glycerol-water solvent also reduces the risk of drying samples. We believe this technology can integrate diverse proteins to contribute to the versatility of microfluidics.

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

“…It is unclear why DMSO improves feature sizes and fabrication quality, although the heat and electron transfer properties of this solvent might play a role, in addition to the attendant increase in the viscosity of the precursor solution. In order to mimic these features, a glycerol-water solvent was employed in a prior work 37 . Because high concentrations of glycerol can be used to preserve proteins 71 , this solvent does not risk damaging the protein at any concentration.…”

“…Because high concentrations of glycerol can be used to preserve proteins 71 , this solvent does not risk damaging the protein at any concentration. When fabricating in glass microfluidic channels, light refraction and aberration due to refractive index changes between device and protein precursor material caused an increase in feature sizes and limited penetration depth 37 . Use of glycerol-water solvents provided an improved refractive index match to glass with n=1.51 from pure water n=1.333 to (a) Laser writing system is composed of three basic components: a laser, a lens and mirror system, and an objective lens.…”

“…33 , with the permission of AIP Publishing, copyright 2015. 37 . Additional investigations of refractive index matching and viscosity optimization in conjunction with proteins could allow glycerol-water solvents to be used to refine the fabrication process 72 .…”

“…The use of effective photoinitiators could improve the fabrication process and also expand the range of applicable proteins. As an example, a benzophenone derivative (BPD) was used to cross-link small amounts of various types of collagen 52 , while the water-soluble compound sodium 4-[2-(4-morpholino)benzoyl-2-dimethylamino]butylben zenesulfonate (MBS) was utilized to cross-link enhanced green fluorescent protein (EGFP) 37 . A three-component system comprising a protein, rhodamine B and flavin adenine dinucleotide (FAD) significantly improved both fabrication speed and feature sizes 66 .…”

“…The non-invasive nature of forming crosslinked proteins in solution enables the fabrication of various complex embedded 3D structures. These structures can be nested on the substrate surfaces 33,34 or embedded into transparent devices such as microfluidic channels [35][36][37] . In addition to 3D fabrication and superior resolution, another advantage of the multi-photon cross-linking of proteins as 3D printing materials is the potential to retain the protein functions after fabrication 35,38,39 .…”