Laser microstructuration of three-dimensional enzyme reactors in microfluidic channels

Abstract

International audienceIn this paper, we report on the fabrication of three-dimensional (3D) enzymatic microreactors within polydimethylsiloxane microfluidic channels through a photocrosslinking mechanism mediated by the two-photon absorption process at the focal point of pulse lasers, i.e., a sub-nanosecond Nd:YAG microlaser or a femtosecond Ti:Sapphire laser. This approach allows the building of localized 3D trypsin structures with submicron resolution. The fabrication of two different trypsin structures was successfully demonstrated using Eosin Y and Flavin Adenine Dinucleotide as biological photosensitizers: (i) arrays of 3D cylindrical rows and (ii) 3D woodpile structure. The enzymatic activity of the fabricated structures was evaluated by fluorescence spectroscopy using BODIPY FL casein as fluorogenic substrate. The real time investigation of the peptide cleavage into the microfluidic channel demonstrated that the fabricated trypsin microstructures maintain their catalytic activity. This approach opens up the way to complex multistep enzymatic reactions in well-localized regions of microfluidic devices, with great importance in health screening and biomedical diagnostics

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

“…Kaehr et al showed that binding fluorescence is proportional to the protein concentration 56 , implying not only retained function but also a non-preferential process. There have also been some reports about the retention of protein enzymatic functions 35,36,38 . However, to date there have been no analyses of protein activity or quantitative assessments of active units.…”

“…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 .…”

In-chip fabrication of free-form 3D constructs for directed cell migration analysis

“…Very recently, the extraordinary ability of 2PP to polymerize and structure 3D objects within a closed microchannel system was reported for use as in-chip filters or as in-chip enzymatic reactors. 26,27 Here, we demonstrate the fabrication of reproducible 3D structures with cell-sized pores by 2PP inside the closed channel of a commercially available disposable polymer microfluidic chip (Fig. 1) designed for chemotaxis analysis in non-structured hydrogel materials.…”

In-chip direct laser writing of a centimeter-scale acoustic micromixer

“…This ensures a good fit and reduces the amount of chemicals required. In-chip DLW was first used for enzymatic 3-D microreactors by Iosin et al 26 Amato et al 16 fabricated a porous filter into an existing microchannel and Serra et al…”