Laser microstructuration of three-dimensional enzyme reactors in microfluidic channels

In 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.