Abstract
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.
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References
Allen R, Nielson R, Wise DD, Shear JB (2005) Catalytic three-dimensional protein architectures. Anal Chem 77:5089–5095
Banu S, Greenway GM, McCreedy T, Shaddick R (2003) Microfabricated bioreactor chips for immobilised enzyme assays. Anal Chim Acta 486:149–157
Basu S, Campagnola PJ (2004) Enzymatic activity of alkaline phosphatase inside protein and polymer structures fabricated via multiphoton excitation. Biomacromolecules 5:572–576
Basu S, Cunningham LP, Pins GD et al (2005) Multiphoton excited fabrication of collagen matrixes cross-linked by a modified benzophenone dimer: bioactivity and enzymatic degradation. Biomacromolecules 6:1465–1474
Campagnola PJ, Delguidice DM, Epling GA et al (2000) 3-Dimensional submicron polymerization of acrylamide by multiphoton excitation of xanthene dyes. Macromolecules 33:1511–1513
Gao J, Xu J, Locascio LE, Lee CS (2001) Integrated microfluidic system enabling protein digestion, peptide separation, and protein identification. Anal Chem 73:2648–2655
Hashimoto M, Kaji H, Kemppinen ME, Nishizawa M (2008) Localized immobilization of proteins onto microstructures within a preassembled microfluidic device. Sens Actuators B 128:545–551
Heo J, Crooks RM (2005) Microfluidic biosensor based on an array of hydrogel-entrapped enzymes. Anal Chem 77:6843–6851
Hill RT, Shear JB (2006) Enzyme–nanoparticle functionalization of three-dimensional protein scaffolds. Anal Chem 78:7022–7026
Holden A, Jung SY, Cremer PS (2004) Patterning enzymes inside microfluidic channels via photoattachment chemistry. Anal Chem 76:1838–1843
Honda T, Miyazaki M, Yamaguchi Y, Nakamura H, Maeda H (2007) Integrated microreaction system for optical resolution of racemic amino acids. Lab Chip 7:366–372
Huber DL, Manginell RP, Samara MA, Kim Bl, Bunker BC (2003) Programmed adsorption and release of proteins in a microfluidic device. Science 301:352–354
Hui AY, Wang G, Lin B, Chan WT (2005) Microwave plasma treatment of polymer surface for irreversible sealing of microfluidic devices. Lab Chip 5:1173–1177
Iosin M, Stephan O, Astilean S, Dupperay A, Baldeck PL (2007) Microstructuration of protein matrices by laser-induced photochemistry. J Optoel Adv Mat 9:716–720
Kaehr B, Allen R, Javier DJ, Currie J, Shear JB (2004) Guiding neuronal development with in situ microfabrication. PNAS 101:16104–16108
Koh G, Pishko M (2005) Immobilization of multi-enzyme microreactors inside microfluidic devices. Sens Actuators B 106:335–342
Křenková J, Foret F (2004) Immobilized microfluidic enzymatic reactors. Electrophoresis 25:3550–3563
Křenková J, Svec F (2009) Less common applications of monoliths: IV. Recent developments in immobilized enzyme reactors for proteomics and biotechnology. J Sep Sci 32:706–718
Mao H, Yang T, Cremer PS (2002) Design and characterization of immobilized enzymes in microfluidic systems. Anal Chem 74:379–385
Peterson DS, Rohr T, Svec F, Fréchet JMJ (2002) Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal Chem 74:4081–4088
Pitts JD, Campagnola PJ, Epling GA, Goodman SL (2000) Submicron multiphoton free-form fabrication of proteins and polymers: studies of reaction efficiencies and applications in sustained release. Macromolecules 33:1514–1523
Pitts JD, Howell AR, Taboada R, Banerjee I, Wang J, Goodman SL, Campagnola PJ (2002) New photoactivators for multiphoton excited three-dimensional submicron cross-linking of proteins: bovine serum albumin and type 1 collagen. Photochem Photobiol 76:135–144
Sakai-Kato K, Kato M, Toyo’oka T (2002) On-line trypsin-encapsulated enzyme reactor by the sol–gel method integrated into capillary electrophoresis. Anal Chem 74:2943–2949
Uhlich T, Hubbell JA (1997) In: 23rd Annual meeting of the society for biomaterials transcripts, New Orleans, LA
Wang C, Oleschuk R, Ouchen F, Li J, Thibault P, Harrison DJ (2000) Integration of immobilized trypsin bead beds for protein digestion within a microfluidic chip incorporating capillary electrophoresis separations and an electrospray mass spectrometry interface. Rapid Commun Mass Spectrom 14:1377–1383
Watts P, Haswell SJ (2005) The application of micro reactors for organic synthesis. Chem Soc Rev 34:235–246
Whitesides GM (2006) The origins and the future of microfluidics. Nature 442:368–373
Yang T, Jung SY, Mao H, Cremer PS (2001) Fabrication of phospholipid bilayer-coated microchannels for on-chip immunoassays. Anal Chem 73:165–169
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This work was supported by Agence Universitaire de la Francophonie (AUF).
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Iosin, M., Scheul, T., Nizak, C. et al. Laser microstructuration of three-dimensional enzyme reactors in microfluidic channels. Microfluid Nanofluid 10, 685–690 (2011). https://doi.org/10.1007/s10404-010-0698-9
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DOI: https://doi.org/10.1007/s10404-010-0698-9