Non-denaturing low-temperature bonding of patterned poly(methyl methacrylate) enzymatic microreactors

A low-temperature solvent bonding system using methanol and water has been developed to bond poly(methyl methacrylate) (PMMA) microchips at 35 A degrees C. The substrate/cover plate adhesion strengths obtained with this bonding protocol peaked at 4,000 kN/m(2) for unmodified PMMA substrates. Nanoindentation measurements performed using atomic force microscopy revealed that only the first 30 nm of the PMMA surface showed a decreased hardness following surface modification and solvent treatment of the PMMA surface allowing the channel architecture to be maintained. The low temperature utilized for bonding enabled both a temperature-robust and temperature-labile enzyme to be facilely patterned prior to bonding with little-to-no loss in enzyme activity. Furthermore, the bonding methodology could be customized and used to fabricate an enzyme microreactor with pepsin (a pH, temperature and solvent sensitive enzyme). The enzyme microreactor performance was characterized by the longevity of the microreactor, as well as the efficiency of the protein digest performed. Enzyme immobilized with WSC decreased over a period of days, whereas the enzyme immobilized with both WSC (water soluble carbodiimide and NHS (N-hydroxysuccinimide) remained active even after a month of use. Short 10 mm column lengths with limited residence time provided high protein sequence coverage.