This paper describes a microfluidic chip wherein the position and order of two immobilized enzymes affects the type and quantity of reaction products in the flowing fluid. Assembly of the chip is based on a self-assembled monolayer presenting two orthogonal covalent capture ligands that immobilize their respective fusion enzyme. A thiol-tagged substrate is flowed over a region presenting the first enzyme-which generates a product that is efficiently transferred to the second enzyme-and the second enzyme's product binds to an adjacent thiol capture site on the chip. The amount of the three possible reaction products is quantified directly on the chip using self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry, revealing that the same microsystem can be spatiotemporally arranged to produce different products depending on the device design. This work allows for optimizing multistep biochemical transformations in favor of a desired product using a facile reaction and analytical format.
This paper describes a method that combines a microfluidic device and self-assembled monolayers for matrixassisted laser desorption/ionization mass spectrometry (SAM-DI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 μL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self-assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are flowed through each of eight channels, where the position along the channel corresponds to reaction time and position across the channel corresponds to the concentration of Ca 2 + . Imaging SAMDI (iSAMDI) is then used to determine the yield for the enzyme reaction at each 200 μm pixel on the monolayer, providing a time course for the reactions. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K 0.5 ) for the Ca 2 +dependent activation of PAD2. This work establishes a highthroughput and label-free method for studying enzymeligand binding interactions and widens the applicability of microfluidics and matrix-assisted laser desorption/ionization mass spectrometry (MALDI) imaging mass spectrometry.
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