Using Microfluidics and Imaging SAMDI-MS To Characterize Reaction Kinetics

Abstract

Microfluidic platforms have enabled the simplification of biochemical assays with a significant reduction in the use of reagents, yet the current methods available for analyzing reaction products can limit applications of these approaches. This paper demonstrates a simple microfluidic device that incorporates a functionalized self-assembled monolayer to measure the rate constant for a chemical reaction. The device mixes the reactants and allows them to selectively immobilize to the monolayer at the base of a microfluidic channel in a time-dependent manner as they flow down the channel. Imaging self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (iSAMDI-MS) is used to acquire a quantitative image representing the time-resolved progress of the reaction as it flowed through the channel. Knowledge of the surface immobilization chemistry and the fluid front characteristics allows for the determination of the chemical reaction rate constant. This approach widens the applicability of microfluidics for chemical reaction monitoring and establishes a label-free method for studying processes that occur within a dispersive regime.

Glycoenzyme Tool Development: Principles, Screening Methods, and Recent Advances†

“…Recently, the Mrksich lab developed a high‐throughput mass spectrometry method termed self‐assembled monolayers for matrix assisted desorption/ionization mass spectrometry (SAMDI‐MS) for the characterization of GT enzymes. [ 135‐137 ] This platform, called glycosylation sequence characterization and optimization by rapid expression and screening (GlycoSCORES), integrates E. coli ‐based cell‐free protein synthesis (CFPS) with SAMDI‐MS. By combining rapid in vitro biosynthesis of GTs in cell‐free extract with SAMDI‐MS, the authors were able to systematically investigate the enzyme's substrate specificity using 3480 unique peptides and 13903 unique reaction conditions, revealing the optimal glycosylation sequon (Figure 5A).…”

Characterizing Enzyme Cooperativity with Imaging SAMDI‐MS

“…The device was assembled with a monolayer floor, where the peptide was immobilized to an otherwise protein resistant surface at a density of approximately 10 % against a background of tri(ethylene glycol)-terminated alkanethiolates. We found in previous work that a peptide density of 10 % maintains inertness towards nonspecific protein adsorption to the surface and provides a sufficient amount of peptide for detection by SAMDI-MS. [8,9] Upon mixing of the solutions, the metal cofactor binds and activates the enzyme, which can then act on the immobilized peptide on the floor of the channel, leading to a conversion of the arginine residue to citrulline. Importantly, this device allows us to study the diffusion of Ca 2 + into the enzyme fluidic channel, which creates a diffusion-mixing region and yields kinetic information on enzyme-metal cooperativity.…”

“…Our approach was based on previous studies that modeled a steady-state reaction-diffusion network that incorporated the velocity pressure-driven fluid front, as well as the diffusion of all soluble species for calculating spatial concentration profiles. [9,23] In this work, we simulated the spatial concentration profiles of Ca 2 + , PAD2, the intermediate PAD2-Ca 2 + complex, and the citrullinated product in an x,y-plane in the center of the channel. It is important to note that we neglected the height of the device,…”

“…This method allows us to perform and analyze thousands of reactions in a single experiment for calculating the Michaelis constant and rate of a chemical reaction. [8,9] In this work, we characterize the Ca 2 + -induced activation of peptidylarginine deiminase type 2 (PAD2), which catalyzes the hydrolysis of an arginine guanidinium group to the corresponding urea to give citrulline. PAD2 is involved in the progression of several diseases, including breast cancer, [10] rheumatoid arthritis, [11] and macular degeneration.…”

Microfluidic Aqueous Two-Phase Focusing of Chemical Species for In Situ Subcellular Stimulation

“…Herein, we report a novel ATPS-based open microfluidic method in which the outward diffusion of target chemical species from its affinitive phase to the surrounding phase was demonstrated to be hindered by the nanoscale interface. A microfluidic probe was developed to control the contact time of two phases via space-time correspondence: – the two phases could always reach the biosamples within 10 –1 s, so the diffusion of species from their preferred phase to the other phase was negligible. Therefore, a chemical flow (the preferred phase with target chemical species) with a sharp boundary and micron-level width was created beneath the microfluidic probe (Figure a).…”