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  • Title: Kinetic Analysis of Enzymes Immobilized in Porous Film Arrays.
    Author: Neira HD, Herr AE.
    Journal: Anal Chem; 2017 Oct 03; 89(19):10311-10320. PubMed ID: 28858525.
    Abstract:
    Measuring the catalytic activity of immobilized enzymes underpins development of biosensing, bioprocessing, and analytical chemistry tools. To expand the range of approaches available for measuring enzymatic activity, we report on a technique to probe activity of enzymes immobilized in porous materials in the absence of confounding mass transport artifacts. We measured reaction kinetics of calf intestinal alkaline phosphatase (CIAP) immobilized in benzophenone-modified polyacrylamide (BPMA-PAAm) gel films housed in an array of fluidically isolated chambers. To ensure kinetics measurements are not confounded by mass transport limitations, we employed Weisz's modulus (Φ), which compares observed enzyme-catalyzed reaction rates to characteristic substrate diffusion times. We characterized activity of CIAP immobilized in BPMA-PAAm gels in a reaction-limited regime (Φ ≪ 0.15 for all measurements), allowing us to isolate the effect of immobilization on enzymatic activity. Immobilization of CIAP in BPMA-PAAm gels produced a ∼2× loss in apparent enzyme-substrate affinity (Km) and ∼200× decrease in intrinsic catalytic activity (kcat) relative to in-solution measurements. As estimating Km and kcat requires multiple steps of data manipulation, we developed a computational approach (bootstrapping) to propagate uncertainty in calibration data through all data manipulation steps. Numerical simulation revealed that calibration error is only negligible when the normalized root-mean-squared error (NRMSE) in the calibration falls below 0.05%. Importantly, bootstrapping is independent of the mathematical model, and thus generalizable beyond enzyme kinetics studies. Furthermore, the measurement tool presented can be readily adapted to study other porous immobilization supports, facilitating rational design (immobilization method, geometry, enzyme loading) of immobilized-enzyme devices.
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