Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

Schie, Morten M. C. H. van; Ebrahimi, Kourosh Honarmand; Hagen, Wilfred R.; Hagedoorn, Peter‐Leon

doi:10.1016/j.ab.2017.12.028

Cited by 11 publications

(5 citation statements)

References 26 publications

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“…The key to increase the throughput is through miniaturization and using microfluidics. The development of miniaturized sensitive calorimeters has produced the field chip calorimetry (Lerchner et al, 2006;Lerchner, et al, 2008;Hartmann et al, 2014;van Schie, et al, 2018). Microfluidic chips in which calorimetric devices are integrated can be used to measure chemical reactions with a very short response time.…”

Section: Discussionmentioning

confidence: 99%

Isothermal Titration Calorimetry in Biocatalysis

Hagedoorn

2022

Front. Catal.

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Isothermal titration calorimetry (ITC) is a popular chemical analysis technique that can be used to measure macromolecular interactions and chemical and physical processes. ITC involves the measurement of heat flow to and from a measurement cell after each injection during a titration experiment. ITC has been useful to measure the thermodynamics of macromolecular interactions such as protein-ligand or protein-protein binding affinity and also chemical processes such as enzyme catalyzed reactions. The use of ITC in biocatalysis has a number of advantages as ITC enables the measurement of enzyme kinetic parameters in a direct manner and, in principle, can be used for most enzymes and substrates. ITC approaches have been developed to measure reversible and irreversible enzyme inhibition, the effects of molecular crowding on enzyme activity, the activity of immobilized enzymes and the conversion of complex polymeric substrates. A disadvantage is that in order to obtain accurate kinetic parameters special care has to be taken in proper experimental design and data interpretation, which unfortunately is not always the case in reported studies. Furthermore, special caution is necessary when ITC experiments are performed that include solvents, reducing agents and may have side reactions. An important bottleneck in the use of calorimetry to measure enzyme activity is the relatively low throughput, which may be solved in the future by sensitive chip based microfluidic enzyme calorimetric devices.

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Section: Discussionmentioning

confidence: 99%

Isothermal Titration Calorimetry in Biocatalysis

Hagedoorn

2022

Front. Catal.

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“…They also reported issues related to the lower volumetric heat generated in the microfluidic calorimeter than in standard isothermal titration calorimeters. [85] Nevertheless, ITC is a promising approach for the label-free characterisation of AP and other enzymes with biologically relevant substrates. The reader is advised to watch this exciting field.…”

Section: Detecting Thermodynamicsmentioning

confidence: 99%

Methods to Characterise Enzyme Kinetics with Biological and Medicinal Substrates: The Case of Alkaline Phosphatase

Harroun

Vallée‐Bélisle

2023

Chemistry Methods

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Alkaline phosphatase (AP) enzymes are of broad interest in fields ranging from biochemistry and medicine to biotechnology and nanotechnology. Characterising the catalytic activity of AP is typically realised by either employing non‐natural signal‐generating substrates that are detectable by absorbance and fluorescence spectroscopy or by quantifying the release of inorganic phosphate by the classic malachite green assay. The latter method is often required for studying “spectroscopically silent” biomolecular substrates, but it does not enable continuous monitoring of kinetics in real‐time. In recent years, newer techniques for studying AP function have been developed to circumvent this limitation, including fluorescent and colourimetric substrate‐specific assays based on supramolecular chemistry, organic probes and nanomaterials, as well as other assays based on isothermal titration calorimetry, direct detection with infrared spectroscopy and mass spectrometry, and monitoring conformational change by fluorescent nanoantennas. Here, we review these strategies and comment on their strengths and weaknesses in the context of AP.

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“…The temperature change range correlated linearly with catalase activity. Recently, van Schie et al showed that a microfluidic calorimeter could be successfully used to determine enzyme activity by developing a new calibration method [114]. Deprotonation of phosphates provided a significant amount of heat during sufficient time for calibration purposes and to determine the sensitivity of each thermopile used.…”

Section: Engineering Aspectsmentioning

confidence: 99%

Multi-Enzyme Systems in Flow Chemistry

Fernandes

Carvalho

2021

Processes

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Recent years have witnessed a growing interest in the use of biocatalysts in flow reactors. This merging combines the high selectivity and mild operation conditions typical of biocatalysis with enhanced mass transfer and resource efficiency associated to flow chemistry. Additionally, it provides a sound environment to emulate Nature by mimicking metabolic pathways in living cells and to produce goods through the systematic organization of enzymes towards efficient cascade reactions. Moreover, by enabling the combination of enzymes from different hosts, this approach paves the way for novel pathways. The present review aims to present recent developments within the scope of flow chemistry involving multi-enzymatic cascade reactions. The types of reactors used are briefly addressed. Immobilization methodologies and strategies for the application of the immobilized biocatalysts are presented and discussed. Key aspects related to the use of whole cells in flow chemistry are presented. The combination of chemocatalysis and biocatalysis is also addressed and relevant aspects are highlighted. Challenges faced in the transition from microscale to industrial scale are presented and discussed.

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Fast and accurate enzyme activity measurements using a chip-based microfluidic calorimeter

Cited by 11 publications

References 26 publications

Isothermal Titration Calorimetry in Biocatalysis

Isothermal Titration Calorimetry in Biocatalysis

Methods to Characterise Enzyme Kinetics with Biological and Medicinal Substrates: The Case of Alkaline Phosphatase

Multi-Enzyme Systems in Flow Chemistry

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