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

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