Table 1: Summarization of flow-based method for enzyme study.

Flow-based techniqueEnzymeMethodologyCommentsReference No.

Continuous flowTyrosine phosphataseUse 3 syringe pumps for delivering solutions with constant combined flow rate of 100 μL/min for reaction with kinetic time scale of a minute or moreGeneral application of flow system based on laminar flow, possible to be applied to faster reactions by reducing the diameter of tubing[7]
Alkaline phosphataseRandomly and site directed immobilized his-tag alkaline phosphatase on beads were studied using FIA-chemiluminescence systemSite directed enzyme had 𝑉 m a x (activity) higher than randomly immobilized enzyme[19]
AcetylcholinesteraseOnline dialysis of product before mixing with chromogenic reagentOnline pretreatment[27]
Acetylcholinesterase and angiotensin-converting enzymeSI-LOV system with microreservoir and fiber optic/stirrerKinetic parameters obtained agree well with literature values[41]
UreaseThermal inactivation, flow colorimetry, and model equationCan conclude about reversibility and irreversibility of denature and native forms[58]
α-amylaseBasic FIA system for measuring enzyme activity at different pHsVery stable at neutral pH 5–8, but loss of activity out of this pH range[59]
GlucanaseFluorescence probe flow injectionConfirm the use of theoretical equation to predict kinetic parameters[60]
Alkaline phosphataseImmobilized enzyme on Sepharose beads and packed in the reactorsStudy effect of orthophosphate inhibitor[61]
α-amylaseImmobilized with glutaraldehyde on polyurethane foam and study activity under magnetic fieldShow oscillatory behavior of enzyme reaction[62]
Alkaline phosphataseOpen-closed flow injection, theophylline as inhibitorKm and inhibitor constant were determined[63]

Stopped flowFructose bisphosphataseLabel free, mid IR detectionAlternative detection method[57]
Glutathione transferasePotentiometric detection for mechanism of different isoenzymesAlternative detection method[18]
ElastaseConventional stopped flow-spectrometric system for slow binding kinetic approachConclude that inhibition is 2 step mechanism and gain insight understanding of the effect of heparin on the inhibition mechanism[65]
5-enolpyruvoyl shikimate-3-phosphate (EPSP) synthase Fluorescence measurement at equilibrium Evaluation of substrate and inhibitor binding[66]
TannaseImmobilized enzyme on glass beads, packed in conductometric flow cellCheck activity of immobilized tannase which is commonly repetitively used in industry[67]
Total and prostatic acid phosphataseDouble injection flow analysisIncrease injection concentration 2 fold to compensate dilution[68]

Protein kinasePhosphorylation of peptide substrate, quench with acetic acidThis is the first chemical observation and characterization of phosphoryl transfer at the active site of protein kinase[69]
Quench flow3-Deoxy-D-manno-2-octulosonate-8-phosphate synthaseAnion exchange HPLC for detection of radiolabelGain conclusion about reaction intermediate[70]
5-enolpyruvoyl shikimate-3-phosphate (EPSP) synthaseRadioactively label the enol moiety and then separate and quantitate products with HPLC after acid quenchObserve tetrahedral intermediate[71]

Zone trapping/Bypass trapped flowHexokinaseCoupled to glucose-6-phosphate dehydrogenase, and monitored production of reduced NADPH fluorometricallyComparable Km and Ki value to the published data obtained from manual techniques[74]

Air segmented flowPeroxidaseLAV with microreservoir with air segmentsMimic manual operation and eliminate dilution/dispersion effect[80]