An electrochemical assay system for peroxidase and peroxidase-couplers reactions based on a fluoride ion-selective electrode.

Abstract

A sensitive and accurate potentiometric reaction-rate method for the assay of peroxidase (EC 1.11.1.7) and peroxidase-coupled reactions is described. The technique is based on the peroxidase-catalyzed rupture of the covalent C--F bond in certain organo-fluoro compounds in the presence of H2O2 and the measurement of liberated F- by a fluoride ion-selective electrode. In a study screening 26 organo-fluoro compounds, 12 were susceptible to C--F bond rupture. However, analytically useful reaction kinetics were observed only with 4-fluoroaniline, 5-fluor-2-methylaniline, 4-fluorophenol, 2,3,5,6-tetrafluorophenol, pentafluorophenol, and 3-fluoro-DL-tyrosine. With this assay system, peroxidase activity as low as 25 mU/L could be precisely measured within 2 min. The reaction was coupled to glucose oxidase and cholesterol oxidase for the determination of glucose and cholesterol. Glucose oxidase activity in solution (to 2 U/L) was measured to demonstrate the feasibility of the assay for oxidase enzymes. Only a few microliters of sample is required and most determinations can be completed within minutes. The assay system offers broad applications and is especially attractive for use in enzyme immunoassays.

H2O2‐ and pH‐sensitive CdTe quantum dots as fluorescence probes for the detection of glucose

“…Thus, 25 min was selected as the optimum reaction time. The pH range of 5.5–6.0 is most suitable for enzymatic reactions involving GOD . In this system, the luminescence intensity of the QDs reached a maximum at about pH 5.6.…”

“…At about pH 5.6, the luminescence intensity reached a maximum, and a further decrease of pH resulted in the decrease in luminescence intensity, and further increase of pH (such as pH 6.0, 6.5 and 7.0) resulted in the decrease in luminescence intensity. The pH‐dependent behavior of the luminescence of CdTe QDs is caused by structural changes at the surface of the QDs . At intermediate pH levels, the interaction between QDs and the capping agent is strong enough such that the capping agent protects QDs more efficiently.…”

Flow‐injection analysis with immobilized oxidase/peroxidase enzymes and fluoride electrode detection

“…In summary, we have demonstrated that the coupled enzymatic-fluoride electrode measurement method originally described Siddiqi [2] can be readily adapted to any one of three different FIA arrangements by using immobilized oxidase and peroxidase enzymes and either p-fluoroaniline or pentafluorophenol as a flowing reagent peroxidase substrate. While the glucose-GOD oxidase system was used merely as a model to investigate the analytical performance of such systems, it is likely that the approaches demonstrated here will be amenable to the design of similar FIA systems for detecting a host of other substrates for which highly selective oxidase enzymes exist (e.g., cholesterol, uric acid, lactate, etc.).…”

“…The peroxidase-catalyzed reaction of parahalogenated aromatic amine substrates with hydrogen peroxide was first reported by Hughs and Sanders [ 11. In a reaction involving transient formation of free radicals, halide ions are liberated. Some 30 years later, Siddiqi [ 2 ] demonstrated that hydrogen peroxide generated from certain highly selective oxidase enzymes could be quantitated by employing such peroxidase-catalyzed reactions in conjunction with direct potentiometric detection of the liberated fluoride ions with a solid-state fluoride ion-selective electrode. For determining various oxidase substrates, the following generic peroxidase coupled enzymatic sequence can be used; ( 2 ) The final products o f the peroxidation reactions (reaction 2) vary considerably depending on the specific fluorocompound used as the cosubstrate for the reaction (e.g., 4-fluorophenol, tetrafluorophenol, 4-fluoroaniline, etc.…”

“…In some cases, the actual products (usually dimers and polymers) have not been identified, although it has been shown that for several fluorocompounds, 1 mol of fluoride is liberated per mole of oxidase substrate (or mole of H,O,) [ 2 ] . The fastest reaction rates have been achieved using penta-and tetrafluorophenol, p-fluorophenol, and p-fluoroaniline.…”