Study of the inhibition effects on glutathione peroxidase immobilized on MNPs using a stopped-flow microfluidic system

• Published in: Analytical and bioanalytical chemistry Vol. 415; no. 11; pp. 2091 - 2100
• Main Authors: Román-Pizarro, Vanesa, Carrión-Escudero, Alba María, Écija-Arenas, Ángela, Fernández-Romero, Juan Manuel
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-05-2023; Springer; Springer Nature B.V
• Subjects: Analysis; Analytical Chemistry; Analytical Nanoscience and Nanotechnology; Biochemistry; Butyl hydroperoxide; Characterization and Evaluation of Materials; Chemical reactions; Chemistry; Chemistry and Materials Science; Copper; Edible oils; Enzymes; Fiber optics; Food Science; Glutathione; Glutathione - metabolism; Glutathione Peroxidase; Homovanillic acid; Hydrogen peroxide; Hydrogen Peroxide - chemistry; Laboratory Medicine; Magnetic properties; Mercury compounds; Microfluidics; Microreactors; Monitoring/Environmental Analysis; Nanoparticles; Optical Fibers; Oxidation-Reduction; Peroxidase; Properties; Research Paper; tert-Butylhydroperoxide; Vanillic Acid - chemistry; Spain; Glutathione peroxidase; Enzyme-magnetic nanoparticles; Stopped-flow microfluidic; Competitive redox reaction; Metallic ions and hydroperoxide as inhibitors; Bifurcated fiber optic fluorimetry
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-023-04521-0

A stopped-flow microfluidic system to monitor glutathione peroxidase (GPx) activity and evaluate potential inhibitors of the enzyme has been developed based on the integration of the microfluidic chip in the reaction/detection zone. This integration supposes the physical alignment at the optimal location of the microfluidic channel, both the magnetically retained enzyme microreactor (MREµR) and the remote luminescence detection using a focused bifurcated fiber optic bundle (BFOB) connected to a conventional spectrofluorometer detector. The method is based on the coupling of two competitive oxidative chemical reactions, in which glutathione (GSH) and homovanillic acid (HVA) competed for their interaction with hydrogen peroxide in the presence of the magnetically retained GPx-MNPs. The biocatalytic reaction was followed by monitoring the fluorescence of the biphenyl-HVA dimer formed. The dynamic range of the calibration graph was 0.45–10 µmol L
−1
, expressed as GSH concentration with a detection limit of 0.1 µmol L
−1
(
r
2
 = 0.9954,
n
 = 10,
r
 = 3). The precision expressed as the relative standard deviation (RSD%) was between 0.5 and 3.9%. The stopped-flow microfluidic system showed a sampling frequency of 25 h
−1
. The method was applied to the study of GPx inhibition provided by three inhibitory compounds, two metallic ions Hg(II) and Cu(II) and t-butyl hydroperoxide, and their presence in liquid samples, as water, milk, and edible oil. Recovery values between 88.7 and 99.4% were achieved in all instances.
Graphical Abstract