Microfluidic sensor using pH gradient with hybrid magnetoliposomes containing laccase immobilized nanocrystals

A method using pH gradient to modify the phospholipid dissociation has been developed using hybrid magnetoliposomes with encapsulated enzyme laccase, which has been immobilized on hydrophilic magnetic nanocrystals (PAA-MNCs) retained in the reaction/detection zone of a microfluidic system. The hybrid magnetoliposomes act as micro containers, providing the safe transfer of the enzyme to the reaction/detection site, where it is retained, and preconcentration of the enzyme in this place occurs, which improves the system's sensitivity. The use of pH change involves the lack of external molecules to release the encapsulated materials. The laccase-mediated redox oxidation of the fluorescent substrate 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to form its non-fluorescent oxide form has been used as the indicator reaction. The method was applied to determine potential laccase inhibitors in beverages and fruits. The study of the inhibitory effect has focused on compounds that follow different mechanisms: cysteine, malic acid, and fumaric acid. The dynamic ranges of the calibration graphs of these compounds were between 0.05-100, 0.02-100, and 0.1-100 μmol L^-1, respectively. The detection limits have been established between 6 and 30 nmol L^-1, and the precision expressed as the percent of relative standard deviation (RSD%) was between 2.5 and 5.1%. The overall microfluidic system showed a sampling frequency of 8 h^-1. The method has been applied to determine fumaric acid as a laccase inhibitor in several juice samples (apple, pear, and grape), with recovery values between 85 and 101%. The results were comparable to those obtained using a LC reference method.