Development of a new glucose colorimetric assay using glucose oxidase immobilized on magnetite-gold-folate nanoparticles

This work investigated a new colorimetric method for glucose detection using glucose oxidase (GOx) immobilized on a glod-coated magnetite nanoparticle core and encased within a conjugated folic acid (FA) cysteine (Cys) shell. Magnetite (Fe3O4) nanoparticles were synthesized by the chemical co-precipitation method of ferrous (Fe2+) and ferric (Fe3+) solution in a mole ratio 1:2 and reacted with sodium hydroxide (NaOH) solution to rapidly form Fe3O4 particles. Gold-coated magnetites (Fe3O4@Au) were produced via the reduction of auric ion (Au3+) to gold nanoparticles (Au0) on the surface of Fe3O4 using sodium citrate as a reducing agent. FA-conjugated Fe3O4@Au nanoparticles were synthesized using Cys as a bi-functional linker which linked to the gold surface through its thiol group (-SH), and conjugated amino group (-NH2) condensed with the carboxylic acid group (-COOH) on folic to form an amide bond. The synthesized Fe3O4@Au-Cys-Fa nanoparticles were used as peroxidase mimetics for sensitive colorimetric detection of glucose. The as-prepared Fe3O4@Au-Cys-FA nanoparticles were used to catalyze the oxidation of a peroxidase substrate, 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)-diammonium salt (ABTS), by hydrogen peroxide (H2O2) to form a colored oxidization product, thereby enabling colorimetric detection of H2O2. The reaction provided a green colored product and showed maximum absorption peak at 418 nm. The catalytic reaction followed Michaelis-Menten kinetics and exhibited strong affinity for H2O2 and ABTS with a molar absorption coefficient of 36,000 M-1cm-1. Fe3O4@Au-Cys-FA nanoparticles were used as a peroxidase mimetic in a colorimetric in a colorimetric method. The developed method provided linearity detection of H2O2 over the concentration range of 5µM to 10 mM, with a detection limit of 2.9 µM (3 S.D.blank/slope). The slope of the calibration plot indicated a sensitivity of 0.1023 (A.U.) mM-1, with a linear correlation of 0.998. An efficient colorimetric sensor was fabricated for glucose detection by mean of GOx immobilized on Fe3O4@Au-Cys-FA nanoparticle using glutaraldehyde vapor (50% w/v) cross-linking. Fe3O4@Au-Cys-FA-GOx nanocomposites were used to catalyze the oxidation of glucose to generate H2O2, coupled with catalyzing the oxidation of ABTS to ABTS+. The proposed nanocomposite exhibited good biocompatibility and provided highly efficient immobilization and retention of GOx. The designed colorimetric method provided a linear dynamic range for glucose detection from 10 µM to 1 mM, a detection limit of 3.8 µM (3 S.D.blank/slope). The sensitivity of the developed glucose detection was 0.4087 (A.U.)mM-1, a linear correlation of 0.997, with a good reproducibility and the nanocomposite was reusable (≥6 times). Moreover, an interference study revealed that maltose, fructose, and sucrose did not affect the colorimetric measurement. The developed method was successfully applied for glucose determination in energy and sports drinks.