Oxidase-functionalized Fe(3)O(4) nanoparticles for fluorescence sensing of specific substrate

This study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe₃O₄ nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H₂O₂, the reaction pH between Fe₃O₄ NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe₃O₄ NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe₃O₄ NPs and oxidase for the quantification of specific substrates through the H₂O₂-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 μM using glucose oxidase–Fe₃O₄, galactose oxidase–Fe₃O₄, and choline oxidase–Fe₃O₄ composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method.     

Fluorescence assay of catecholamines based on the inhibition of peroxidase-like activity of magnetite nanoparticles

We report a fluorescence approach for the highly selective and sensitive detection of catecholamines using magnetite nanoparticles (Fe₃O₄ NPs) in the presence of Amplex UltraRed (AUR) and H₂O₂. Fe₃O₄ NPs catalyze H₂O₂-mediated oxidation of AUR. The resulting product fluoresces (excitation/emission maxima, ca. 568/587 nm) more strongly, relative to AUR. When catecholamines bind to Fe₃O₄, the complexes that are formed induce decreased activity of Fe₃O₄ NPs, mediated through the coordination between Fe³⁺ on the NP surface and the catechol moiety of catecholamines. As a result, Fe₃O₄ NPs-catalyzed H₂O₂-mediated oxidation of AUR is inhibited by catecholamines. The limits of detection for dopamine (DA), l-DOPA, norepinephrine, and epinephrine were 3 nM, 3 nM, 3 nM, and 6 nM, respectively. The Fe₃O₄ NPs-H₂O₂-AUR probe exhibited high selectivity (>1000-fold) toward catecholamines over other tested biomolecules that commonly exist in urine. Four catecholamines had similar sensitivity because the inhibition of the Fe₃O₄ NPs activity relies on the presence of the catechol moiety. This approach also allowed the determination of tyrosinase activity because tyrosinase catalyzes the conversion of l-tyrosine to l-DOPA. We validated the practicality of the use of the Fe₃O₄ NPs-H₂O₂-AUR probe for the determination of the concentrations of DA in urine samples.     

A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts

The SIEFED (“Specific Immunological Extraction Followed by Enzymatic Detection”) method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H₂O₂ and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.