title: Fluorescence enhancement aided by metal ion displacement.
abstract: Immunosensors are one of the most common platform used in clinical laboratories, in particular the class based on Enzyme Linked Fluorescent Assays (ELFA) takes advantage of the amplification step of the enzyme, usually the alkaline phosphatase, that catalyzes the hydrolysis of a fluorescent substrate leading it to fluoresce. Anyway, they suffer in sensitivity if compared to molecular diagnostic or more modern in vitro diagnostic devices. In our work, a simple and effective mechanism to enhance the fluorescent signal, and hence the sensitivity of the system, is presented. It is based on the metal ion displacement principle in which a second fluorophore, in our case Calcein Blue, quenched by a cobalt ion is add to the first one (4-MUP), and, in presence of inorganic phosphate, it will be progressively activated by the inorganic phosphate itself leading to the metal displacement. In this way Calcein Blue, newly free to fluoresce, contributes to global fluorescent signal generated by 4-MU. We have tested our proof of principle on a currently used immunoanalyzer, that is VIDAS® system (bioMérieux, Marcy l'Etoile, France) obtaining a fluorescence enhancement of about 50% for each concentration of hydrolyzed 4-MUP tested.
authors: University of Florence, Department of Chemistry Ugo Schiff, Via della Lastruccia 13, 50019 Sesto Fiorentino, Florence, Italy; Consiglio Nazionale delle Ricerche-Istituto di Chimica dei Composti di OrganoMetallci (CNR-ICCOM), Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy.
authors: Vanessa
authors: V
authors: Susini
authors: Consiglio Nazionale delle Ricerche-Istituto di Chimica dei Composti di OrganoMetallci (CNR-ICCOM), Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy. Electronic address: andrea.ienco@iccom.cnr.it.
authors: Andrea
authors: A
authors: Ienco
authors: bioMérieux Italia Spa, Via di Campigliano, 58, 50012 Bagno a Ripoli, Florence, Italy.
authors: Veronica
authors: V
authors: Lucia Rossi
authors: Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, via Paolo Savi 10, 56126 Pisa, Italy.
authors: Aldo
authors: A
authors: Paolicchi
authors: bioMérieux Italia Spa, Via di Campigliano, 58, 50012 Bagno a Ripoli, Florence, Italy. Electronic address: antonio.sanesi@biomerieux.com.
authors: Antonio
authors: A
authors: Sanesi
mesh_terms: D015374:Biosensing Techniques; D003035:Cobalt / Q000737:chemistry / Q000302:isolation & purification*; D057075:Enzyme Assays; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D011933:Reagent Kits, Diagnostic
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D011933:Reagent Kits, Diagnostic; D003035:Cobalt; C007740:fluorexon
keywords: Fluorescence enhancement; Immunosensors; Metal ion displacement