title: Functionalized gold nanoclusters as fluorescent labels for immunoassays: Application to human serum immunoglobulin E determination.
abstract: A quantitative immunoassay for the determination of immunoglobulin E (IgE) in human serum using gold nanoclusters (AuNCs) as fluorescent label was developed. Water soluble AuNCs were synthesized using lipoic acid and then thoroughly characterized. The obtained AuNCs have a particle size of 2.7 ± 0.1 nm and maximum fluorescence emission at 710 nm. The synthesized AuNCs showed very good stability of the fluorescent signal with light exposure and at neutral and slightly basic media. A covalent bioconjugation of these AuNCs with the desired antibody was carried out by the carbodiimide reaction. After due optimization of such bioconjugation reaction, a molar ratio 1:3 (antibody:AuNCs) was selected. The bioconjugate maintained an intense luminescence emission, slightly red-shifted as compared to the free AuNCs. Two typical immunoassay configurations, competitive and sandwich, were assayed and their performance for IgE determination critically compared. After the different immunoassay steps were accomplished, the fluorescence emission of the bioconjugate was measured. While the sandwich format provided a detection limit (DL) of 10 ng/mL and a linear range between 25 and 565 ng/mL of IgE, the competitive format revealed a DL of 0.2 ng/mL with a linear range between 0.3 and 7.1 ng/mL The applicability of the more sensitive competitive fluorescent immunoassay was assessed by successful analysis of the IgE in human serum and comparison of results with those from a commercial kit. The main advantages of the proposed AuNCs-based fluorimetric method include a low DL and a simple immunoassay protocol involving few reagents.
authors.affiliation: Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería, 8, 33006 Oviedo, Spain.
authors.forename: María Cruz
authors.initials: MC
authors.lastname: Alonso
authors.affiliation: Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería, 8, 33006 Oviedo, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Trapiella-Alfonso
authors.affiliation: Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería, 8, 33006 Oviedo, Spain. Electronic address: jcostafe@uniovi.es.
authors.forename: José M Costa
authors.initials: JM
authors.lastname: Fernández
authors.affiliation: Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería, 8, 33006 Oviedo, Spain. Electronic address: mrpereiro@uniovi.es.
authors.forename: Rosario
authors.initials: R
authors.lastname: Pereiro
authors.affiliation: Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julián Clavería, 8, 33006 Oviedo, Spain.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Sanz-Medel
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation*; D001774:Blood Chemical Analysis / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D006046:Gold / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D007073:Immunoglobulin E / Q000097:blood*; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D058726:Nanoconjugates / Q000737:chemistry / Q000648:ultrastructure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D058726:Nanoconjugates; D007073:Immunoglobulin E; D006046:Gold
keywords: Competitive immunoassay; Fluorescence; Gold nanoclusters; Human serum; Immunoglobulin E