title: Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors.
abstract: The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot.
authors.affiliation: Nanobiotechnology & Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain. joseplluis.acero@urv.cat.
authors.forename: Josep Ll
authors.initials: JL
authors.lastname: Acero Sánchez
authors.affiliation: Nanobiotechnology & Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain.
authors.forename: Alex
authors.initials: A
authors.lastname: Fragoso
authors.affiliation: Department of Chemistry, University at Albany, 1400 Washington Ave, Albany, NY 12222, United States.
authors.forename: Hamdi
authors.initials: H
authors.lastname: Joda
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle-upon-Tyne, NE2 4HH, UK.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Suárez
authors.affiliation: Institute of Cellular Medicine, Newcastle University, Newcastle-upon-Tyne, NE2 4HH, UK.
authors.forename: Calum J
authors.initials: CJ
authors.lastname: McNeil
authors.affiliation: Nanobiotechnology & Bioanalysis Group, Departament d'Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007, Tarragona, Spain. ciara.osullivan@urv.cat.|Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, 08010, Barcelona, Spain. ciara.osullivan@urv.cat.
authors.forename: Ciara K
authors.initials: CK
authors.lastname: O'Sullivan
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000276:immunology*; D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis; D003217:Conductometry / Q000295:instrumentation*; D004220:Disulfides / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry*; D007118:Immunoassay / Q000295:instrumentation*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000295:instrumentation*; D013499:Surface Properties
chemical_list: D000906:Antibodies; D020099:Coated Materials, Biocompatible; D004220:Disulfides; D006046:Gold
keywords: Neuron-specific enolase (NSE); SPR and electrochemical immunosensors; Self-assembled monolayer (SAM); Site-directed protein modification; Surface chemistry