title: A sulfhydryl-reactive ruthenium (II) complex and its conjugation to protein G as a universal reagent for fluorescent immunoassays.
abstract: To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2'-dipyridine Ruthenium bis (hexafluorophosphate). The synthesized Ru(II) complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II)-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The emission peak wavelength of the Ru(II)-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II) complex, indicating that Ru(II)-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG) binding assay was conducted. The result showed that Ru(II)-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II)-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II)-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays.
authors.affiliation: Graduate Institute of Systems Biology and Bioinformatics, National Central University, Jhongli City, Taoyuan Country, Taiwan.
authors.forename: Jing-Tang
authors.initials: JT
authors.lastname: Lin
authors.forename: Po-Chung
authors.initials: PC
authors.lastname: Chen
authors.forename: Thirumani Venkatshwar
authors.initials: TV
authors.lastname: Goud
authors.forename: Bor-Rong
authors.initials: BR
authors.lastname: Huang
authors.forename: Tzu-Chau
authors.initials: TC
authors.lastname: Lin
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Biellmann
authors.forename: Chien-Sheng
authors.initials: CS
authors.lastname: Chen
mesh_terms: D000906:Antibodies / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D056831:Coordination Complexes / Q000138:chemical synthesis / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000737:chemistry; D006639:Histidine / Q000276:immunology; D007118:Immunoassay; D007074:Immunoglobulin G / Q000378:metabolism; D009842:Oligopeptides / Q000276:immunology; D011485:Protein Binding; D012428:Ruthenium / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013440:Sulfides / Q000737:chemistry
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D056831:Coordination Complexes; D005456:Fluorescent Dyes; C471213:His-His-His-His-His-His; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D009842:Oligopeptides; D013388:Succinimides; D013438:Sulfhydryl Compounds; D013440:Sulfides; D006639:Histidine; C039011:N-hydroxysuccinimide S-acetylthioacetate; D012428:Ruthenium