title: Improved activity of immobilized antibody by paratope orientation controller: probing paratope orientation by electrochemical strategy and surface plasmon resonance spectroscopy.
abstract: Electrochemical method and surface plasmon resonance (SPR) spectroscopic analysis are utilized herein to investigate antibody immobilization without and with orientation control for site-positioning paratopes (antigen binding site) of the antibody molecules. Biotin and its antibody were selected in current study as model. Such an approach employed thiophene-3-boronic acid (T3BA) as paratope orientation controller, (i) enabled site orientation of the antibody molecules reducing the hiding of paratopes, and (ii) maintained the activity of the captured antibodies, as confirmed by electrochemical and SPR analysis. Anti-biotin antibody (a glycoprotein) was covalently bound to a self-assembled monolayer of T3BA modified on a nanogold-electrodeposited screen-printed electrode through boronic acid-saccharide interactions, with the boronic acid units specifically binding to the glycosylation sites of the antibody molecules. The immunosensor functioned based on competition between the analyte biotin and biotin-tagged, potassium hexacyanoferrate(II)-encapsulated liposomes. The current signal produced by the released liposomal Fe(CN)6(4-), measured using square wave voltammetry, yielded a sigmoidally shaped dose-response curve that was linear over eight orders of magnitude (from 10(-11) to 10(-3)M). Furthermore this biosensing system fabricated based on T3BA approach was found to possess significantly improved sensitivity, and the limit of detection toward biotin was calculated as 0.102 ng mL(-1) (equivalent to 6 μL of 4.19 × 10(-10)M biotin).
authors.affiliation: BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan; Department of Chemistry, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
authors.forename: Wei-Ching
authors.initials: WC
authors.lastname: Liao
authors.affiliation: BioAnalytical Chemistry and Nanobiomedicine Laboratory, Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan; Department of Chemistry, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan. Electronic address: jaho@ntu.edu.tw.
authors.forename: Ja-An
authors.initials: JA
authors.lastname: Annie Ho
mesh_terms: D000906:Antibodies / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D001666:Binding Sites, Antibody / Q000276:immunology; D015374:Biosensing Techniques / Q000295:instrumentation*; D001710:Biotin / Q000032:analysis* / Q000737:chemistry / Q000276:immunology*; D001897:Boronic Acids / Q000737:chemistry; D003217:Conductometry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation*; D011487:Protein Conformation; D020349:Surface Plasmon Resonance / Q000295:instrumentation*; D013876:Thiophenes / Q000737:chemistry
chemical_list: D000906:Antibodies; D001897:Boronic Acids; D013876:Thiophenes; C533772:thiophene-3-boronic acid; D001710:Biotin
keywords: Boronate affinity; Electrochemistry; Immunosensor; SPR analysis; Site-oriented immobilization