title: In situ microliter-droplet anodic stripping voltammetry of copper stained on the gold label after galvanic replacement reaction enlargement for ultrasensitive immunoassay of proteins.
abstract: We report a new protocol for ultrasensitive electrochemical sandwich-type immunosensing, on the basis of signal amplification by gold-label/copper-staining, galvanic replacement reactions (GRRs), and in situ microliter-droplet anodic stripping voltammetry (ASV) after an enhanced cathodic preconcentration of copper. First, a sandwich-type immuno-structure is appropriately assembled at a glassy carbon electrode. Second, copper is selectively stained on the catalytic surfaces of second antibody-conjugated Au nanoparticles through CuSO4-ascorbic acid redox reaction, and the GRRs between HAuCl4 and the stained copper are used to amplify the quantity of copper. Finally, the corresponding antigen is determined based on simultaneous chemical-dissolution/cathodic-preconcentration of copper for in-situ ASV analysis directly at the immunoelectrode. Cyclic voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance and scanning electron microscopy are used for film characterization and/or process monitoring. Under optimized conditions, ultrasensitive analyses of human immunoglobulin G (IgG) and human carbohydrate antigen 125 (CA125) are achieved. The limits of detection are 0.3 fg mL(-1) (equivalent to 7 IgG molecules in the 6 μL sample employed) for IgG (S/N=3) and 1.3 nU mL(-1) for CA125 (S/N=3), respectively, which are amongst the best reported to date for the two proteins. The theoretical feasibility of such a single-molecule-level amperometric immunoassay is also discussed based on the immunological reaction thermodynamics.
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Qin
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China.
authors.forename: Aigui
authors.initials: A
authors.lastname: Xu
authors.affiliation: Liuzhou Traditional Chinese Medicine Hospital, Liuzhou 545001, China.
authors.forename: Linchun
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China.
authors.forename: Long
authors.initials: L
authors.lastname: Chao
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China.
authors.forename: Fang
authors.initials: F
authors.lastname: He
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China.
authors.forename: Yueming
authors.initials: Y
authors.lastname: Tan
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.affiliation: Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China. Electronic address: xieqj@hunnu.edu.cn.
authors.forename: Qingji
authors.initials: Q
authors.lastname: Xie
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry; D015374:Biosensing Techniques / Q000295:instrumentation*; D018394:CA-125 Antigen / Q000032:analysis / Q000097:blood*; D003300:Copper / Q000737:chemistry*; D004566:Electrodes; D004867:Equipment Design; D006046:Gold / Q000737:chemistry*; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation*; D007074:Immunoglobulin G / Q000032:analysis / Q000097:blood*; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000737:chemistry
chemical_list: D055910:Antibodies, Immobilized; D018394:CA-125 Antigen; D007074:Immunoglobulin G; D006046:Gold; D003300:Copper
keywords: Electrochemical sandwich-type immunosensor; Galvanic replacement reactions; Human carbohydrate antigen 125; Human immunoglobulin G; Signal enhancement