Simultaneous detection of four biomarkers with one sensing surface based on redox probe tagging strategy

• Immunosensor for simultaneous detection of four biomarkers on identical interface.
• Aq, Thi, Fc and Co(bpy)33+ were adopted as signal tags.
• Preparing Au/PDDA/PTCA/CNTs/redox-probe@D-Ab as signal tracer.
• The immunosensor exhibited excellent sensitivity and selectivity.

In this paper, a simple and sensitive amperometric immunosensor for simultaneous detection of four biomarkers by using distinguishable redox-probes as signal tags was proposed for the first time. In sandwich immunoassay format, four kinds of capture antibodies (C-Ab) were immobilized by gold nanoparticles (AuNPs) electro-deposited on the surface of glass carbon electrode (GCE); four kinds of detection antibodies (D-Ab) labeled with different redox probes (including anthraquinone 2-carboxylic acid (Aq), thionine (Thi), ferrocenecarboxylic acid (Fc) and tris(2,2’-bipyridine-4,4’-dicarboxylic acid) cobalt(III) (Co(bpy)33+)), were combined with 3,4,9,10-perylenetetracarboxylic acid (PTCA), poly(diallyldimethylammonium chloride) (PDDA) and AuNPs functionalized carbon nanotubes, and served as signal tracer. When four target antigens were present, differential pulse voltammetry (DPV) scan exhibited four well-resolved peaks, each peak indicated one antigen, and its intensity was quantitative correlational to the concentration of corresponding analyte. To verify the strategy, four biomarkers for diagnosis of colorectal carcinoma, including carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9 CA125, and CA242, were used as model analytes, the immunosensor exhibited high selectivity and sensitivity, and peak current displayed good linear relationship to logarithm concentration in the ranges from 0.016 to 15 ng mL−1 for CEA; 0.008 to 10 ng mL−1 for CA19-9; 0.012 to 12 ng mL−1 for CA125; 0.010 to 10 ng mL−1 for CA242, and low detection limits of 4.2, 2.8, 3.3 and 3.8 pg mL−1, respectively.

In this work, an amperometric immunosensor for simultaneous detection of four biomarkers by using distinguishable redox-probes as signal labels was proposed for the first time. Four kinds of redox probes including anthraquinone 2-carboxylic acid (Aq), thionine (Thi), ferrocenecarboxylic acid (Fc) and tris(2,2(-bipyridine-4,4(-dicarboxylic acid) cobalt(III) (Co(bpy)33+ were adopted to label four detection antibodies (D-Ab) through amide reaction, then integrated with functionalized carbon nanotubes (CNTs), and served as tracer in sandwich-typed immunoassay format. Differential pulse voltammetry (DPV) scan exhibited four well-resolved peaks at different potential position, each peak indicated one target antigen, and the current intensity was quantitative to the concentration of corresponding analyte illustrates the fabricated procedure and the principle of sandwich-type immunoassay.Figure optionsDownload as PowerPoint slide