Eelectrochemical Immunosensors For Multianalyte Immunoassay Based On Metallic-encoded Probes

This paper developed a series of simple, highly sensitive and low-cost multianalyte electrochemical immunoassay for the simultaneous determination of multiple cancer biomarkers(AFP, CEA and CA19-9) in a single run, based on metallic-encoded probes,sandwich immunoassays and electrochemical stripping analysis. Multianalyte electrochemical immunoassay in a single run developed through single-analyte assay. The electrochemical stripping analysis of the metal components from the nanocomposite provides a means for discriminating multiple targets based on the peak potential of metal.The stripping currents of metal are proportional to the concentration of corresponding targets. The capture probes including Dynabeads and magnetic molecularly imprinted polymers(MMIPs) not only simplified the separation step, but also enriched target analytes. The signal tags were prepared by loading metallic-encoded labels including quantum dots, recombinant apoferritin(r Apo) and apoferritin(Apo) on matrix(Envision,graphene and poly-L-lysine), respectively. After a sandwich-type immunoreaction and magnetic separation, the subsequent electrochemical stripping analysis from the immunocomplex provides a means for quantification of targets based on the peak currents of metal. The detailed study was stated as follows:1. Signal amplification for multianalyte electrochemical immunoassay with bidirectional stripping voltammetry using metal-enriched polymer nanolabelsA novel bidirectional(anodic and cathodic) stripping voltammetric immunoassay was designed for the simultaneous determination of multiple model cancer biomarkers(AFP,CEA and CA19-9) in a single run, based on the integration of Envision complex loaded metal nanoparticles as signal tags and immunomagnetic beads as capture probes. The Envision complex, which is a long branching polymer consisting of numerous secondary antibodies and horseradish peroxidase(HRP), was employed for signal amplification by labeling corresponding detection antibodies and further loading metal nanoparticles(Cd S,Pb S and gold) to prepare distinguishable metal signal tags. Herein, the generation of immunosensing probes involved the co-immobilization of three types of primary anti-AFP,anti-CEA, and anti-CA19-9 antibodies onto a single magnetic Dynabead. After a twobinding step sandwich-type immunoassay, the Envision/Cd S, Envision/Pb S and Envision/Au signal tags were introduced onto the surface of the Dynabeads. The subsequent bidirectional voltammetric analysis of stripping metal components fromimmunocomplexes for quantification of tumor biomarkers was performed in a microcell with minimum capacity of 50 μL. Experimental results showed the immunoassay enabled the simultaneous determination of multiple biomarkers over a broad range of concentrations(AFP, 1 pg m L-1-50 ng m L-1; CEA, 1 pg m L-1-50 ng m L-1; CA19-9, 5 pg m L-1-100 ng m L-1) with detection limits reaching 0.02 pg m L-1 for AFP, 0.05 pg m L-1 for CEA, and 0.3 pg m L-1 for CA19-9.2. Simultaneous electrochemical immunoassay using graphene-Au grafted recombinant Apoferritin-encoded metallic labels as signal tags and dual-template magnetic molecular imprinted polymer as capture probesA novel electrochemical multiplexed immunoassay was designed for simultaneous determination of alpha-fetoprotein(AFP) and carcinoembryonic antigen(CEA) using recombinant apoferritin-encoded metallic nanoparticles(r Apo-M) as labels and dualtemplate magnetic molecularly imprinted polymers(MMIPs) as capture probes. The labels were prepared by loading recombinant apoferritin(r-Apo) and separately immobilize primary antibodies(anti-AFP and anti-CEA) via Au nanoparticles of in site growth on graphene(G). The capture probes were synthesized by self-polymerization of dopamine(DA) on the Fe3O4 nanoparticles(Fe3O4 NPs) and using AFP and CEA as the template proteins, which were used to enrich the targets simultaneously. After a sandwich-type immunoreaction, the labels were captured to the surface of MMIPs. The subsequent electrochemical stripping analysis of the metal components from the immunocomplex provide a means for quantification of targets based on the peak currents of Cd and Pb.Experimental results showed the immunoassay enabled the simultaneous determination of AFP and CEA in a single run with wide dynamic ranges of 0.001-5 ng m L-1. And the detection limits of AFP and CEA were 0.3 and 0.35 pg m L-1(S/N=3), respectively. These results suggested that the proposed multiplexed immunoassay would be applied for clinical screening of other biomarkers.3. Multiplexed electrochemical immunoassay of biomarkers based on bio-based polymer encoded nanotags for signal amplificationA novel sandwich-type electrochemical multiplexed immunoassay was designed for simultaneous determination of multiple model cancer biomarkers(AFP and CEA) using bio-based polymer nanotags and dual antibody labeled immunize-magnetic beads as capture probes. The bio-based polymer nanotags were prepared by co-immobilizing encoded metallic apoferritin(Cd-Apo and Pb-Apo) and primary antibodies(anti-AFP and anti-CEA) on poly-L-lysine(PLL) via gold nanoparticles, respectively. The preparation procedures were very simply through self-assembly technology without using coupling agent. After a sandwich-type immunoreaction, the labels were captured to the surface ofDynabeads. The subsequent electrochemical stripping analysis of the metal components from the nanocomposite provide a means for discriminating dual targets based on the peak potential of Cd and Pb. The currents of Cd and Pb were proportional to the concentration of AFP and CEA, respectively. Experimental results showed the immunoassay enabled the simultaneous determination of AFP and CEA in a single run with dynamic ranges of 0.01-50 ng m L-1. And the detection limits of AFP and CEA were 4 pg m L-1(S/N=3),respectively.