| Biomarkers are playing increasing important role in cancer risk assessment and screening, refine prognosis and estimate response to biological therapy. Various detect methods based on electrochemical technology have attracted great attention because they are economical, fast and easily quantifiable. Amperometric immunosensors, by immobilization of bioactive materials on the electrode surface, used in detecting the change in current caused by an antibody-antigen reaction, have proved to be a sensitive, rapid, robust and cost effective quantitative method. As for the fabrication process of electrochemical immunosensors, emphasis was placed on the step of immobilizing biomolecules on the sensing surface. Nanomaterials usually a play key role in sensors construction and performance improvement. Here, a series of researches focus on the preparation of multi-functionalized nanomaterials, and the construction of the amperometric immunosensors and simultaneous multianalytes immunoassays technology are presented. The detail contents are as follows:1. A novel amperometric immunosensor constructed with gold-platinum nanoparticles and horseradish peroxidase nanoparticles as well as nickel hexacyanoferrates nanoparticlesIn this work, we were taking full advantage of the merits of gold-platinum nanoparticles (Au-PtNPs), nickel hexacyanoferrates nanopartcles (NiHCFNPs) and horseradish peroxidase nanoparticles (HRPNPs) to fabricate a simple, highly sensitive and stable AFP immunosensor. Initially, Au-PtNPs were electrodeposited on the glass carbon electrode (GCE) by a potentiometric stripping analysis technique, then NiHCFNPs were assembled onto the Au-PtNPs through a gold-cyanide bond. Afterwards, the second layer of Au-PtNPs was deposited, subsequently, anti-fetoprotein (anti-AFP) was immobilized through the reaction of amino and mercapto groups with the Au-PtNPs. HRPNPs were immobilized as a blocking reagent. In this project, NiHCFNPs provided excellent redox peaks for detection, and the response signal could be further amplified by the synergistic catalysis action of Au-PtNPs and HRPNPs towards the reduction of H2O2, we employed HRPNPs as a catalysis and blocking substance instead of HRP, the former can load much more HRP molecules on limited binding sites than the latter, and it was beneficial to improve the response signal. In addition, the fabrication method was very simple and the nano-materials adopted in this project were highly stable, so it was apt to improve the stability of the immunosensor.2. Novel amperometric immunosensor for detection of Streptococcus suis serotype 2 using nickel-gold nanocomposite as traceMost of the conventional methods of detection of streptococcus suis serotype 2 (SS2) were time-consuming, costly and needing laborious procedures. We attempted to develop a simple, quick and sensitive electrochemical immunosenosor for detection of SS2. As the CN" in nickel hexacyanoferrates NiHCF could easily bind with Au ion, this characteristic could be used to preparation of gold-nickel nanocomposite through simple mixing the two kinds of materials. To construct the biosensor, Au nanoparticles (AuNPs) were simply electrochemicaldeposited on the surface of GCE, and immobilized protein A (PA) to integrate with capture antibody (Ab1). Au nanocages (AuNCs) and NiHCFNPs complex were used to combination of detection antibody (Ab2) as trace, which not only provided redox peak for detection, also performed catalysis of ascorbic acid (AA) to amplify the response signal. The resulted immunosensor was very simple, highly sensitive and low cost.3. Amperometric immunosensor for simultaneous detection of three analytes in one interface using dual functionalized grapheme sheets integrated with redox-probes as tracer matrixesBased on sandwich-type amperometric assay, three kinds of Abl were immobilized onto PA/Nafion modified GCE, and three kinds of Ab2, primarily crosslinked with three redox-probes, were integrated with 3,4,9,10-perylenetetracarboxylic acid/Au/grephene sheet (PTCA/Au/GS) nano-composites through AuNPs and carboxyl group and adopted as tracer. This protocol presents several advantages. Firstly, each molecule of PA contains five homologous Ig-binding domains, and each domain can bind one protein molecule, using PA/Nafion as base layer can not only immobilize a great deal of Ab1, but also benefited lowering system background current. Secondly, functionalized GS with AuNPs and carboxyl group can fully integrate redox-probe@Ab2 composites, thus greatly improved the signal intensity of tracer. To test the feasibility, we selected three biomarkers of AFP, CEA and SS2 as model analytes. The resulted immunosensors exhibited low background current and excellent sensitivity, also wide detection range and low detection limit.4. Simultaneous detection of four biomarkers with one sensing surface based on redox probe tagging strategyIn clinical diagnosis, the measurement of single cancer marker is usually not sufficient, and combinatorial markers measurement has proved to be an effective method in improving diagnostic value. In this study, we presented the progress of redox probe tagging strategy that simultaneous determination of four biomarkers with one biosensing surface. To achieve this, four redox probes, including anthraquinone 2-carboxylic acid (Aq), Thi, Co(bpy)33+and Fc, were adopted as signal tags. The relatively broad potential window between neighbor tags implied there was minimal peak overlap. In sandwich immunoassay format, four kinds of capture antibodies were immobilized by AuNPs electrodeposited on the surface of glass carbon electrode (GCE); four kinds of detection antibodies, first labeled with different redox-probes, were immobilized by AuNPs wrapping on the surface of CNTs modified with 3,4,9,10-perylenetetracarboxylic acid (PTCA) and poly(diallyldimethylam-monium chloride (PDDA), and act as tracer. When using CEA, CA125, CA19-9 and CA242 as model analytes, the resulting immunosensors exhibited low background current, excellent sensitivity and selectivity.5. Ultrasensitive simultaneous detection of four biomarkers based on hybridization chain reaction and biotin-streptavidin signal amplification strategyNano-materials usually played key role in sensors construction and performance improvement. Recently, one-step codeposition of GS and GS-Au methods have attracted much attention due to the remarkable traits of simple, fast, and green. Owning to the presence of oxygen functionalities on GS surface provide reactive sites for the nucleation, AuNPs could uniformly grow at GS surfaces without agglomeration. Hybridization chain reaction (HCR) has been used to increase the amount of signal tags in various sensors. In this work, GS-Au nanomaterial, HCR and biotin/streptavidin (B/SA) signal amplification methods were adopted to synergistically improve the response performance of the multi-analytes immunosensor. In addition, magnetic nanomaterials provided great convenience in the preparation of Ab2 bionconjugates through magnet separation. When using four kinds of classic biomarkers (AFP, CEA, PSA and CA125) as model, the resulted immunosensor exhibited wide detection range and ultralow detection limit.
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