| Electrochemical immunosensors, which combine electrochemical technology with immunoassay, are well suitable for aiding in the diagnosis of cancer disease duo to their high target specificity, rapid detection, inexpensive fabrication and operation. During recent years, the research of design and fabrication of electrochemical immunosensor continues to remain a vibrant area of research and shows no signs of slowing down, and mainly focuses on the sensitivity enhancement and signal amplification. Indeed, thanks to the continued development and incorporation of nanoscale materials into the construction of sensitive electrode interface for analyte recognization and the design of electroactive nanoprobe for signal amplification due to their large specific surface area and good biocompatibility in comparison with traditional macroscale materials, nanomaterials have enabled significant improvements in the analytical performance and utility of biosensors, such as improving detection limits, selectivity, and stability of electrochemical immuneosensors. In this thesis, we mainly focus on both the preparation of the sensitive immunoreaction interface and the enhancement of the signal of the electrochemical immunosensors. The detail contents are as follows:1. Amperometric immunosensor based on multi-walled carbon nanotubes/prussian blue/nano-Au modified electrode for determination ofα-fetoproteinA conspicuously simple and highly sensitive reagentless amperometric immunosensor based on the sequential electrodeposition of prussian blue (PB) and gold nanoparticles (GNP) on multi-walled carbon nanotubes (MWCNT) modified glassy carbon electrode (GCE) surface has been proposed for the detection ofα-fetoprotein (AFP). By comparison with PB, the MWCNT/PB composite film had been proved to show much better electrochemical stability and larger response current. The electrodeposited GNP film can be used not only to immobilize biomolecles and keep their activity, but also to avoid the leakage of PB and to prevent shedding of MWCNT/PB composite film from the electrode surface. The performance and factors influencing the performace of the immunosensor were investigated. Under optimal experimental conditions, the proposed immunosensor for AFP was observed with an ultralow limit of detection (LOD) equal to 3 pg·mL-1 (at 38), and the linear working range spanned the concentrations of AFP from 0.01 ng·mL-1 to 300 ng·mL-1. Moreover, the immunosensor as well as a commercially available kit, was examined for use in the determination of AFP in real human serum specimens. More significantly, the proposed immunosensor exhibited good accuracy, high sensitivity and stability, indicating the developed immunoassay could be a promising alternative approach for detection of AFP and other tumor markers in the clinical diagnosis.2. Amperometric immunosensor based on Nafion-multwalled carbon nanotubes-Ferrocenecarboxylic acid nanosphere composite modified electrode for determination of human chorionic gonadotropinA novel reagentless immunosensor was proposed based on Nafion-Multiwalled carbon nanotubes-Ferrocenecarboxylic acid nanosphere composite (Nafion-MWCNT-FcCOOH) for determination of human chorionic gonadotropin (HCG). Ferrocenecarboxylic acid nanospheres were successfully prepared by the ultrasonic-solvent-substitution method. The composite was fabricated on the surface of the bare glassy carbon electrode and used as substrate for construction of reagentless amperometric immunosensor. In this work, Ferrocenecarboxylic acid nanospheres were used as redox-active species for HCG detection. Under optimal experimental conditions, the proposed immunosensor exhibited a good response to HCG, the linear working range spanned the concentrations of HCG from 0.05 mlU·mL-1 to 200 mIU·mL-1 with a detection limit of 0.015 mIU·mL-1. Moreover, the proposed immunosensor exhibited good accuracy, high sensitivity and stability.3. A novel sandwich-type immunosensor based on electroactive multibioconjugates as signal amplification labels for highly sensitive detection of tumor biomarkersWe have reported a novel electrochemical amplification immunoassay using electroactive multibioconjugates as signal amplification labels for highly sensitive detection of tumor biomarkers. In this method, silica nanoparticles with good monodispersity and uniform round structure were employed as the carriers for immobilization of electroactive mediator, HRP and HRP labeled carcinoembryonic antigen (HRP-anti-CEA). Greatly enhanced sensitivity was achieved by introducing covalent bonding of electroactive thionine onto the surface of silica nanoparticles by using y-glycidoxypropyltrimethoxysilane (GPMS) as the linkage and coimmobilization of HRP and HRP-labeled anti-CEA (HRP-anti-CEA, HRP-Ab2) onto the thionine-modified silica surface at high HRP/HRP-Ab2 ratio through EDC/NHS. The amount of captured SiO2/Thi/HRP-Ab2 by sandwiched immunoreaction was related to the concentration of CEA (as a mode analyte) in the incubation solution, the calibration range for CEA detection was found to be 0.02 ng·mL-1 to 10 ng·mL-1 with an ultralow limit of detection of 7 pg·mL-1 that is well-below the threshold value of 2.5 ng·mL-1 for clinical diagnosis. Compared with traditional sandwich immunoassay, the detection sensitivity of the presented approach was greatly enhanced due to the large surface area of silica nanoparticle carriers, which increased in Thi and HRP loading per sandwiched immunoreaction.4. A novel electrochemical immunoassay based on diazotization-coupled functionalized bioconjugates as trace labels for ultrasensitive detection of carcinoembryonic antigenA novel sandwich electrochemical immunoassay immunosensor based on the signal amplification strategy of diazotizati on-coupling concept for ultrasensitive detection carcinoembryonic antigen (CEA) was reported. Diazo-4Atp-coupled-thionine (Thi)-conjugated gold nanoparticles were prepared for immobilization of horseradish peroxidase (HRP) and secondary anti-CEA to form core-shell bioconjugates (denoted as HRP/anti-CEA/AuNPs/Thi-4Atp@AuNP), which were used as electrochemical signal amplification reagent. The sensitivity of the immunosensor was greatly amplified by a dual amplification. One is that a large number of Thi and HRP was introduced on the electrode surface through sandwich immunoreaction, and the other is that HRP as enhancer could catalyze the oxidation reaction of thionine by H2O2, which results in great enhancement of the reduction peak current. Thus, the bioconjugates-based assay provided an amplification approach for detecting CEA at trace levels, leading to a detection limit as low as 0.7 pg·mL-1.5. Aamperometric immunosensor based on magnetic beads with electroactive polymer brush as signal amplification elements for highly sensitive detection of CA125A novel strategy for signal amplification of ultrasensitive sandwich amperometric immunosensor was proposed for the first time based on the amplification by atom transfer radical polymerization (ATRP) of glycidyl methacrylate (GMA) combined with the signal amplification of electroactive multielectron-polytyrosine. Polyglycidyl methacrylate (PGMA) chains were grafted on SiO2@Fe3O4 magnetic nanoparticles by ATRP. Similar to nanoparticles, long chain of PGMA provides numerous epoxy groups for chemical immobileization of electroactive multielectron-polityrosine in an attempt to further amplificate signal as well as to enhance detection sensitiveity. Under optimal experimental conditions, the proposed sandwich amperometric immunosensor exhibited a good response to CA125 with an ultralow detection limit of 0.3 U·mL-...
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