Studies On Electrochemical Immunosensor Based On Multifunctional Nanomaterials As Labels And Platforms For Signal Amplification

This thesis focuses on constructing a series of sensitive electrochemical immunosensors for tumor markers detection through screening some unique properties of nanometer materials as labels and platforms for sinal amplification. Despite of many advances in this field, It is still a challenge to exploit new approaches that can improve the sensitivity and decrease the detection limit of single tumor markers of the immunosensor, develop multiple tumor markers detection in the single run, and make portable and affordable devices for online detection to meet the requirement of modern medical diagnostics and biomedical research application. There are six chapters in this thesis, and main contents are listed as follows:Charpter 1:the definition, classification and principle of immunoassay and immunosesors were introduced in brief, and applications of multifunctional nanomaterials in electro-immunosensors were reviewed in detail. Meanwhile, the research purpose and signification of this thesis were summarized.Charpter 2:A feasible and practicable amperometric immunoassay strategy for sensitive screening of carcinoembryonic antigen (CEA) in human serum was developed using carbon nanotube (CNT)-based symbiotic coaxial nanocables as labels. To construct such a nanocable, a thin layer of silica nanoparticles was coated on the CNT surface by sonication and sol-gel methods, and then colloidal gold nanoparticles were assembled on the amino-functionalized SiO2/CNTs, which were used for the label of horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA-Au/SiO2/CNT). In the presence of analyte CEA, the sandwich-type immunocomplex was formed on an anti-CEA/Au/thionine/Nafion-modified glassy carbon electrode by using HRP-anti-CEA-Au/SiO2/CNTs as detection antibodies. Under optimal conditions, the cathodic peak currents of the electrochemical immunosensor were proportional to the logarithm of CEA concentration over the range from 0.01 to 12 ng mL-1 in pH 5.5 HAc-NaAc containing 5 mM H2O2, the detection limit (LOD) is 5 pg mL-1 CEA at 3σ.Charpter 3:This charper demonstrates a facile and feasible strategy toward the development of advanced electrochemical immunosensors based on chemically functionalized magnetic mesoporous organic inorganic hybrid nanomaterials, and the preparation, characterization, and measurement of relevant properties of the immunosensor for detection of carcinoembryonic antigen (CEA, as a model analyte) in clinical immunoassays. The as-prepared nanomaterials composed of a magnetic mesoporous NiCo2O4 nanosheet, an interlayer of Nafion/thionine organic molecules and a nanogold layer show good adsorption properties for the attachment of horseradish peroxidase-labeled secondaryanti-CEA antibody (HRP-anti-CEA). With a sandwich-type immunoassay format, the functional bionanomaterials present good analytical properties to facilitate and modulate the way it was integrated onto the electrochemical immunosensors, and allows the detection of CEA at a concentration as low as 0.5 pg mL"1 at 3a.Charpter 4:A class of novel redox-active hybrid nanostructures comprising of metal platinum and ordered mesoporous carbon (Pt-MSC) was designed as molecular tags for sensitive electrochemical immunoassay (alpha-fetoprotein, AFP, as a model analyte) on the cyclodextrin-functionalized graphene sensing platform. To construct such a molecular tag, a layer of positively charged thionine was initially adsorbed onto the MSC through negatively charged carboxyl groups, and [PtCl6]2- ions were then conjugated onto the thionine/MSCs, followed by in situ reducing to metal platinum. Finally, horseradish peroxidase-labeled anti-AFP (HRP-anti-AFP) was conjugated onto the metal platinum surface. A sandwich-type immunoassay format was employed for determination of AFP by using the labeled HRP toward the reduction of H2O2 with the aid of thionine in pH 6.5 acetic acid-buffered solution. Under optimal conditions, the developed immunoassay displayed a wide linear range from 2.0 pg mL-1 to 10.0 ng mL’1 toward AFP standards with a low detection limit (LOD) of 1.5pgmL-1 at 3σ.Charpter 5:A sensitive and feasible multiplexed immunoassay protocol for simultaneous electrochemical determination of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) was developed using multifunctionalized graphene nanotags on a cyclodextrin-modified immunosensor. The sensor was fabricated via the guest-host chemistry between the immobilized cyclodextrin and anti-CEA/anti-AFP antibodies. Two nanotags including cyclodextrin-thionine-graphene and cyclodextrin-ferrocene-graphene nanostructures were synthesized based on the wet chemistry method, respectively. The synthesized nanotags were employed for the labelling of horseradish peroxidase (HRP)-anti-AFP and HRP-anti-CEA conjugate, respectively. With a sandwich-type assay format, the signal was monitored through the labelling HRP toward catalytic reduction of H2O2 at various peak potentials in the presence of thionine and ferrocene, respectively. Under optimal conditions, the multiplexed immunoassay enabled the simultaneous determination of CEA and AFP with wide working ranges of 0.001-60 ng mL-1 for AFP and 0.003-40 ng mL-1 for CEA. The detection limits were 0.5 pg mL-1 for AFP and 0.8 pg mL-1 for CEA (at 3σ). No obvious nonspecific adsorption and cross-talk were observed during a series of analyses to detect target analytes. Intra-and inter-assay coefficients of variation (CV) were below 9.5%.Charper 6:A new magneto-controlled microfluidic device for direct electrochemical determination of squamous cell carcinoma antigen (SCC-Ag) in serum was designed by using anti-SCC antibody (SCC-Ab)-functionalized magnetic mesoporous nanogold/thionine/NiCo2O4 hybrid nanostructures as immunosensing probes (P1-Ab) and horseradish peroxidase-SCC-Ab conjugates-labeled nanogold/graphene nanosheets as signal tags (P2-Ab). In the presence of the analyte SCC-Ag, the sandwich immunocomplex was formed between the immunosensing probes and the signal tags. With the aid of an external magnet, the formed immunocomplex was attached to the microfluidic device. The assay was implemented in newborn calf serum (NBCS) containing 2.5 mM H2O2 based on the labeled peroxidase on the P2-Ab toward the catalytic reduction of H2O2. Under optimal conditions, the increase in the current was proportional to the concentration of SCC-Ag from 2.5 pg mL-1 to 15 ng mL-1. The detection limit (LOD) was 1.0 pg mL-1 SCC-Ag at 3σ. The electrochemical immunoassay displayed an acceptable precision, selectivity and stability. Clinical serum specimens were assayed with the method, and the results were in acceptable agreement with those obtained from the referenced electrochemiluminescent method. Importantly, the method can be suitable for on-line use in the mass production of miniaturized lab-on-a-chip devices and open a new opportunity for protein diagnostics and biosecurity.