Study On Signal Enhanced Immunosensor For Electrochemical Determination Of Tumor Markers

The determination of tumor marker plays an important role in clinical diagnoses, prognosis, and cure evaluation for the patients with certain tumor associated diseases. Electrochemical immunosensor, based on the highly specific molecular recognition of antigen by its antibodies and the electrochemical technique, has become one of the predominant analytical teniques for tumor associated diseases due to its low cost, high sensitivity, high selectivity, simple and fast. For the successful development of an electrochemical immunosensor, the immobilized strategy of biomolecules and the amplification of elelctrochemical signal should be two key issues. Above all, the signal amplified strategy plays a key role in improving the sensitivity of the electrochemical immunosensor. In this thesis, we report the proof-of-concept of several new electrochemical immunsensors for the determination of tumor marker by coupling with the signal amplification strategies including enzyme catalyzed signal amplification, nano signal amplification, molecular biological amplification, and redox cycling amplification. The thesis consists of 6 chapters as follows:In the first chapter, a general introduction of tumor marker (the definition, categories, and clinical significant) and the present detection method was made. Then, the detection principle and categories of the electrochemical immunosensor was described in brief, and the signal amplified strategies was introduced in detail. In the meanwhile, a general introduction of multiplex immunosensor was made. In addition, the purpose and signification of this thesis were summarized also.In the second chapter, a facile and sensitive electrochemical immunosensor was developed for determination of alpha-fetoprotein (AFP) with a sandwich-type mode by using silver nanowire-graphene hybrid nanocomposites as label, nanogold doped-chitosan film as immunosensing probe. The as-prepared AgNW-GO with highly loading capacity and good biocompatibility possesses the ability to load multiple horseradish peroxidase and biomolecules. The assay was carried out by using differential pulse voltammetry measurement method. Under optiamal conditions, the electrochemical immunosensor exhibited a wide linear range of 0.05-400 ng mL’1 with a low detection limit of 5 pg mL-1 AFP (at 3sB). In addition, the selectivity, stability and reproducibility of the immunosensor were acceptable.In the third chapter, a novel flow-through multiplexed immunoassay protocol in biological fluids was designed using biofunctionalized magnetic graphene nanosheets as immunosensing probes and multipunctional nanogold hollow microspheres as distinguishable signal tags. The probes were fabricated by means of co-immobilization of primary anti-CEA and anti-AFP antibodies on the Fe3O4 nanoparticles-coated graphene nanosheets. The revers-micelle methods was used for the synthesis of distinguishable signal tags by encapsulation of horseradish peroxide-thionine and HRP-ferrocene into nanogold hollow microspheres, respectively, which were ultilized as labels. Experimental results revealed that the multiplexed electrochemical immunosensor enabled the simultaneous monitoring of carcinoembryonic (CEA) and alpha-fetoprotein (AFP) in a single run. The developed immunoassay exhibited good electrochemical behaviors toward CEA in the working range of 0.01～80 ng mL-1, and AFP in the working range 0.01～200 ng mL-1. In addition, the selectivity of the immunoassay was acceptable.In the fourth chapter, a new electrochemical immunoassensor based on molecular biological amplification strategy was proposed. Initially, the capture antibodies were immobilized on the surface of β-cyclodextrin (β-CD) modified glass carbon electrode. After a typical sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which triggered the formation of long DNA contacamers with auxiliary DNA (DNAzyme molecules) and signal DNA (ferrocene molecules). Due to the long DNA contacamer label carries with a large number of signal molecules and DNAzymes, there is substantial signal amplification. Under optimal conditions, the electrochemical signal increased as the IgGl (as a model) concentrations increased. The dynamic concentration range spanned from 0.1 pg mL-1 to 100 ng mL-1 with a detection limit of 0.1 pg mL-1 IgGl. Moreover, the stability, reproducibility and selectivity of the immunosensor were acceptable. Importantly, the methodology was evaluated for the analysis of clinical serum specimens, receiving a good correlation between the electrochemical immunoassay and commercially available enzyme-linked immunosorbent assay (ELISA) for determination of IgGl.In the fifth chapter, a non-enzyme sandwich-type electrochemical immunosensor was designed by using nanocatalyst and redox cycling signal amplification. Platinum-cerium oxide hybrid nanomaterials were designed as bionanolabels for highly electrochemical detection of tumor markers (CEA, used as a model). Initially, the p-nitrophenol (NP) molecules were reduced to p-aminophenol (PAP) by the catalysis of the immobilized platinum nanoparticles labels on the platinum-cerium oxide with the aid of NaBH4, then the generated PAP molecules were electrochemical oxidized to p-quinone imine (PQI) by the immobilized ferrocene on the glassy carbon electrode, and then the obtained PQI was reduced back to PAP by NaBH4. The electrochemical immunsensor was evaluated by differential pulse voltammetry. It was found that the peak currents had a good linear response to the logarithm of CEA concentration in the range of 0.5 pg mL-1～20 ng mL-1 with a detection limit of 0.5 pg mL-1.In the sixth chapter, multifunctionalized thionine-modified cerium oxide nanostructures (Thi-CeO2) with redox ability and catalytic activity were designed as the bionanolabels for a non-enzyme electrochemical immunosensor of tumor marker (carcinoembryonic antigen, CEA, used as a model). The cerium oxide can not only be utilized as the substrate for nanocomposites preparation, but also can be used as a catalyst with highly catalytic efficiency. In the article, the carried CeO2 nanoparticles autocatalytically hydrolyzed the phosphate ester bond of L-asorbic acid 2-phosphate (AAP) to produce a new reactant (L-ascorbic acid, AA), then the generated AA was electrochemical oxidized by the assembled thionine on the Thi-CeO2, and the resultant produce was then reduced back to AA by the added tris (2-carboxyethy) phosphine (TCEP). The catalytic cycling could be re-triggered by the thionine and TCEP, resulting in amplification of the electrochemical signal. Under the optimal conditions, the sensitivity and dynamic range of the electrochemical immunosensor were evaluated by using differential pulse voltammetry (DPV). The DPV peak current increased with increasing CEA concentration, a linear dependence between the peak current and the logarithm of CEA was obtained in the range from 0.1 pg mL to 80 ng mL with a detection limit of 0.08 pg mL estimated at a signal-to-nosie ratio 3.