Highly Sensitive Electrochemical Immunosensors Using DNA Functionalized Nanomaterials For Signal Amplification

Electrochemical immunosensors, which combine the specific antigen-antibody immunoreaction, signal amplification protocal and electrochemical detection technique, have shown great application prospectives in the fields of clinical diagnosis and pharmaceutical alanalyses due to their advantages of high sensitivity and portability. Among the various signal amplification techniques, DNA based signal protocols have attracted great attentions in recent years due to their good stability, high solubility and specific coding ability which make them can be enginieered as either template for the synthesis of highly catalytic nanoclusters or as initiator to trigger a polymerase chain reaction and a hybridized chain reaction for signal molecular loading. Based on these advantages, this thesis focuses on the preparation and characterization of DNA functionalized nanoprobes and their application in the development of highly sensitive electrochemical biosensors for tumor maker detection. Specific work is listed as follows:1. Ultrasensitive enzyme-free electrochemical immunosensor based on hybridization chain reaction triggered double strand DNA@Au nanoparticle tag.An ultrasensitive enzyme-free electrochemical immunoassay was developed for detection of the fg mL-1level carcinoembryonic antigen (CEA) by using a double strand DNA@Au nanoparticle (dsDNA@AuNP) tag and hexaammineruthenium(III) chloride (RuHex) as the electroactive indicator. The dsDNA@AuNP was synthesized by one-pot hybrid polymerization of dsDNA on initiator DNA modified AuNPs via hybridization chain reaction. The immunosensor was prepared by covalently cross-linking capture antibody on chitosan/AuNP nanocomposite modified glass carbon electrode. The AuNPs accelerated the electron transfer and led to high detection sensitivity. With a sandwich-type immunoreaction and a biotin-streptavidin affinity reaction, the dsDNA@AuNP tag was conjugated on the immunocomplex to bring a high amount of RuHex to the electrode surface via electrostatic interaction, resulting in an amplified electrochemical signal. Under optimal conditions, the proposed sensing platform showed a wide linear detection range from10fg mL-1to10ng mL-1, and the detection limit of CEA was3.2fg mL-1.2. Ultrasensitive electrochemical immunoassay based on silver nanoclusters loaded graphene oxide. An ultrasensitive electrochemical immunoassay method was developed for detection of tumor marker by employing a silver nanocluster (DNA/AgNCs) and graphene oxide nanosheet (GO) as signal amplification tag. The DNA/AgNCs synthesized by using DNA as a template and NaBH4as a reducing reagent could be stably adsorbed on the interface of the GO via π-π stacking. After cross-linked with Ab2antibody, the nanocomposite was used as a signal amplification tag for sensitive detection of tumor marker through its highly catalytic activity toward H2O2, which could generate significantly amplified electrochemical signal. Under optimal conditions, the as-prepared immunosensor showed high sensitivity towards carcinoembryonic antigen (CEA), a model analyte related with many cancers, with a wide linear detection range of0.1pg mL-1to100ng mL-1and a detection limit of0.037pg mL-1.