Enzyme-functionalized Nanoprobes And Their Applications For Electrochemical Immunoassay Of Tumor Biomarkers

Utrasenstive immunoassay of tumor biomarkers is of great importance for tumor early diagnosis. By combination with the high label loading ability of nanomaterials and the catalysis of enzyme biomolecules, various enzyme-functionalized nanoprobes can be prepared and used for the signal tracing of sandwich immunoassays. They can not only greatly enhance the signal responses of the immunorecognition events but also enable the possibility to develop a great variety of signal transduction strategies with excellent performance through enzymatically catalytic reations, thus gaining considerable attentions in the ultrasensitive immunoassay field recently. This thesis combines the enzyme-functionalized nanoprobes prepared from the nanocarriers such as graphene oxide, gold nanorod and nanoporous gold nanosphere with the constructed electrochemical immunosensors to perform three researches in the field of ultrasensitive immunoassay of tumor biomarkers which are listed as follows: 1. Amplified inhibition of the electrochemical signal of ferrocene by enzyme functionalized graphene oxide for ultrasensitive immunosensingA nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene(CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase(HRP) on the graphene oxide(GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles(Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis. 2. Enzymatically catalytic deposition of gold nanoparticles by glucose oxidase-functionalized gold nanoprobe for ultrasensitive electrochemical immunoassay.A novel ultrasensitive immunoassay method was developed by combination of the enzymatically catalytic gold deposition with the prepared gold nanoprobe and the gold stripping analysis at an electrochemical chip based immunosensor. The immunosensor was constructed through covalently immobilizing capture antibody at a carbon nanotube(CNT) modified screen-printed carbon electrode. The gold nanoprobe was prepared by loading signal antibody and high-content glucose oxidase(GOD) on the nanocarrier of gold nanorod(Au NR). After sandwich immunoreaction, the GOD-Au NR nanoprobe could be quantitatively captured onto the immunosensor surface and then induce the deposition of gold nanoparticles via the enzymatically catalytic reaction. Based on the electrochemical stripping analysis of the Au NR nanocarriers and the enzymatically produced Au NPs, sensitive electrochemical signal was obtained for the immunoassay. Both the GOD-induced deposition of Au NPs by the nanoprobe and the sensitive electrochemical stripping analysis on the CNTs based sensing surface greatly amplified the signal response, leading to the ultrahigh sensitivity of this method. Using carcinoembryonic antigen as a model analyte, excellent analytical performance including a wide linear range from 0.01 to 100 ng/mL and a detection limit down to 4.2 pg/mL was obtained. In addition, this immunosensor showed high specificity and satisfactory reproducibility, stability and reliability. The relatively positive detection potential excluded the conventional interference from dissolved oxygen. Thus this electrochemical chip based immunosensing method provided great potentials for practical applications. 3. Enzymatically catalytic signal tracing by glucose oxidase and ferrocene dually functionalized nanoporous gold nanoprobe for ultrasensitive electrochemical measurement of a tumor biomarker A nanoporous gold nanosphere(pAu NS) was synthesized to load high-content glucose oxidase(GOD) and ferrocene(Fc) for successful preparation of a new gold nanoprobe. After specific recognition of the tumor biomarker of carcinoembryonic antigen(CEA) at a gold electrode based aptasensor, this GOD and Fc dually functionalized pAu NS nanoprobe was further used for sandwich immunoreaction and signal tracing. Based on the Fc-mediated GOD-catalytic reaction, the gold nanoprobes quantitatively captured onto the electrode surface produced sensitive electrochemical signal corresponding to the protein recognition events, which led to the development of a new biosensing method for CEA measurement. Both the high loading of GOD and Fc on the pAu NS nanocarrier and the enzymatically catalytic reaction of the nanoprobe greatly amplify the electrochemical signal; meanwhile, the immobilization of the Fc mediator on this enzyme nanoprobe and the highly specific aptamer recognition drastically decrease the background current, resulting in the achievement of ultrahigh sensitivity of the method. Under optimum conditions, this method shows excellent analytical performance including a wide linear relationship of five-order of magnitude and a low detection limit down to 0.45 pg/mL. Thus this pAu NS based gold nanoprobe and the proposed immunoassay method provide great potentials for practical applications.