Analytical Application of Biosensing Technology Based on Nano Materials in the Bioassay and Clinical Diagnosis
There has been considerable interest in studying biosensors because they have potential applications intreatment, bioengineering, environmental protection and food industry. A large number of researches indicate that applying nanomaterials for the fabrication of biosensors will greatly improve the performance of the resulting biosensors. Developing biosensors to detect the meaningful bio-markers for the accurate and rapid diagnosis of clinical diseases as well as for the field screening and mass monitoring of the epidemic diseases has been a novel, attractive and hot topic in the current studies. This dissertation focuses on using a variety of nanomaterials and appropriate detection technique to develop a series of nanoparticules-based biosensors for the diagnosis of some clinical serious diseases including prostate , Vibrio cholera, liver diseases and so on, by probing their specific markers. The details are summarized as follows:(1)A highly sensitive immunosensing method for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome (Chapter 2). Horseradish peroxidase (HRP) encapsulated and antibody-modified liposome acts as the carrier of a large number of markers and specific recognition label for the amplified detection of PSA. The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the signal of HRP-catalyzed luminol/peroxide/enhancer system. The”sandwich-type”immunoassay provides the amplification route for the PSA detection in ultratrace levels. The CL emission intensity exhibits dynamic correlation to PSA concentration in the range from 0.74 pg/mL to 0.74μg/mL with readily achievable detection limit of 0.7 pg/mL.(2) An ultrasensitive biosensor was developed for the detection of CT based on a supported lipid membrane as sensing surface and the HRP/GM1-functionalized liposome as detection probe (Chapter 3). The supported lipid based biosensing surface could be renewed easily and rapidly. The application of enhanced chemiluminescence reaction in the detection of HRP-bearing liposome afforded a further signal amplification and background alleviation. The developed biosensor was shown to give chemiluminescence signal in linear correlation to CT concentration within the range from 1 pg/mL to 1 ng/mL with readily achievable detection limit of 0.8 pg/mL.(3) A highly sensitive and specific electrochemical immunosensor by using gold nanoparticle label for enzymatic catalytic amplification was developed with (AFP) as the model analyte (Chapter 4). Monoclonal mouse anti-human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti-human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti-rabbit IgG conjugated with alkaline phosphatase, dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold-based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng/mL to 500 ng/mL with a detection limit of 0.8 ng/mL.(4) An electrochemical label-free detection technique was worked out for the detection of nucleic acids based on the precipitation of single-walled (SWNTs) by DNA hybridization (Chapter 5). The DNA hybridization between target DNA and the DNA probe coating on the nanotubes could actively remove DNA probe from the SWNTs surface. The precipitation of SWNTs onto the n-octadecyl mercaptan (C18H37SH) modified Au electrode substantially restores heterogeneous electron transfer between bare Au electrode and redox species in solution phase which was almost totally blocked by the SAM of C18H37SH, and as a result, the electrical signal of the electrode was correlated with the concentration of target DNA in the range from 6.4 pM to 50 nM with a detection limit of 3.2 pM.