Novel Immobilization Material-Based Piezoelectric And Impedance Sensing Techniques For The Detection Of Cancer And Other Disease Markers

Use of chemo/biosensing techniques to detect the bio-markers for the accurate and rapid diagnosis of clinical diseases as well as the screening in situ and the ensemble control of epidemic diseases has been a novel, attractive and hot topic in the current medical research. Piezoelectric acoustic wave immunosensing technique, which has high sensitivity of signal response and high specificity of immunoreaction, is sensitive to multiple parameters such as the surface mass, and the density, viscosity, conductivity and dielectric effect of the test system( solution). This kind of technique shows an attractive future of potential applications in the biochemical diagnosis of clinical diseases. In addition, the electrochemical impedance biosensors have been widely used for the assay of biological analytes due to the advantages including their simple-design, high-sensitivity and low-cost. However, the method of immobilization, the reproducibility and the reusability still remain to be solved in the design and applications of these sensors. Among these problems presented, the key step for the fabrication of biosensors with excellent properties is the immobilization of bio-species on the surface of transducers. In the past few years, nanomaterials have been widely researched and used. Applying nanomaterials for the fabrication of biosensors will greatly improve the performance of the resulting biosensor. This research prepared many nanomaterials with different morphology, taking advantage of the good physical, chemical, electrocatalytic and biocompatibility of these nanomaterials and using them for the preparation of sensors. The details are shown as follows:(1) An antibody immobilization strategy based on the nanogold-hydroxyapatite (GNP-HA) hybrid material has been proposed for the fabrication of piezoelectric immunosensor (Chapter 2).The detection performances of the resulting immunosensor were investigated by use of the antibody-antigen model system ofα-Fetoprotein (AFP). The hybrid material was characterized by the UV-vis spectroscopy,the scanning electronic microscope (SEM) and transmission electric microscope (TEM) measurements. Furthermore, the frequency and electrochemical impedance response characteristics for the processes of immobilization and immunoreaction of anchored AFP antibodies were studied in detail. The immunoresponses of the proposed immunosensor were compared with those with antibodies immobilized by using HA or GNP alone. It was found that the developed sensing interface has some advantages such as the activation-free immobilization and the high antigen-binding activities of antibodies. The as-prepared immunosensor can allow for the determination of AFP in the concentration range of 15.3～600.0 ng mL-1. (2) A high sensitivity piezoelectric immunoassay has been developed for the detection of carbohydrate antigen 19-9 (Chapter 3). The poly-L-lysine/ hydroxyapatite/ carbon nanotube (PLL/HA/CNT) nanocomposite has been first synthesized according to the chemical co-precipitation method and a general design strategy for immunosensing platform has been proposed on the basis of PLL/HA/CNT nanocomposite adsorption of antibodies. The as-prepared immunosensor can allow for the specific determination of CA19-9 in the concentration range of 12.5～270.0 U mL-1. (3) A reversible piezoelectric immunosensing diagnosis technique has been proposed for the detection of tumor maker of CA125 (Chapter 4). The hydroxyapatite /chitosan nanocomposite(HA/CS)has been first synthesized and characterized by the SEM and TEM measurements. The piezoelectric crystals were modified with the HA/CS nanocomposite and further assembled nanogold for removable adsorption of CA 125 antibody. By use of BSA to minimize the possible nonsepecific adsorptions from background, this technique has been developed for determination of CA125 in the concentration range of 15.3～440.0 U mL-1. (4) A biomolecular immobilization strategy based on the adsorption of GNP-CaCO3 hybrid material has been proposed (Chapter 5). Gold nanoparticles (GNP) were assembled on the surface of porous calcium carbonate microparticles (CaCO3) in a neutral aqueous solution through electrostatic interaction. The resulting GNP-CaCO3 hybrid material was expected to offer a promising template for CA15-3 antibody immobilization because of the satisfactory biocompatibility and the synergic effect of CaCO3 microparticles and GNP, the antibodies immobilized on the sensing surface can be maintained good immunoactivity. It is found that a linear range of 8.0～266.0 U mL-1of CA 15-3 can be determined with this developed system.(5) A sensitive piezoelectric immunoassay has been advanced on the basis of cell immobilization strategy (Chapter 6). The crystal was modified with cysteamine to deposite yeast cells, on which PSA antibodies were immobilized. In contrast to the traditional glutaraldehyde immobilization approach, results indicate the yeast cells could provide appropriate bio-surroundings for antibodies and allow antibody molecules bound with higher bioactivity and achieve better immunoreaction capability. A PSA serum concentration in the range of 5.0 ? 604.0 ng mL-1 can be determined by this new system.(6) A novel program for the orientation-controlled immobilization of antibodies has been proposed using nanogold-protein A (Chapter 7). Nanogold particles were employed to be labeled with protein A (PA). Mediated by gold nanoparticle-labeled protein A, the oriented immobilization of antibodies on the surface of the quartz crystal was carried out using C1q antibody as a model test system. This strategy can effectively avoid the possible protein denaturation resulting from the flat gold electrode traditionally used so that it may favor PA molecules a large amount and a well-retained bioactivity to antibodies. The immunoreaction process and piezo-response features of the created immunological interface were real-time monitored by using the piezoelectric sensing technique. Moreover, the kinetic characterizations of cyclic voltammograms and electrochemical impedance spectroscopy were performed. It is found that the developed nanogold-PA immobilization procedure may be popularized as a general alternative method of designing various immunosensing platforms.(8) A novel piezoelectric and electrochemical impedance sensing technique based on enzyme-catalyzed depositing amplification has been proposed for the detection of toxoplasma gondii-Specific IgG(Tg-IgG)(chapter 8). The HRP-labeled second antibody (anti-Tg-IgG-HRP) was first immobilized on the electrode surface by sandwich method (TgAg/Tg-IgG/anti-Tg-IgG-HRP). Then the undissolving deposition was formed on the electrode surface by using HRP catalyzing the oxidation of 3, 3'-diaminobenxidine by hydrogen peroxide. So the piezoelectric and electrochemical impedance signal of the electrode is amplified greatly. It was found that the amount of the precipitate accumulated on the conductive electrod surface was determined by the concentration of the target analyte Tg-IgG and the time employed for the biocatalyzed precipitation. A linear response in the electron transfer resistance measured by EIS experiments for the dynamic dilution range of Tg-IgG was observed between 1: 8000 and 1: 200 with a detection limit of 1: 9600. (9) An iridium oxide film-enhanced impedance immunosensor has been developed for rapid detection of carcinoembyronic antigen (Chapter 9).Gold electrode was electrochemically modified with IrOx and simultaneously functionalized with protein A (PA) thin film to bind CEA antibodies in an orientated way. It has been found that the antibody loading amount was dependent on the PA concentration and the deposition time of IrOx matrix. Under the optimized experimental conditions, the electron transfer resistances obtained were linearly related to the CEA concentrations range of 36.2 ng mL-1～460.0 ng mL-1, with a detection limit of 28.0 ng mL-1. Analytical results of clinical samples from cancer patients show that the proposed immunoassay is reasonably comparable with the chemiluminescence immunoassay (CLIA), indicating the feasibility of using the proposed method for CEA immunoassay in clinical diagnosis.