Studies On The Application Of Nanotechnique And Biotechnique In Amplified Electrochemical Immunoassay

Immunoassay is an analytical method which combines the specific immune-reorganization and modern analytical technique. Compared with the traditional immunoassay, electrochemical immunosensor, a collectivity of immunoreactions, signal occurrence and signal detection, displayed various advantages:simple manipulation, low cost. high sensitivity, rapidly measurement, et al. Recently, amplified electrochemical immunosensor has been received much attention, which achieved by employing enzymes, nanoparticles and nanocontainers as amplifiers or biotechnique amplification. However, high cost for separate sample detection and time waste for batch samples detection detection make against for clinical diagnoses, specially for the diagnoses of acute or virulent disease. Therefore, as a low cost assay to rapidly measure proteins with high selectivity and sensitivity, the electrochemical immunosensor has aroused much interests. This research focuses on the preparation of the multi-functionalized nanomaterial to fabricate the immunoactive interface and the preparation of high/ultra sensitivy immunoassay based on the amplified technique combination:nanotechnique based amplification, enzyme associated signal enhancement and biotechenology amplification. The research contents are mainly as follows:1. Multilayer structured amperometric immunosensor built by self-assembly of a redox multi-wall carbon nanotube compositeA reagentless amprometric immunosensor was built on the conductive multilayer, comprised of Nafion-coated multi-wall carbon nanotubes (MWCNT), thionine (Thi) and gold nanoparticles (nano-Au). Firstly, Nafion-MWCNT homogeneous composite was dropped on the surface of the glass carbon electrode (GCE). Then Thi was chemisorbed by both carboxylic MWCNT and cation exchanger Nafion. Furthermore, the negative-charged nano-Au, which was used to immobilize biomolecules, was chemisorbed onto Thi film through the electrostatic force with the amino groups of Thi. The stepwise self-assembly process of the immunosensor was characterized by means of cyclic voltammetry (CV), and the microstructure of modified film was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Using α-1-fetoprotein (AFP) as a model, this immunosensor presented amplified sensitivity, good stability, and a broader linear response as well as good selectivity and storage stability.2. Sandwich nanohybrid of functionalized multi-wall carbon nanotube for reagentless amperometric biosensing towards tumor markerIn this part, a novel self-assemble sandwich nanohybrid of functionalized multi-wall carbon nanotube (MWCNTs) was proposed. Fistly, MWCNT functionalized by Nafion (N-MWCNTs) was dropped on the surface of glass carbon electrode. Based on the iron exchange reaction, Thionine (Thi) could be absorbed stably by Nafion. Following that, thionine covalently linked the nanocomposite of MWCNT functionalized by gold nanoparticles (GNP-MWCNTs) to form the sandwich self-assemble film, which was utilized to prepare the immunorsensor towards the detection of tumor marker: a-1-Fetoprotein (AFP). The anti-AFP was immobilized on the double enlarged specific surface area by GNP and MWCNTs. This regentless immunosensor displayed a sensitive detection owning to the conductive electronic transformation channel constructed by the sandwich film of functionalized MWCNTs. Cyclic voltammetry was ultilized to characterize the self-assembly process of the immunosensor. Towards the detection of α-1-fetoprotein (AFP), the proposed immunosensor presented amplified sensitivity, high stability, good selectivity.3. Enzyme-nanoparticle conjugates at oil-water interface for amplification of electrochemical immunosensingThe preparation of biocatalytic capsule (Ab2-catalytic capsule) was based on utilizing "oil"(organic compound) to entrap the nano-Au/HRP/HAFP bioconjugates, which were produced by horseradish peroxidase (HRP) and HRP labeled a-1-Fetoprotein (HAFP) co-conjugated on gold nanoparticles (nano-Au). A signal amplification strategy was introduced by the prepared biocatalytic capsule as labels for electrochemical immunodetection of α-1-fetoprotein (AFP). The transmission electron microscopy (TEM) images indicated that the biocatalytic capsules were successfully synthesized. The cyclic voltammetry (CV) experiments provided the evidence that the prepared capsule exhibited high catalytic activity for the reduction of H2O2. Besides, a biocompatible monolayer (collagen-TiO2) was introduced to immobilize a nano-Au layer to fabricate the primary antibody on the electrode surface. After the prepared biocatalytic capsule was captured to the electrode surface to form the sandwich type immunoassay, the enhanced sensitivity was obtained with a detection limit of29ng-mL"1. The proposed immunoassay also presented good stability, selectivity and storage stability.4. Preparation of ferrocenemonocarboxylic nanospheres/grapherne hybrid nanomaterial for electrochemical detection of Streptocooccus suis serotype2A rapid detection strategy for Streptococcus suis serotype2(SS2) detection was proposed in this part. As the electroactive material, ferrocenemonocarboxylic nanospheres (nFc) was prepared firstly and colvantly anchored onto graphene nanosheet (GN) to form the hybrid material GN/nFc, which was bioconjugated with detection antibody as labels for sandwich format immunoassay. Graphene nanosheet provides high surface areas to link the ferrocenemonocarboxylic nanospheres plentifully via EDC/NHS as linkage. The enhanced electrochemical signal could be recorded due to the large amount of immobilized nFc. Meanwhile, for the high electrocatalytic efficiency of nFc towards the oxidation of ascorbic acid, the multiple amplified electrochemical signals could be obtained by using the proposed immunoassay towards the detection of SS2. The experimental results demonstrated that the proposed immunosensor displayed wide linear range (0.05-100ng-mL-1), low detection limit (0.013ng-mL-1) and could be as a new method to detect SS2quickly and precisely.5. Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensingA multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H2O2. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of1.7ng-mL'...