Studies On Electrochemical Immunosensor For Pebrine Disease Diagnosis Based On Nanomaterial And Signal Amplification Strategies

Pebrine disease, a unique kind of silkworm infection disease, could infect the next generation through mother moth transovarial transmission, resulting in a severe damage to the industry of silkworm. It has been counted as the only object of quarantine at silkworm egg production, and the pathogen of Pebrine disease is Nosema bombycis（N.b）. Through long-term research and development, the spore wall protein of Nosema bombycis（SWP N.b） was determined as a related marker of Pebrine disease. Therefore, the development of reliable, cost-effective, accurate detection strategies for SWP N.b detection is particularly vital. So far, some strategies have been applied for the diagnosis of Pebrine disease, such as serological diagnosis, enzyme linked immunosorbent assay and the PCR diagnosis technology. However, they are often suffered from time-consuming testing cycle, special equipment and professional operation. Electrochemical immunosensor, taking the simple transduction of electrochemical signals from the specific antibody-antigen interaction, combines the merits of electrochemical technology and immunoassay, which holds great potential as a next-generation biomolecules detection strategy due to its inherent advantages of high sensitivity, simple instrumentation, preeminent compatibility and low cost. Therefore, the aim of our present work is to construct simple, reliable, cost-effective and high-sensitivity electrochemical immunosensor for the diagnosis of Pebrine disease. The detail contents are as follows:In chapter one, electrochemical immunosensor for detecting the Pebrine disease related spore wall protein of Nosema bombycis（SWP N.b） was fabricated based on the amplification of hemin/G-quadruplex functionalized Pt@Pd nanowires（Pt@PdNWs）. The synthesized Pt@PdNWs possessed large surface area, which could effectively improve the immobilization amount of hemin/G-quadruplex DNAzyme concatamers produced via hybridization chain reaction（HCR）. In the presence of SWP N.b, the hemin/G-quadruplex labeled Pt@PdNWs bioconjugations was captured on electrode surface and thus obtained electrochemical signal. Furthermore, Pt@PdNWs with excellent electrocatalytic performance could also amplify electrochemical signal. With these amplification factors, the electrochemical immunosensor exhibited a wide linear range from 0.001 ng mL^-1 to 100 ng mL^-1with a detection limit（LOD） of 0.25 pg mL^-1, providing a new promise for the diagnosis of Pebrine disease.Based on the surprise discovery that the hemin/G-quadruplex could also catalyze oxidation of L-cysteine to L-cystine with the concomitant generation of H₂O₂ from the dissolved oxygen, a pseudobienzyme electrocatalytic amplificated electrochemical immunosensor was designed with fullerene supported Pt-Pd nanoparticles(C₆₀@Pt-Pd nanoparticles) as nanocarrier. And experimental results demonstrated that adopting L-cysteine as substance to generate fixed H₂O₂ could largely amplify the electrochemical signal and with better catalytic efficiency than NADH, which has not been studied to the best of our knowledge. Furthermore, the method for the synthesis of C₆₀@Pt-Pd nanoparticles provided a new way to improve the solubility and biocompatibility of C₆₀, which could promote its further application in biology.For electrochemical immunosensor, signal amplification is crucial for obtaining low detection limit and high sensitivity, the general one of signal amplification strategies is enzyme involved electrocatalytical amplification. However, the signal amplification strategy was still involved in the drawbacks of using easy decomposed and high cost protein enzyme and far interaction distance between catalyst and substance, which limited the catalytic efficiency in signal amplification. Therefore, we constructed a simple electrochemical proximity immunoassay（ECPA） system for the total protein of Nosema bombycis（TP N.b） detection based on a new amplification strategy. The desirable amplification strategy was achieved via co-immobilizing catalyst（Fe₃O₄NPs） and substance（methylene blue, MB） in electrode surface. Considering the desirable sensitivity and specificity, as well as the novel and simple features, this signal amplified ECPA system opened an opportunity for quantitative analysis of many other kinds of protein biomarker.