| Respiratory syncytial virus (RSV), an enveloped, nonsegmented, negative, single stranded RNA virus in the family of Paramyxoviridae, genus Pneumovirus, is one of the most important cause of lower respiratory tract infection. RSV infection is a common contributor causing bronchiolitis, pneumonia, and chronic obstructive pulmonary infections and affects mainly children and elderly along with other immunodeficiency patients leading to high mortality and morbidity. So far, there has been no convincing and safe vaccine. For effective early diagnosis, prevention, treatment and control of RSV disease, the development of simple, rapid and sensitive methods to diagnose RSV is of great significance. In addition, to discover new drug targets and develop new RSV drugs, investigating the interaction between RSV and the host cell is also very important. In this thesis, gold nanoparticles were introduced to the traditional immunoassay for signal amplification, and the immuno-analysis of RSV and its cell membrane receptor were investigated. The details are as follows:1. Gold nanoparticles-based immunoassay for RSV1) An enhanced RSV immunosaasy was developed by taking gold nanoparticles (AuNPs) as the carrier of multiple enzymes for signal amplification. RSV antibody and alkaline phosphatase (ALP) were coupled to the AuNPs surface via electrostatic adsorption to form dual labeled AuNPs conjugates. The capture antibody was conjugated to the strep tavidin-functionalized magnetic microbeads through the biotin molecules. In the presence of target RSV, the sandwiched immunocomplex was formed. The quantification of RSV was based on the signal generated by the conversion of the substrates into colored molecules. The linear range is from 0.5 to 80 pg mL-1. This sensitive immunoassay for RSV can be further extended to other pathogens in clinical diagnostics.2) An ultrasensitive plasmonic immunoassay with dual signal amplification strategy for RSV was described by taking AuNPs as signal report molecules. Negative charged adenosine triphosphate (ATP) can induce the aggregation of positive charged AuNPs while ALP will keep AuNPs dispersed by converting ATP into adenosine via an enzymatic reaction of dephosphorylation. Based on this, the plasmonic immunoassay was developed by monitoring the absorbtion properties of AuNPs. Furthermore, since magnetic beads were used to load many ALP molecules and metal ions were applied to accelerate the enzymatic reaction, dual signal amplification can be achieved to significantly improve the sensitivity. Compared to conventional immunoassay, this method resulted in a higher sensitivity with the linear range of 0.1-30 pg/mL and the detection limit of 0.02 pg/mL, showing great potential for clinical diagnosis.2. Gold nanoparticles amplified in situ immuno-analysis of cell membrane receptor for RSVUpon infecting the host cells, the binding of RSV to the toll-like receptors (TLR) produces a signal and activates the transcription factor associated with inflammation. Such signal transduction induces inflammation and stimulates the cytokine secretion. The serial effects would further enhance receptor expression. Taking host cell as the sensing element, TLRs expressions were detected based on antigen-antibody bindings. The results showed that the TLR4 expression of HEp-2 cells was not proportional to the MOI of RSV. Since cells are hypersensitive to the external invasion of microorganisms, the AuNPs amplified in situ immuno-analysis of TLRs on the cell membrane surfaces could reveal the interaction between RSV and the host cells.In summary, we have successfully developed immunoassay for RSV and its cell membrane receptor by combining AuNPs with traditional immunoassays. The detection sensitivity was considerably enhanced by signal amplification. Besides, the applications of nanomaterials in immunoassay were broaden. Additionally, the in situ immuno-analysis of cell membrane receptor provides new ideas for scientific research of the interaction between pathogenic microorganisms and the host cells. |
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