Semi-synthesis Of Lactose Functionalized Magnetic Iron Oxide Nanoparticles And Their Application In The Analysis Of Ricin,

Ricin, as a ribosome-inactivating protein, is constructed by two chains A and B, in which chains A and B are linked by a disulfide bond. Ricin toxin B chain (RTB) is a lectin with two galactose binding sites and can bind to cell-surface galactose and carry the toxicity chain–ricin toxin A chain (RTA) into the cells. As a highly efficient N-glycosidase, RTA can inactivate 28S ribosomal RNA, block protein synthesis and lead to cell death. Due to its strong toxicity and easy purification, ricin can be easily used as a biological warfare agent in the terrorism attacks. So it has been listed in Chemical Weapons Convention for its controlled use. To develop a rapid ricin detection method means a lot to the military and also to the treatment of the potential threat caused by ricin.By far, the assays of ricin mainly focus on two ways: the antibody-based immunological method and the instrument analysis, such as biological mass spectrum. This thesis included the synthesis of magnetic iron-oxide nanoparticles, construction of new immunological method based on the enrichment of galactose-functionalized magnetic iron-oxide nanoparticles, and the instrument analysis based on the enrichment of galactose-functionalized magnetic iron-oxide nanoparticles and LC-MS/MS assay. On the basis of the above-mentioned content, this thesis is composed of three chapters.In the first chapter, it makes a brief introduction on the biological characteristic of ricin and its detection. Also, a mini-review of the synthesis, stabilization, functionalization and biomedical application of magnetic iron-oxide nanoparticles is provided. Based on such information, we summarize the objective basis, the significance and methodology of the thesis.In the second chapter, the determination method based on the enrichment of galactose-functionalized magnetic iron-oxide nanoparticles is established and applied forthe detection of ricin in buffer and serum. First, bare Fe₃O₄ nanoparticles are prepared by co-precipitation method and coated with silanization agent. Then, the modified galactose is covalent linked to the surface of obtained amino magnetic nanoparticles, and several methods are used to characterize the synthesized nanoparticles. The amount of galactose-loaded and ricin-binding on final nanoparticles is about 30μg/mg and 29μg/mg, respectively. For ricin in physiological buffer and serum, the linear calibration curves of established method are ranged from 2 to 64 ng/mL and 4 to 64 ng/mL, respectively; the corresponding limit of detections (LODs) are 2 ng/mL and 4 ng/mL. This method has shorter time (about 3 hours), little interference in serum, and can be used for rapid detection of ricin samples.In the third part, the determination method of ricin is constructed by the LC-MS/MS based on the enrichment of galactose-functionalized magnetic iron-oxide nanoparticles. First, the mass spectrum parameters are optimized according to the specific peptide obtained from the trypsin digest of ricin. The linear calibration curve ranges from 5 ng/mL to 1μg/mL. The LOD can reach to 2 ng/mL. This method has well selectivity, accuracy, and can be used for traceability detection of ricin.