Purification Of Endogenous Brassinosteroids From Plant Tissues Using Novel Boronate Affinity Magnetic Nanoparticles

Brassinosteroids (BRs), an important endogenous phytohormone, play an important regulatory role in a variety of physiological processes including stress tolerance, cell elongation and division, photomorphogenesis, and so on. Besides, BRs have been proved to be effective in increasing crop yields and improving fruit quality. Therefore, the biosynthesis, signaling and molecular mechanisms of BRs function become a hotspot in the research field of phytophysiology. Quantitative analysis of endogenous level and organ-specific distribution is a key issue in clarifying the physiological role of BRs. However, due to the extremely low level, unique physicochemical properties of BRs and the complexity of plant matrix, little progress has been made on developing high-sensitivity BRs analysis method, especially sample pretreatment technique. At present, BRs are most isolated based on non-specific adsorption mechanisms, which make the pretreatment process highly sample-consuming, tedious and labor-intensive. Therefore, it is in urgent need to develop a highly selective and sensitive purification strategy for BRs analysis. Considering the cis-diol sub-structure of most bioactive BRs and the specific boronate affinity of immobilized boronic acid, this dissertation describes the preparation of a novel type of boronate affinity magnetic nanoparticles (BA-MNPs) and explores its application in the purification of endogenous BRs and the purification mechanism. The main progress can be listed as follows:1. A novel type of BA-MNPs was synthesised by surface polymerization of4-vinylphenyl boronic acid (VPBA) onto Fe3O4magnetic nanoparticles. The magnetic performance, surface morphology,particle size and distribution, adsorption and desorption behavior of BA-MNPs were investigated in detail. BA-MNPs had a high saturation magnetization of11.2emu/g. The adsorption of BRs on BA-MNPs followed Langmuir isotherm with a high capacity determined to be27mg/g, in accordance with the chemisorption nature of boronate affinity.2. BA-MNPs could improve the mass spectrometry response of endogenous 24-epiBL dramatically through adsorbing some impurities in plant extracts. Further experiments demonstrated that the sensitivity improvement was achieved by removal of cis-diol containing compounds and fatty amine through comprehensive mechanisms including boronate affinity, reversed-phase, and charge transfer interactions. Combining SPE pretreatment and BA-MNPs adsorption, a simple and convenient purification method was developed to reduce the minimum consumption of plant tissues to17mg, one percent of reported minimum.3. BA-MNPs were utilized to selectively enrich endogenous BRs from the SPE passthrough and4BRs were detected in500mg of real plant materials with signal to noise (S/N) above90except teasterone. In order to simplify the handling process, a simple protocol was developed for direct enrichment of BRs from plant materials, which provided a new idea for selective purification of BRs and some reference value for other hydrophobic cis-diol molecules.