Application Of Functionalized Magnetic Beads As Adsorbent In Enrichment Of Malachite Green From Aquatic Products

Malachite green (MG), belongs to the triphenylmethane dye family, which has been used worldwide for fungicide and ectoparasiticide in the aquaculture industry as strong antibacterial dyes. MG can be easily transferred to humankind through food chain because of residues in fish tissues. The usage of MG in aquaculture industries is strictly banned in many countries for its carcinogenic and genotoxic properties. Therefore, it is a challenge for research and regulatory agencies to build a quickly MG separation and determination method which is suitable for complex systems. In this study, a direct competitive MG ELISA method is established for MG and LMG, which is good for rapid on-site screening in a semiquantitative sense.The study contents four parts, as follows:1) Synthesis of Sulfo-Functionalized Iron Oxide Magnetic BeadsCarboxyl-functionalized magnetic beads were prepared by precipitation method. Then Sulfo-functionalized magnetic beads were prepared by covalently conjugating aminobenzene-sulfonic acid onto carboxyl surface of magnetic beads with 1-(3-dimethylamino)propyl]-3-ethylcarbo-diimide(EDC)/N-hydroxysulfo-succinimide sodium salt(Sulfo-NHS) method. The size of nano-magnetic beads was observed by TEM at 80 kV. The sulfonic acid group bound on nano-magnetic beads was determined by Fourier transform infrared spectrum and Zetasizer Nano ZS. All the results were supported that the sulfonic acid group has been coated on the surface of the nano-magnetic beads successfully.2) Enrichment of Malachite Green in Fishpond Water by Sulfo-Functionalized Iron Oxide Magnetic BeadsSulfo-functionalized iron oxide magnetic beads were used as dispersive solid phase extraction sorbent, and hence, a high efficient extraction procedure was developed for enriching trace amounts of malachite green (MG) in fishpond water. Parameters of absorption and desorption of MG including pH value of fishpond water and elution solution were optimized. Results showed that sulfo-functionalized magnetic beads exhibited the high efficiency on enrichment of MG in fishpond water at pH 3.0-6.0 and the highest efficiency achieved at pH 4.0 (82.4±5.48%, N=3). Acetonitrile was effective in eluting MG on the magnetic beads. At pH 4.0 and 25℃, the adsorption behavior followed the Langmuir adsorption isotherm with a maximum adsorption capacity of 66.67 ng mg-1 and a Langmuir adsorption equilibrium constant of 0.375 mL ng-1.3) Enrichment of Leucomalachite Green in Fish muscles by Sulfo-Function-alized Iron Oxide Magnetic BeadsLMG was extracted by adding MaIlainve buffer and acetonitrile in fish muscles, and then it was enriched by magnetic beads from the extract. The extraction conditions, oxidative conditions and pH were optimized before this high efficient extraction procedure was developed for enriching trace amounts of leucomalachite green (LMG) in fish muscles. However, the average recovery was only around 30% when this method was applied in spiked samples. Further researchs for application of this method in fish muscles were needed.4) Synthesis of oleinic acid functionalized Magnetic Beads and enrichment of LMG in fish muscles by this magnetic beadsOleinic acid functionalized magnetic beads were prepared by covalently conjugating Oleinic acid and ethylenediamine onto carboxyl surface of magnetic beads with EDC/Sulfo-NHS method. Those beads were used as dispersive solid phase extraction sorbent, and hence, a high efficient extraction procedure was developed for enriching trace amounts of leucomalachite green (LMG) in fish muscles. Parameters of absorption were optimized. Results showed that oleinic acid functionalized magnetic beads exhibited the high efficiency on enrichment of LMG in fish muscles, and the highest efficiency achieved at pH 10.0, concentration of acetonitril was 10%(v/v) and NaCl was 2.0 mol/L(84.5±1.55%, N=3). Acetonitrile was effective in eluting MG on the magnetic beads. At pH 4.0, concentration of NaCl was 2.0 mol/L and temperature of reaction was 25℃, the adsorption behavior followed the Freundlich adsorption isotherm. Finally, application of this method in spiked fish muscles and compared it with the Chinese standard method, Results showed that this method is better than the Chinese standard method.