| With the expansion of aquaculture in China, it is necessary to use fish drugs to prevent and cure fish diseases. Some prohibited drugs, such as nitrofurans and malachite green(MG), are added illegally into fish feed or water due to their low cost and effectiveness against fish diseases. These artificial compounds and drug residuals not only affect to the quality and safety of aquatic products, but also pose a threat to human health and the environment.The traditional methods used to detect prohibited drugs are HPLC or LC-MS, which have advantages of high sensitivity and accuracy, but they are time-consuming, costly and require severe sample preparation, making it impossible to screen a large quantity of samples. So it has always been a hot research area to find rapid and sensitive methods to replace those common practices. Raman spectroscopy involves the use of fingerprint spectrum of molecular vibration, and the metal substrates with rough surface can result in significant Raman enhancement. The size, shape, dispersity and surface charge of nanoparticles(NPs) affect the affinity of target molecule onto the substrate surface, and thus affect the Raman enhancement effect. In this study, a variety of metal colloids were synthesized and used as SERS active substrates in detection of trace amount of furazolidone and MG.Firstly, furazolidone standards were analyzed using two commercial substrates including KlariteTM and Q-SERS and laboratory-synthesized Au colloid as SERS substrate. The result showed that different analytes may require different substrates for maximum surface enhancement effect. The use of KlariteTM and Au colloid as SERS substrate resulted in similar enhancement effect for furazolidone with the minimum detectable concentration of 0.1 mg L-1 as well as similar quantitative analysis capabilities. The use of Au colloid led to the optimal SERS signal at following conditions: p H≈3, the ratio of Au colloid and furazolidone was 5:1, and mixing time was 2 second. In addition, different spectral acquisition conditions, such as laser source and laser power, have greater impact on the sensitivity of SERS.Secondly, KlariteTM and Au colloid were further tested for analysis of furazolidone in fish feed. Since matrix interfered with the adsorption of target analytes onto surfaces of NPs, sample pretreatment was necessary to minimize the matrix effects. The methylene chloride extracts of fish feed were poured through column filled with anhydrous sodium and neutral alumina to remove water and fat. To get optimal SERS signal, the ratio of Au colloid and furazolidone extracts was 5:2, and mixing time was 5 min. With the use of KlariteTM and Au colloid as SERS substrates, sensitive detection of furazolidone in fish feeds at as low as 0.5 and 1 mg Kg-1 could be achieved.Thirdly, an easy-to-prepare super-paramagnetic Fe3O4/Ag hybrid NPs were synthesized and characterized by transmission electron microscopy(TEM) and superconducting quantum interference device(SQUID), and then optimized by adjusting the ratio of silver particles aggregated with APTMS-modified Fe3O4 NPs. The size of optimal hybrid NPs was 73±9 nm. The hybrid NPs were assembled under an external magnetic field before being used as the substrate for SERS analysis, leading to reproducible and stronger SERS signals. The SERS spectral features of furazolidone standard solution were clearly identified at concentrations as low as 40 ng m L-1, and furazolidone in fish feeds could be detected at 500 ng g-1. The results indicated that the Fe3O4/Ag hybrid NPs as SERS substrates had a great potential for detection of trace amount of furazolidone and other prohibited or restricted antibiotics in the animal and fish feeds.Finally, Au colloid with particle size of about 55 nm was synthesized. The Au NPs at neutral p H resulted in significant SERS enhancement for MG and its metabolite leucomalachite green(LMG) in muscles from seven different types of fish. Through simplifying sample pretreatment, the analysis could be reduced to 60 min compared to 3 ~ 4 h of traditional methods. The minimum levels in fish muscle could be detected ranged from 2 to 10 ng g-1, and the method had the highest sensitivity for tilapia and bighead carp had the highest sensitivity. Hence, it is meaningful to use SERS coupled with Au colloid for detecting MG and LMG in fish samples to ensure the safety of aquatic products.In summary, a series of metal colloids were applied as SERS substrates in analysis of furazolidone in fish feeds as well MG and LMG in fish muscles from seven different species. Magnetic composite NPs were synthesized and used for sample extracts from simplified sample preparation protocol, which filled in the gaps between SERS theory and application in determining nitrofurans in actual samples. Moreover, easy-to-prepare Au colloid combined with simple sample pretreatment made it possible for screening a large amount of MG and LMG in various fish muscles. This study confirmed that the surface morphology of metal colloid and sample pretreatment method affected the adsorption of target molecule onto the substrate, thus affecting the SERS enhancement. Therefore, in future studies more research should be conducted on designing and synthesizing different types of metal colloids, as well as simplifying pre-treatment methods suitable for SERS analysis. |
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