Analyses Of Malachite Green And Leucomalachite Green In Tilapia Fillets With Surface-enhanced Raman Spectroscopy Coupled With Gold Substrate

China is the world’s largest producer and exporter of aquatic products. In recentyears, with its rapid growth of fishing industry, the safety issue of China fish productsbecomes a growing concern to consumers abroad and at home. Malachite green is anindustry dye that can effectively prevent and treat various fungus caused fish diseases.However, Malachite green and its metabolites pose health risks to human bodies. Manycountries such as European Union, United States, Japan and China have already bannedthe use of malachite green in aquaculture. Yet due to its low cost and strong features ofefficacy, malachite green is still used in a relatively large scale in aquaculture practice.So far, the routine methods used to detect trace amounts of drug residues in aquaticproducts are mainly chromatography or chromatography mass spectrometry-basedapproaches. They are of high sensitivity and good accuracy, while those methodsusually require high purity of sample extracts. For example, LC-MS/MS is the officialmethod for detecting malachite green and its metabolites residues in fish products. Thismethod required complex sample preparation, and it is time-consuming andlabor-intensive. Therefore, developing simple and rapid method for detecting malachitegreen and its metabolites in fish products is of significant importance to ensure thesafety of fish products. In this study, SERS technology coupled with gold substrates wasapplied to explore the qualitative and quantitative possibility for analyses of malachitegreen and its metabolites in fish products. The possibility for simplifying samplepreparation was also explored.Firstly, standard solution of malachite green (MG) was used to gain a basicunderstanding of the potential of SERS technology for qualitative and quantitativeanalyses of MG. Different gold substrates, Q-SERS with gold nanoparticlesnon-uniformly distributed on the surface, Klarite substrate with inverted pyramid nanostructure on the surface, and self-assembly gold nanoparticle suspension werecompared. Spectral collection conditions, such as different laser light source, objectiveand laser power were also compared. Experimental results showed that differentsubstrates and spectral collection conditions will lead to different detection limit andaccuracy of quantitative analyses. The lowest detectable concentrations were rangingfrom0.5-100ng/mL, and the R2of PLS model were ranging from0.915-0.98, showinggreat potential to apply SERS for analyses of fish drugs.Secondly, SERS was also applied in analyses of MG and its metabolitesleucomalachite green (LMG) in contaminated fish samples. With the US FDA samplepreparation method, the sensitivity of SERS technology in contaminated fish samplescould reach1ppb levels and the PLS models showed good predictability (R2=0.984,RMSEV=1.97ng/g), indicating great potential of using SERS with multivariateanalyses as an alternative method for determination of MG residues in fish fillets.In addition, the possibility for simplifying sample preparation based on US FDAstandard method was also explored. This study confirmed that the more residues of fishcomponents in the extracts, the less sensitive of the SERS method for detection of MGin tilapia fillets. The lowest detectable concentrations of MG were2and5ng/g forextracts prepared involving one and no cartridge, respectively. For quantitative analyses,the more residues of fish components in the samples, the lower the predictability of therelevant PLS model. The simplified version of US FDA sample preparation using oneless cartridge, the PLS models showed good predictability (RMSEV=2.21ng/g, R2=0.988), The method involving no cartridge significantly simplify the sample preparation,reduce the amount of chemical solvents, and eliminate the cost for cartridges. Althoughthe quantitative result based on this method involving no cartridge (RMSEV=9.32ng/g,R2=0.788) was not ideal, it could be used to detect fish contaminated with MG at5ppblevel. Based on the research results above, a suitable sample preparation of LMG andMG residues in fish fillets for SERS analysis was developed. This strategy enablesminimal sample preparation with analysis time less than60minutes and asuper-sensitive detection of total LMG and MG extracts in tilapia fish fillets at as low as2ng/gbased on PCA was obtained. Such a strategy is of great significance in the foodsafety and defense area, particularly when screening a large number of samples isnecessary.In conclusion, SERS is a very complex process, with many influencing factors. Different spectral acquisition conditions will greatly affect the detection sensitivity andthe accuracy of quantitative analysis. SERS spectra combined with multivariateanalyses could achieve an accurate quantitative analyses of trace amount of drugresidues in a certain concentration range. The sensitivity of SERS technology indetection of MG and its metabolites LMG in contaminated fish samples could reachsingle-digit ppb levels ranging from1to5ng/g, depending upon the complexity ofsample preparation methods. Therefore, in future study, the development of suitablesample preparation as well as different surface patterned SERS-active substrates forSERS analyses is urgently needed.