A New Method For Detecting Cyrimazine And Melamine In Milk Based On Aptamer Biosensor

Cyromazine(Cyr)is a kind of triazine organic compound,which is widely used as a feed additive to inhibit the growth of maggot flies in animal husbandry industry due to its inhibition of insect growth and killing of parasites.However,overuse of cyromazine will cause contamination of animal-derived food,and even potential environmental problems,which will harm human health.For example,it has been shown to cause breast tumors in mice.Cyromazine in animals and plants can be dealkylated into melamine(Mel),melamine will combine with its metabolites can precipitate in kidney cells,eventually leading to renal failure.Therefore,long-term ingestion of cyromazine or melamine can cause irreversible damage to the kidneys of both animals and humans.Cyromazine and melamine have been implicated in many food safety incidents in recent years,including pet food incidents in the United States in2007 and milk powder incidents in China in 2008.The occurrence of related events to the countries on the food cyclimazine and melamine residue detection standards more and more stringent.For example,in the United States,the maximum residue standard of cyromazine in milk is 0.05mg/kg;In China,it is stipulated that the residual amount of cyromazine and melamine should be tested before the raw milk leaves the factory,and the limit of melamine in food should be strictly stipulated:1.0mg/kg in baby's food;2.5mg/kg in adult foods.In the face of severe food safety problems and strict detection requirements,it is of great significance to develop a highly sensitive,simple and rapid detection technology.In this study,based on the high specificity of nucleic acid aptamer,three different detection techniques were established to realize the simultaneous detection of Cyclopromazine and melamine.The specific methods are as follows:(1)A colorimetric method of 3,3',5,5'-tetramethylbenzidine(TMB)/H₂O₂ catalyzed by aptamer-coated gold nanoparticles(Au NPs)was established to achieve the simultaneous detection of cyclimazine and melamine in dairy products:A simple and economical colorimetric sensor was designed for the simultaneous detection of Cyr and Mel in milk by using gold nanoparticles to catalyze the oxidation of 3,3',5,5'-tetrapethylbenzidine(TMB)/H₂O₂instead of catalase and the specific combination of the aptamer and target substance.Under the optimal conditions,the linear detection range of Cyr was 0-1.0 ppm,the linear regression equation was y=-0.4416x+0.8933,and the linear correlation coefficient was R²=0.9989.Under the optimal conditions,the linear detection range of Mel in this detection system is 0-1.0 ppm,the linear regression equation is y=-0.6579x+0.8059,and the linear correlation coefficient R²=0.9967.(2)A series of logic switches have been developed based on aptamer-modified nanocrystalline gold to realize simultaneous detection of Cyr and Mel in dairy products:Using aptamer,cetyltrimethylammonium bromide(CTAB),Mel and cyclopromazine to control the aggregation and dispersion of gold nanoparticles,a series of logic switches were designed and a rapid detection method was established based on these switches.Firstly,both Mel and cyclopromazine could induce the aggregation of Au NPs.According to this characteristic,OR logic gate was designed to detect whether Cyr or Mel was present in the system.Secondly,based on the fact that aptamer T31 can specifically bind to Mel to prevent the aggregation of AUNPs,we also developed an improved inhibition gate to detect the presence of Mel.The detection limit for Mel was 0.12 ppm by naked eye and 2.2 ppb by spectrophotometer.Third,due to the adsorption of the aptamer Tcy1 on Au NPs and the strong coordination between Tcy1 and cyclopromazine,the addition of cyclopromazine and CTAB immediately led to the aggregation of Au NPs.The detection limit was 0.17 ppm by naked eye and 9.0 ppb by spectrophotometry.The design of this detection system provides a foundation for the development of functional logic switches.(3)Detection of Cyr and Mel in milk by surface-enhanced Raman technique based on Cyr and Mel specific aptamer:The specific aptamer was first bound to Cyr and Mel,and then nano-silver was controlled to be reduced in situ on the surface of the aptamer to form Cyr/Mel-Apt-Ag NPs complex,and the Raman enhancement effect was achieved.The linear detection range of Cyr was 0?0.5ppm,and the linear fitting equation was y=2437.8x+424.67,correlation coefficient R²=0.998,and the detection limit was 13.6ppb.Under the optimal conditions,there was a good linear relationship between the concentration of Mel in 0-0.5 ppm under this detection system.The linear regression equation was y=479.8.8X-0.91,R²=0.998,and the detection limit(LOD)was 43.5ppb.In conclusion,the three detection methods established in this study have the characteristics of rapid,convenient,high specificity,high sensitivity and adaptability to high throughput detection in the detection of cyclopromazine and melamine,which is of great significance for maintaining food safety.