Research And Application Of Rapid Detection Methods For Three Harmful Substances In Mackerel

In order to improve food safety,our research group has carried out a preliminary study on the rapid detection of some harmful substances such as biogenic amines in mackerel and the evaluation of mackerel freshness.To further establish and improve the detection methods of harmful substances in mackerel,three rapid detection methods for enrofloxacin,cadaverine and histamine were established and applied in mackerel samples.The main contents are as follows:（1）Using square wave voltammetry and Alizarin red S（ARS）as electrochemical probe,a sensitive and easy-to-use method for measuring enrofloxacin was established and successfully applied to the determination of enrofloxacin in mackerel samples.The conditions of the pH value of the test solution and the electrochemical measurement method were optimized,and the binding mechanism between alizarin red and enrofloxacin was explored.The experimental results show that the response current difference（-ΔI,μA）has a good linear relationship with enrofloxacin concentration in the range of 2.19×10^-99 to 1.0×10^-77 mol/L with a detection limit of7.3×10^-1010 mol/L.The results of the spiked recovery of enrofloxacin in mackerel by this method were 90.6%¹00.7%.At the same time,the method was validated by liquid chromatography-tandem mass spectrometry（LC-MS/MS）.The detection method has good stability and selectivity,high sensitivity and good accuracy,and is suitable for the determination of enrofloxacin in mackerel samples.（2）The unfolded hemoglobin was immobilized on the surface of the clay-gold nanoparticles（Clay-AuNPs）modified glassy carbon electrode（GCE）by physical adsorption method,and then the diamine oxidase（DAO）was immobilized with 2.5%glutaraldehyde as a crosslinking agent.The resulting electrochemical biosensor had good detection performance for cadaverine.The electrode modification system,the concentration of DAO,the type of peroxidase,the stability of the modified electrode and the test conditions were optimized.The modified electrode was characterized by cyclic voltammetry（CV）,electrochemical impedance spectroscopy（EIS）and transmission electron microscopy（TEM）.Furthermore,the method was used to detect cadaverine content in mackerel during storage.The results showed that under the optimized conditions,the response current of the sensor and the concentration of cadaverine had a good linear relationship between 2.0×10^-121.0×10^-11mol/L（r=0.991）,and the detection limit（S/N=3）is 6.7×10^-1313 mol/L.The spiked recovery of cadaverine in mackerel samples by this biosensor method was 82.2%¹09.1%.The method has good reproducibility and stability,high sensitivity,and is suitable for the determination of cadaverine content in mackerel samples.（3）Histamine is the most toxic substance in biological amines,and most food poisoning incidents are related to the consumption of histamine excess food.In view of the fact that histamine can agglomerate nano-gold and cause its color and spectrum to change,a simple,rapid and sensitive visual colorimetric detection method for the determination of histamine in mackerel was established by using nano-gold as the main chromogenic substances and azo reagents as background reagents to increase the color rendering effect.In order to obtain the best measurement results,the chromogenic conditions such as the volume ratio of nano-gold and azo,the type of the test base solution were optimized.The color rendering effect of the standard colorimetric solution was investigated,which indicated that the visual colorimetric method could quickly identify histamine in the range of 1.0×10^-51.0×10^-22 mol/L,and the detection limit of the visual colorimetric method was 1.0×10^-55 mol/L.The characterization and properties test of standard colorimetric solution were performed by ultraviolet-visible spectrophotometry.The experimental results show that the absorbance and histamine concentration in this color-developing system have a linear relationship in the range of 1.0×10^-6to 5.0×10^-4mol/L.The detection limit of histamine is 3.3×10^-77 mol/L.At the same time,in order to prove the accuracy and reliability of the detection method,the recovery experiments of histamine in mackerel samples were performed to obtain recovery rate of 90.9%¹02.1%.It shows that the detection method is highly accurate and suitable for the determination of histamine content in mackerel samples.