Detection Methods for Main Risk Factors of Sea Fishes Using Colorimetric Sensor Array
Author:Lv Ri Qin
Supervisor:huang xing zuo
Sea fishes are popular delicacy all over the world due to their nutrition and delicious tastes.Fishing is a traditional industry which plays an important role in the national economy in China.However,sea fishes often have to undergo a series of links including rapid freezing,transportation,storage and so on before consumed.During these links,a large number of risk factors may be introduced.Therefore,the safety monitoring of sea fishes and their products is very necessary to ensure their edible quality and safety before sales.At present,the safety of sea fishes is investigated by applying different traditional physical and chemical analysis according to different risk factors,these kinds of analysis methods are cumbersome,time-consuming,and require large-scale instruments and equipment,which cannot meet the requirement of rapid detection.According to the difference of traits of sea fishes safety,our research combined olfactory visualization with taste visualization to achieve detection of risk factors of sea fishes.For each risk factor,sensitive chromogenic materials were selected by analyzing the mechanism of reaction between the chromogenic materials and the risk factor.After the selection of the chromogenic materials,the colorimetric sensor arrays(CSA)were established for detection of the risk factor.The main contents of this research are as follows:(1)Screening and detection of main risk factors for sea fishesMackerel and large yellow croaker were selected as the samples of this study.The changes of total volatile basic nitrogen(TVB-N)and histamine in the storage at 4℃ of mackerel samples were determined by Semi-micro nitrogen determination and UV-Vis Spectroscopy method.During the storage,with the extension of storage time,the TVB-N content and histamine content showed a gradual upward trend.After five days of storage,the TVB-N content exceeded the national standard limit of 30 mg/100 g.However,the histamine content exceeded the national standard limit of 40 mg/100 g after seven days of storage.The freshness of the mackerel dropped gradually during its storage,and after five days,the fish body was completely corrupt and inedible.The heavy metals content of large yellow croaker samples,including arsenic(As),lead(Pb)and cadmium(Cd),were determined by inductively coupled plasma mass spectrometry(ICP-MS)method.The As contents of all large yellow croaker samples tested exceeded the national standard.And the number of samples in which Pb and Cd contents exceeded the national limit standard exceeded 20%.In view of the results obtained by conventional methods,the TVB-N,histamine and heavy metals were selected as the risk factors of sea fish.The TVB-N content and histamine content in the mackerels and the heavy metals content in the yellow croakers measured by conventional methods were regarded as the measured values,which were used to establish relationship with the signals of colorimetric sensor array.(2)Determination of characteristic volatile gases during storage of sea fishThe sea fish freshness was detected by colorimetric sensor array based on the reaction of chromogenic materials with the volatile gases of sea fish.The compositions of volatile compounds of mackerel samples were analyzed by combining headspace solid phase microextraction and gas chromatography-mass spectrometry during storage.A total of 53 different volatile compounds were drawn from the mackerel samples tested,including 6 kinds of hydrocarbons,a benzene,11 kinds of alcohols,6 kinds of ketones,11 kinds of aldehydes,5 kinds of esters,4 kinds of nitrogenous compounds,1 kind of acid and 8 kinds of other gases.Of all the volitaile gases compounds,there are five volatile gases which’s relative contents varied regularly,namely: trimethylamine(TMA),n-hexanal,n-octanal,furfural and methyl ketone.The relative content of volatile gas was analyzed by cluster analysis.The results of the cluster analysis were consistent with the freshness of the samples,and the adjacent days were also close in the clustering map,which indicates that the volatile gases of the mackerel can be used to characterize the freshness of the fish.The relative content of TMA increased continuously during spoilage not only for mackerel but also in butterfish and large yellow croaker.Because TMA was an important omponent of the freshness index TVB-N detected in this study.Therefore TMA was selected as characteristic gas for the whole storage stage of mackerel.(3)Analysis of reaction mechanism between colorimetric sensor array and main risk factorsIn order to select chromogenic materials for the detection of risk factors,this study applied quantum chemistry and UV-visible spectroscopy method to study the reaction mechanism of chromogenic materials and risk factors.In order to screen chromogenic materials for TVB-N detection,Quantum chemistry was applied to clarify the detection mechanism of chromogenic materials with volatile gas of fish.The combined display of TPP,CoTPP,CuTPP,FeTPP,MnTPP,NiTPP and ZnTPP before and after reaction with TMA were calculated by DFT method at the B3LYP/6-31G(d)level.The binding capacity of porphyrins with TMA was ordered: MnTPP>NiTPP>ZnTPP>FeTPP>CoTPP>CuTPP>TPP,The results showed that the binding energy of MnTPP was 17.00 kcal/mol and the binding energy of TPP was 1.42 kcal/mol,which means the binding ability of MnTPP and TMA was the strongest and TPP had the least binding ability when combined with TMA.And the metal porphyrins had a stronger binding ability to TMA than TPP.In that regard,metal porphyrins could be added when constructing colorimetric sensor array for TVB-N detection.In order to screen chromogenic materials for histamine detection,UV-visible spectroscopy method was applied to clarify the detection mechanism of chromogenic materials with histamine.When the azo reagent solution reacted with histamine,there was a strong absorption peak between 400 and 450 nm,and the absorbance solution gradually increased with the increase of histamine concentration.The result revealed the selected azo reagent can be used to construct a colorimetric sensor array for histamine detection.For selecting chromogenic materials for the detection of heavy metals,four pyridine azo compounds(PA1-PA4)and four porphyrin compounds(P1-P4)were selected to analyze their reaction mechanism with heavy metals including As、Pb and Cd.The chemical shift of proton signals was calculated by DFT method at the B3LYP/genecp level such as(C,H,O,N)and Pb were at 6-31G(d)and LANL2 DZ levels respectively.The binding energy of pyridine azo materials to As,Pb and Cd ranged from 620 to 690,180 to 210 and 200 to 230 kcal/mol,respectively.The binding energy of porphyrins to As,Pb and Cd ranged from 690~770,190~220 and 220~250kcal/mol,respectively.Pyridine azo compounds and porphyrins were not significantly different in their ability to detect the same heavy metal.Of the three heavy metals,results showed that the pyridine azo materials and porphyrins have the strongest binding ability to As,and the binding energy was ranked as As>Cd>Pb.Therefore the pyridine azo materials: PA1-PA4 and porphyrins: P1-P4 were selected as chromogenic materials for building CSA for heavy metals detection in fish.For selecting chromogenic materials for the detection of enrofloxacin,the reaction mechanism of selected pyridine azo materials and porphyrins B1-B9 with enrofloxacin was detected by UV-visible absorption spectroscopy.The results showed that the spectral response values of B1,B4 and B5 and the concentration of enrofloxacin solution were close to a linear relationship and there was no relationship between the response of B7 and the concentration of enrofloxacin.Judging from the results obtained,the results showed that B7 cannot be used as a chromogenic material for the detection of enrofloxacin,thus it was eliminated when constructing the sensor array for detection of enrofloxacin.(4)Detection of main risk factors of sea fishes based on colorimetric sensor arrayThe main risk factors of sea fishes were detected by the CSA technique.For the detection of TVB-N content of mackerel samples,the TVB-N content detected by semi-micro kjeldahl method was taken as a comparison,support vector machine(SVM)was applied to establish the relationship between the signals of CSA and the values of TVB-N of mackerel samples.In the optimal case of parameter c=11.3137,g=0.088388,the correlation coefficient of prediction set reached 0.8155 and the root mean square error was 2.5887.The results showed that the constructed CSA could be used to determine the TVB-N content in the mackerel samples evaluated.For the determination of histamine content in mackerel,the sensitive unit was constructed by selecting azo reagent as the chromogenic material.The gray values of sensors were chosen for statistical and quantitative analysis.The relationship between gray values and the histamine content were established based on linear regression.The results of the regression showed a high correlation and the calibration correlation coefficient was 0.9926.For the determination of the content of heavy metals including As,Pb and Cd in large yellow croaker samples,four kinds of pyridine azos and four porphyrins were selected to construct the colorimetric sensor array.The heavy metals content detected by ICP-MS method was taken as a comparison.SVM was applied to establish the relationship between the signals of CSA and the values of heavy metals of large yellow croaker samples.In the optimal case of parameter c=8,g=0.25,the correlation coefficient of prediction set reached 0.76249 and the root mean square error was 1.2723.The results showed that the constructed CSA has better prediction ability for As,and has low detection ability for Pb and Cd.For wider application,it is necessary to screen other suitable chromogenic materials to construct a targeted sensor array to improve the detection ability.For the determination of enrofloxacin,a pyridine azos and seven porphyrins were selected to construct the colorimetric sensor.SVM was applied to establish the relationship between the signals of CSA and the concentration of enrofloxacin solution.In the optimal case of parameter c=8,g=0.35355,the correlation coefficient of prediction set reached 0.93836 and the root mean square error is 9.0434.The results showed that the constructed CSA has better prediction ability for enrofloxacin.The results showed that the homemade CSAs could be applied for rapid and easy determination of sea fishes main risk factors.The study results provide a new method for sea fishes safety detection,which has positive significance for improving detection technology level of the food and agricultural products safety,and the study is of great significance to control the quality of China’s fishery products and promote the development of China’s fishes industry.