Research On The Method Of Detecting Clenbuterol Hydrochloride In Pork Using The Portable Glucose Meter

Clenbuterol Hydrochloride(CLB),which is also called as Clenbuterol,have stable chemical structure.When heating temperature reached to 172 ?,it can be decomposed.CLB was so easy to residues in animal tissues that it was danger for human.If consumers eated the meat which contains CLB,it could result in acute poisoning and chronic poisoning.CLB is banned in feeding the animals in our country,and there are usage standard in many countries and international organizations.At present,the main method of detection the CLB were the enzyme-linked immunosorbent assay,AuNPs test strip for rapid detection,high performance liquid chromatography,gas chromatography-mass spectrometry,Biosensing Technology,and so on.But these methods are suitable for different test objects,each has its advantages and disadvantages.Therefore,it is necessary to develop a sensitive and convenient method for detecting the residues of CLB in pork.In this study,a method was builed to detect CLB residue which was based on portable glucose meter.Through establish a new detection technology of magnetic immunobiosensing probe which was using the test of magnetic separation technology,at the same time,the portable glucose meter was used to promote reaction to produce the signal which is invertase catalyzed by detection of immune response so that the accurate quantitative CLB residue.The method has the advantages of simple operation,high sensitivity and low detection cost.The results are as follows:(1)A magnetic immune-sensing probe is prepared by the CLB-BSA,which is carried of monodisperse carboxylic beads.Through the single factor experiment to study the influence of CLB-BSA addition,reaction temperature,reaction time on the coupling rate.Through the Box-Behnken test design to established the variation of coupling rate with the CLB-BSA addition,reaction temperature,reaction time regression model: Y=-151.943+7.700X2+6.095X3-0.0175X1X2+0.0125X1X3-0.01745X12-0.13625X22-0.2325X32.The regression model is highly significant,the lack of fit is not significant,the decision coefficient is high(R2=0.9909).The model can be used for actual prediction.The order of impact effect on the independent variable coupling rate are as follows: the reaction temperature,the reaction time,CLB-BSA addition.Through optimizing the CLB-BSA addition,reaction temperature and reactiontime in the way of response surface methodology,the optimal parameters for the preparation of magnetic immunosensor were 41 ?g,reaction temperature of 25 ?,reaction time 13 h.Under the optimal conditions,the five tests were carried out.The average coupling rate was 15.22%,and the difference was only about 0.25%.(2)The optimum AuNPs solution pH for preparation IgG-AuNPs-Invertase was8.5,the concentration of invertase was 120 ?g/mL,the concentration of anti-goat IgG was 12 ?g / mL,and the ratio of IgG-AuNPs-Invertase to rabbit IgG was 10:1.The UV-Visible absorption spectra show that the prepared Au NPs has good homogeneity.The secondary antibody and invertase can be successfully labeled on the surface of AuNPs by using the labeling method of this experiment.Transmission electron microscopy shows that the prepared IgG-Au NPs-Invertase particles evenly dispersed and the particles around a circle of visible halo,while the particle size becomes larger which indicated that Ig G and invertase were successfully labeled to the surface of Au NPs.(3)The optimization of test conditions for CLB residue was based on portable glucose meter.The results showed that the dosage of immunomagnetic beads was 25?g,the CLB antibody was diluted by 1:800,the dilution of IgG-Au NPs-Invertase was1:500,and the invertase was involved in the hydrolysis of CLB.The optimum temperature for the reaction was 55 ? and the time was 30 min.(4)This study established a method based on glucose meter to detect CLB.The experiments proved that the method is sensitive and the detection limit of this method is 0.1 ng/mL and the CLB concentration is in the range of 0.1~100 ng/m L,it has a good linear relationship,and the linearity of the regression equation is 0.9969.The recoveries were 85%~94% and the standard deviations were less than 5% at 1 ng /g(low),5 ng/g(middle),15 ng/g(high),indicating that the accuracy of method was high;The intra-assay CV was 1.2% ~ 1.6%,and the inter-assay CV was between1.1% ~ 8.8%,indicating good reproducibility and stability.There was a very low cross-reactivity to salbutamol and ractopamine,high specificity.(5)CLB was added to 6 samples without CLB,the final concentration of CLB in sample was 0.2 ng/g,5 ng/g,10 ng/g,15 ng/g,20 ng/g,50 ng/g,and the results were statistically analyzed and linear regression analysis,and the result was not significant.The linear equation:Y=0.8787X+0.0668,R2= 0.9981,with the HPLC of detection of high compliance.It indicated that this method had lower detection limit.