Analysis Of Food Tetracycline Residues Using Paper-based Molecularly Imprinted Composite Material With Hydrolyzed Gelatin As A Functional Monomer

Background:An increasing attention is paying to the food safety issues c aused by tetracycline residues;however,there wasn’t extremely convenient and effective rapid detection method of food tetracycline residues.The present study explored to controllably build selective paper-based molecularly imprinted composite material,which the specific recognition and tetracycline antibiotics functional molecular imprinting cavity was made by reverse addition-fragmentation transfer process（RAFT）.Method:Hydrolyzed gelatin was produced using the hydrolysis of gelatin from pig skin and fish skin treated with enzyme,acid or alkali,and the hydrolysis conditions（temperature,p H and time）were optimized.Additionally,the relative molecular weight,isoelectric point,and protein denaturation temperature of the hydrolyzed peptides were also analyzed.Furthermore,the synthetic conditions such as paper-based materials,Rhodamine B concentration,solvent,template molecule,functional monomer,and crosslinking agent were optimized with single factor experiment,in order to select an optimal preparation of paper-based molecularly imprinted composite.Meanwhile,paper-based molecularly imprinted composite material built with optimal scheme was characterized using scanning electron microscopy（SEM）,infrared spectrometer,static adsorption experiment,and dynamic adsorption experiment.And the binding site of tetracycline target molecules in paper-based molecularly imprinted materials was analyzed by Scatchard method.Besides,Paper-based molecularly imprinted composite material was used for detection of tetracycline in dai ry products,and limit of detection（LOD）and sensitivity were calculated.Result:The optimal preparation of hydrolyzed gelatin was that,using collagen enzyme under the condition of 37℃,p H=7.4,enzymolysis for 60 min.Meanwhile,the basic properties of hydrolyzed gelatin from optimal preparation was analyzed,found that the molecular weight of hydrolyzed gelatin was under 50000,the main amino acids were isoleucine and cysteine,the isoelectric point was 4.51,and hydrolyzed gelatin didn’t showed thermal denaturation analyzed with DSC method.According to optimized analysis,the optimal preparation plan of paper-based molecularly imprinted composite material was the following,the paper-based material was made of the A4 print paper attached with scotch tape and then peeled off,the fluorescence quantitative stain of Rhodamine B solution was 0.1 mg/ml;with 40 ml of acetonitrile as the solvent,1 mmol tetracycline as template molecule,8 mmol MAA+0.3 g hydrolyzed gelatin as the functional monomer,and 28 mmol EGDMA as crosslinking agent,and the template molecule elution time was 36 h.Paper-based molecularly imprinted composite material built with optimal scheme was characterized using scanning electron microscopy（SEM）and infrared spectrometer,confirmed that the molecularly imprinted polymer hole was successfully synthesized on paper fiber surface.The adsorption capacity in tetracycline solution reach maximum in 200 mg/L,respectively 40.80 and 18.12 mg g^-1,thus the imprinting index was 1.912.And th ere were two different types of binding sites between MIPs and tetracycline,investigated with Scatchard method.Additionally,the paper-based molecularly imprinted composite material had specific selectivity recognition to tetracycline.The paper-based molecularly imprinted composite material was used to detect the content of tetracycline in commercial dairy,the standard curve was set up within 0.5-50 mg L^-1,and the detection limit of 0.48 mg L^-1.This method did not check out tetracycline in commercial dairy,and sample added recovery ranged from78.8%to 104.1%.Conclusion:The result showed that hydrolyzed gelatin as functional monomer improved the specificity adsorption capacity of paper-based molecularly imprinted materials to tetracycline.The optimal paper-based molecularly imprinted composite material showed high selectivity to tetracycline.There were two different types of binding sites between MIPs and tetracycline,the maximum adsorption capacity in tetracycline solution was 40.80 mg g^-1,showing strong specificity adsorption performance.The curve linear of paper-based molecularly imprinted composite material in tetracycline solution was greater within the scope of 0.5-50 mg L^-1,and had higher sensitivity and detection limit.This paper can be used for tetracycline residues screening test in livestock and poultry products with fast,convenient,cheap,and sensitive.