Study On Identification Of Irradiated Food Containing Fat By Immunoassays

The irradiation of food products by ionizing radiation to control microorganisms and enhance shelflife has become a popular preservation technique worldwide. Although this process is considered a safefood preserving method, consumers have demanded the right to be informed about the food theypurchase. In addition, the labeling of foods containing an irradiated ingredient should be a legalrequirement in several countries. Therefore, reliable and convenient methods must be available for theauthentication of irradiated foods to meet labeling requirements and regulate international trade. Most ofirradiated food containing fat, therefore, it is very important and of great significance to study onidentification of irradiated food containg fat by detecting unique radiolytic products2-Dodecylcyclobutanone （2-DCB）.The purpose of this investigation is to develop a new rapid extraction and purifaction method for2-DCB in irradiated food containg fat, prepare highly sensitive and specific polyclonal antibodies to2-DCB, develop an indirect competitive enzyme-linked immunosorbent assay （cELISA） for efficientlyscreening and quantifing2-DCB in irradiated lipid-containing foods, and develop a novel biosensorregulated by the "rotator" ε-subunit of F0F1-ATPase which can be used as a reliable, rapid andcost-effective screening technique for the determination of irradiated lipid-containing foods. The maincontent and results of this investigation were showed as follows:1. A new rapid extraction and purifaction method based on direct solvent extraction （DSE）/gaschromatography-mass spectrometry （GC-MS） was developed to provide a rapid and simple samplepreparation method for identification of irradiated food containg fat by analysis of2-DCB.2-DCB wasextracted from irradiated beef by direct blending with acetonitrile and then purified on a silica SPEcartridge without prior fat removal. The whole procedure was about60minutes to90minutes. Theprocedure was evaluated with a recovery test in three samples spiked with2-DCB at0.01and0.5mg/kgof ground beef, resulting in88.7%to90.4%recoveries with mostly less than5%RSD, demonstratedhigh efficiency of the extraction method.2. A highly sensitive and specific polyclonal antibodies against2-DCB was obtained in this study.2-Oxo-cyclobutane undecanoic acid, was used as an alternative to2-DCB and conjugated to BSA andOVA via a conventional carbodiimide condensation reaction to prepare the immunogen and the coatingantigen of2-DCB. The coupling ratios were9.9:1and1.4:1, respectively. The monoclonal antibodyagainst2-DCB was obtained by immunizing New Zealand white rabbits, with a high specificity and lowcross-reactivity for2-tetradecylcyclobutanone （2-TCB;<8%） and other structurally related compounds（<0.1%）.3. An indirect competitive enzyme-linked immunosorbent assay （ELISA） was developed for thedetection of2-DCB, a chemical marker of irradiated lipid-containing foods in this study. The cELISAmethod was applicable at optimal experimental conditions of0.001μg/mL to100μg/mL2-DCB in thebuffer solution, with an IC50value of0.25μg/mL and a limit of detection （defined as the IC20） ofapproximately0.004μg/mL. The recovery ratio of2-DCB from ground beef patties ranged from84.4% to109.8%. The intra-assay and inter-assay coefficients of variation were <10.0%and <12.0%,respectively, which demonstrated the high accuracy and precision of the assay. The proposed methodwas validated by gas chromatography-mass spectrometry with high correlation. The same method wasused to detect2-DCB in ground beef patties irradiated at0.5,1.0,3.0,5.0, and7.0kGy; the2-DCBconcentration linearly increased with the radiation dose. The proposed ELISA-based technique could bea feasible quantitative screening method for the2-DCB analysis of irradiated ground beef and otherlipid-containing foods because of its high sensitivity, simplicity, rapid reaction, low cost, and highsample throughput.4. A novel biosensor （immuno-rotary biosensor） was developed by conjugating2-DCB polyclonalantibodies with the "rotator" ε-subunit of F₀F₁-ATPase within chromatophores with an ε-subunitpolyclonal antibodies–biotin–avidin–biotin linker to capture2-DCB residues. The conjugationconditions were then optimized. The limit of detection （LOD） of the optimized biosensor was10^-8μg/mL. Interferences caused by sample matrix were easily overcome by a simple dilution step beforeanalysis. The whole detection process requires only about10min. The recovery ratio was75.1%to109.2%, whereas the intra-assay and inter-assay coefficients of variation were both<13.0, therebydemonstrating the high accuracy and precision of the assay. The proposed method was validated by itsgood correlation with GC-MS results. The proposed method was likewise used to detect2-DCB inground beef patties irradiated at0.5,1.0,3.0,5.0, and7.0kGy. The concentration of2-DCB linearlyincrease with the irradiation dose. The proposed biosensor has high sensitivity, high simplicity, rapidreaction times, low cost, and high sample throughput. Thus, the biosensor could be used as a feasiblequantitative screening method for the2-DCB analysis of irradiated lipid-containing foods and othersmall molecular compounds.