Study On The Fluorescence Detection Method Of β-lactoglobulin In Food Based On Aptamer

β-lactoglobulin（β-Lg）,a type of whey protein in milk,is the main allergic protein in milk,which can cause allergic reactions such as skin urticaria,gastrointestinal vomiting and respiratory discomfort,and even cause anaphylactic shock in severe cases.Therefore,it is necessary to establish an effectiveβ-Lg detection method to protect public health.In this thesis,based on the specific recognition ofβ-Lg byβ-Lg aptamer,two simple and sensitiveβ-Lg fluorescence detection methods were established,and the methods were successfully applied to the determination ofβ-Lg content in real foods.The details of the study are as follows:In the first part,a fluorescence method based on tungsten disulfide（WS₂）nanosheets and FAM-labeledβ-Lg aptamer was established to detectβ-Lg.In this method,due to the van der Waals force between DNA bases and WS₂ nanosheets,FAM labeledβ-Lg aptamers were adsorbed on the surface of WS₂ nanosheets.The fluorescence resonance energy transfer（FRET）phenomenon occurred between FAM dye and WS₂ nanosheets,resulting in the fluorescence of FAM being quenched.When theβ-Lg was present,specific binding occurred between the aptamer and theβ-Lg,which induced the aptamer to form a more rigid conformation and weakened the van der Waals force between the aptamer and WS₂ nanosheets.Therefore,the FAM-labeled aptamer was released from the surface of the nanosheet and the fluorescence signal was restored.Under the optimal detection conditions,the linear detection range of the method was 0.1-100μg m L^-1,and the detection limit was 20.4 ng m L^-1.The method was further applied to the detection ofβ-Lg in milk and infant formula,and the results were consistent with the ELISA results with the relative standard deviation（RSD）between 2.6%and 3.9%.The recoveries of added standard in infant formula were 98.1%-103.5%.This method can be used for the sensitive detection ofβ-Lg in milk and infant formula.In the second part,a fluorescence signal amplification method based on G-quadruplex and exonuclease Ⅲ was established to detectβ-Lg.In this method,a recognition probe（HP1）containingβ-Lg aptamer sequence and a signal probe（HP2）containing G-quadruplex sequence were designed.In the absence ofβ-Lg,HP1 and HP2 were in a hairpin state,and they were independently stable and do not interfere with each other.HP2 was not cleaved by nucleic acid exonuclease Ⅲ.Whenβ-Lg was present,HP1 bound toβ-Lg and undergoed a conformational change.HP1 complements the prominent sticky end of HP2 to form a double-stranded structure,and exonuclease Ⅲ degraded the double-stranded structure from the 3’end and produced the G-quadruplex.The G-quadruplex forms the G-quadruplex DNAzyme with hemin,which can catalyze the reaction of H₂O₂ with o-phenylenediamine（OPD）to produce the fluorescent substance 2,3-diaminophenoxazine（DAP）.In addition,we had also developed a fluorescent test paper using this system to detectβ-Lg according to the change of fluorescence color.Finally,we successfully detected theβ-Lg content in actual food by using the photo function of smart phone and Image J software.Under the optimal detection conditions,the linear detection range ofβ-Lg was 0.01-1μg m L^-1 and 1-100μg m L^-1,and the detection limit was 0.58 ng m L^-1.This method was used for the determination ofβ-Lg in infant formula.The spiked recoveries ranged from 94.0%to 100.3%,which were consistent with the results of ELISA.The results indicate that this method can be used for sensitive and accurate detection ofβ-Lg in infant formula.