Research On Detection Method Of Antibiotic Residues In Milk Based On Fluorescent Aptasensor

Aminoglycoside antibiotics(AGs)are commonly used to treat bacterial infections in dairy cows,but their misuse can result in excessive residues in milk.This can cause allergic reactions and disrupt the balance of intestinal flora in humans.Therefore,it is crucial to develop a detection method that is fast,simple,and efficient.Compared to traditional AGs detection methods,the fluorescent aptasensor approach offers several advantages,including low cost,high sensitivity,and rapidity.Silver nanoclusters(DNA-Ag NCs)were synthesized by using DNA templates containing AGs aptamers,which have excellent fluorescence properties and can specifically recognize AGs.Moreover,fluorescent aptasensors for sensitive AG detection can be constructed using fluorescence quenching materials and DNA-Ag NCs.In this study,DNA-Ag NCs were synthesized in a region with minimal interference from the milk matrix using a Kanamycin(KAN)aptamer sequence template.This resulted in reduced background interference and enabled the detection of KAN.The study also examined the effect of guanine(G)on hairpin DNA-Ag NCs and the impact of palladium shell thickness on the quenching efficiency of core-shell gold-palladium nanoparticles(Au@Pd NPs)for constructing a fluorescent probe for detecting Streptomycin(STR).Furthermore,a ratiometric fluorescent sensor was developed using ZIF-8-encapsulated palladium nanoclusters(ZIF-8@Pd NCs)as the reference signal and DNA-Ag NCs containing Tobramycin(TOB)aptamers as the response signal.By using 3D printing to develop supporting detection equipment,it became possible to achieve visual rapid detection of TOB based on changes in fluorescent color.The specific work is as follows:(1)To detect KAN in milk and reduce interference from the milk background matrix,we constructed an aptasensor based on DNA-Ag NCs that emits red fluorescence in the visible wavelength range.We used a DNA template strand containing KAN aptamer to synthesize DNA-Ag NCs that can emit red fluorescence in a region with minimal interference.The strong penetrating red fluorescence can further reduce background interference.A fluorescence sensing method was established using fluorescence resonance energy transfer(FRET)that occurs between DNA-Ag NCs and gold nanoparticles(Au NPs).The electrostatic interaction between DNA-Ag NCs and Au NPs produces FRET,which results in the quenching of DNA-Ag NCs fluorescence.The aptamer and KAN can specifically bind,leading to the weakening of the electrostatic interaction between DNA-Ag NCs and Au NPs and the recovery of DNA-Ag NCs fluorescence.Quantitative detection of KAN can be achieved by measuring the change in fluorescence signal of DNA-Ag NCs.The sensor demonstrated a detection limit as low as 22.6 n M for KAN,with satisfactory specificity and sensitivity.(2)A fluorescent aptasensor for detecting STR was constructed based on novel G base-regulated hairpin DNA-Ag NCs and Au@Pd NPs.By adding different numbers of G bases in the hairpin loop,the red shift of emission wavelength and the increase of fluorescence intensity can be realized.Au@Pd NPs were prepared by seed growth method and deposition method,and the Au@Pd NPs composite not only solved the problem of weak quenching ability of palladium nanoparticles(Pd NPs)in the long wavelength range but also effectively expanded the fluorescence quenching range of Au NPs.The designed sensor has a good response to STR in the range of 50-1250 n M,with a detection limit of18.7 n M.In addition,the sensor has been successfully applied to STR detection in milk,indicating its promising application in animal-derived food detection.(3)A novel ratiometric fluorescence visualization aptasensor was developed to detect TOB,utilizing ZIF-8@Pd NCs as the reference signal for emitting blue fluorescence and DNA-Ag NCs containing TOB aptamers as response signal for emitting yellow fluorescence.The sensor was coupled with 3D printing technology to create supporting equipment for TOB detection.In the absence of TOB,DNA-Ag NCs and ZIF-8@Pd NCs combined via π-π stacking,which quenched the fluorescence of DNA-Ag NCs.However,when TOB was present,it specifically bound to the aptamer,weakening the π-π stacking interaction between ZIF-8@Pd NCs and the aptamer,resulting in the desorption of DNA-Ag NCs from the ZIF-8 surface and recovery of their fluorescence.The developed sensor has a linear range from 50 n M to 500 n M,with a detection limit of 15.3 n M.Upon exposure to UV light,the fluorescent color changes visibly from blue to yellow with increasing TOB concentration.An instrument identification device was also created using 3D printing technology,ultraviolet lamp beads,and RGB identification components to provide a fast reading of TOB concentration.By programming with Python and establishing an RGB standard curve,direct reading of TOB concentration can be achieved.This portable,rapid detection equipment offers an economical,efficient,reliable,and time-saving method for detecting TOB.