Detection Of Antibiotics In Milk By Surface Enhanced Raman Spectroscopy Based On Functional AgNPs Substrate

Antibiotics are widely used as antimicrobial drugs for dairy disease treatment.The irrational use of antibiotics can cause excessive residues in milk and threaten human health.Therefore,there is an urgent need to establish a rapid and reliable method to detect trace amounts of antibiotics in milk.Surface-enhanced Raman spectroscopy（SERS）is a sensitive and efficient tool for detection and analysis,and the key to SERS technology is the development of suitable SERS substrates.Compared with conventional Ag NPs substrates,functionalized Ag NPs substrates can significantly improve the sensitivity,stability and specificity of the detection,which in turn is important for the detection of trace antibiotics in milk.In this study,three antibiotics in milk were used as examples.Based on the nature and structure of antibiotic molecules,suitable functionalized silver nanosubstrates were developed:β-CD-Ag NPs,Na Cl and Mg SO₄ two-step functionalized Ag NPs and DNA aptamer functionalized Ag NPs,for the SERS detection of norfloxacin,amoxicillin and gentamicin,respectively.The details are as follows:1.A highly sensitive method for detection of norfloxacin（NFX）was developed based onβ-CD-Ag NPs SERS substrate.The unique spatial size and hydrophilicity ofβ-CD on the surface of Ag NPs could selectively capture the target molecule NFX through some weak interactions,including hydrogen-bond interaction,electrostatic interaction,etc.The interactions were characterized by the UV-Vis absorption spectroscopy,fluorescence spectroscopy,Zeta potential and DLS.The Raman signal of NFX is largely enhanced when anchored byβ-CD on the surface of Ag NPs.Through a series of experiments,the limit of detection（LOD）in standard solution and spiked milk were calculated to be 3.214 pmol/L and5.327 nmol/L.For milk sample,the recovery was 101.30%to 104.01%with the relative standard deviation（RSD）from 2.986%to 9.136%.To sum up,this developed SERS strategy is sensitive and specific to detect norfloxacin,it has practical application value and prospects.2.An efficient and sensitive amoxicillin（AMX）identification method based on a two-step functionalized SERS substrate:Ag NPs-Na Cl-analyte-Mg SO₄ was designed.Na Cl increases the probability of electron transfer through activation and Mg SO₄ increases the likelihood of local surface plasmon resonance through aggregation.Therefore,the significantly enhanced Raman signal of AMX can be directly identified.These mentioned CE and EM mechanisms were verified by UV-Vis absorption spectroscopy,SEM,Zeta potential,dynamic light scattering and density functional theory calculations,revealing the interaction between substrate and AMX.After condition optimization and quantitative detection experiments,the limits of detection of the standard solution and milk were 0.025 n M and 0.30 n M,respectively.In milk assays,recovery rate ranged from 88.02 to 106.73%,with RSD of 0.737 to 2.343%.In conclusion,the simple,reliable and sensitive SERS method proposed in this study can realize the quantification detection of AMX in milk.3.The detection method of gentamicin（GEN）in milk based on DNA aptamer-functionalized Ag NPs SERS substrates was developed.4-MBA is fixed on Ag NPs as a Raman signal molecule,and gentamicin DNA aptamer is modified on the surface of Ag NPs.DNA will increase the distance between Ag NPs and weaken the SERS signal of 4-MBA.When the target analyte gentamicin is present,DNA and gentamicin are specifically bound due to base pairing,DNA leaves the Ag NPs surface,the Raman"hot spot"is released,and the SERS intensity is restored.And the recovery degree of intensity is positively correlated with the concentration of gentamicin to be measured.The feasibility and anti-interference of the"weakening-recovery"detection scheme were verified by SERS spectral analysis,Zate potential and DLS analysis.By detecting the change of SERS intensity of signal molecule 4-MBA,the SERS spectrum experiment established the curve equation between gentamicin concentration and SERS intensity of signal molecule,and realized the quantitative detection of gentamicin The minimum detection concentrations were 10^-11 M and 10^-10 M in standard solution and milk,respectively.And the R² was 0.994 and 0.987,respectively.For milk samples,the recoveries were 101.95-108.28%with RSDs of 4.597-8.083%.Therefore,the method is sensitive,specific and of practical significance for the detection of trace gentamicin in milk.