Construction And Application Of Lateral Flow Strip For Tobramycin Based On Aptamer And Platinum-decorated Gold Nanoparticles

At present,food safety issues caused by food-borne antibiotic residues have aroused the attention of the whole society.Countries all over the world have issued strict maximum residue limits for antibiotics in animal-derived products.Tobramycin has been widely used for the treatment of infections caused by gram-negative bacteria and other microorganisms.However,the over-use in clinical and animal medicine leads to the existence of residues in animal-derived foods,which has a huge potential threat to human health and even the entire ecological environment.Traditional detection methods for antibiotics are mostly limited by high requirements of equipment,professional operation,long analysis time,etc.They are not suitable for on-site applications.Therefore,it is necessary to establish rapid and sensitive method for on-site detection of tobramycin residues.In this thesis,a bifunctional lateral flow strip was constructed by using aptamer and novel nanomaterials with catalytic activity for the rapid and sensitive detection of tobramycin.In this paper,a dual-function lateral flow chromatography test strip was constructed using aptamers and new nanomaterials for the rapid and sensitive detection of tobramycin.The platinum-modified gold nanoparticles(Au@Pt NPs)with a core-shell structure retain almost all the characteristics of gold nanoparticles(AuNPs),and at the same time have the ultra-high peroxidase-like catalytic activity of the Pt shell.In the presence of the substrate 3,3',5,5'-tetramethylbenzidine(TMB)and H₂O₂,a blue product can be quickly formed.The blue signal produced by the catalytic reaction is more prominent than the inherent red intensity of AuNPs.During the construction process,the hybrid duplex of tobramycin-specific aptamer(Apt)and its complementary probe(cDNA)was fixed on the surface of Au@Pt NPs by sulfhydrylation at the 5' end of cDNA to form Au@Pt NPs-cDNA-Apt complex.When tobramycin is present in the test sample,Apt preferentially binds tobramycin and dehybridizes with cDNA.Au@Pt NPs-cDNA migrates with the liquid,and the free cDNA is further detected by the detection line(T line)and control line(C line)is captured by the fixed probes DNA1 and DNA2,causing Au@Pt NPs to accumulate on the T line and C line.Among them,DNA1 and DNA2 are partially complementary probes and fully complementary probes of cDNA,respectively.This test strip provides two different detection schemes:one is red only produced by the intrinsic color of AuNPs("low sensitivity"mode),and the other is more sensitive produced by Au@Pt NPs catalytic substrate Dark blue("High Sensitivity"mode).Firstly,Au@Pt NPs were prepared by a three-step synthesis method of seed-mediated growth,and their bifunctional properties were studied.Then,test the construction and assembly of the test strip,and after the probe design verification of the detection principle is passed,the key conditions that affect the test strip detection are further optimized.Finally,the detection performance of the test strip and the simulation of actual sample detection are investigated.The main findings are as follows:(1)The Au@Pt NPs with core-shell structure are prepared,with good dispersibility,uniform particle size,and a size of 40±2 nm,which has good retention of the plasma optical properties of the original AuNPs,and also has strong enzymatic catalytic properties;(2)Optimal testing conditions for the test strips:the concentration of T-line probe DNA1is 5?mol·L^-1,the concentration of C-line probe DNA2 is 7.5?mol·L^-1,the fixed amount of gold standard is 35?L,NC membrane type is Vivid 170,the flow buffer is 1%BSA+15×SSC,and the reaction time is 5 min;(3)The test strip has passed the sensitive inspection,and the naked eye detection limits in the two modes are 60 nmol L^-1 and 5 nmol L^-1 respectively.The colloidal gold scanner or Image J software is used for quantitative analysis,and the detection limits of the two modes are calculated(LOD)are respectively 0.09 nmol L^-1 and 0.02 nmol L^-1;(4)The developed test strip has good detection specificity and storage stability,and has been successfully applied to the detection of tobramycin in different artificial simulated samples,and the detection limit is lower than the maximum residue limit stipulated by the EU.This thesis provides a highly sensitive,rapid and economical solution for the on-site detection of tobramycin residue.The test strip can run in two modes to realize on-demand adjustment of detection performance,showing better universality and broader application prospects.