Fluorescence Detection Of Tetracycline In Foods By Magnetic Beads Based On Aptamer And DNA Tetrahedron

Tetracycline(TET)is a broad-spectrum antibiotic with the advantages of low price and good antibacterial performance.It is widely used in the treatment of animal diseases or feed additives.However,the abuse of TET will inevitably lead its residual in the human body through the biological chain,which causes some allergic reactions,nephrotoxicity,gastrointestinal dysfunction and other hazards to the human body.Therefore,the detection of TET has great significance to food safety supervision and protection of human health.Aptamer is a single-stranded DNA or RNA that can bind to the target molecule with high affinity.It has a wide range of applications in the fields of medical diagnosis,environmental monitoring and biological analysis.This work aims to synthesize aptamer-modified DNA tetrahedral nanostructures and assemble it on magnetic beads(MBs)to construct magnetic beads detection probes for realize simple,rapid and sensitive the TET detection.This work is divided into three parts:Section 1,aptamer-modified DNA tetrahedral nanostructures(TETapt-tet)and single-stranded aptamer(TETapt)were assembled on MBs,and a ROX-c DNA was designed as the fluorescent signal probe to hybridize with aptamer,then two magnetic beads detection probes,ROX-TETapt-tet MBs and ROX-TETapt MBs,were constructed.Next,the step was to compare the aptamer density,TET recognition efficiency and anti-interference ability of the two magnetic beads detection probes in complex media.The method of using DNA tetrahedrons immobilized on MBs could reduce the density of aptamers and improve the binding efficiency of aptamers for TET detection.The reaction time was 30 min.The detection results of the two magnetic beads detection probes in fetal bovine serum show that ROX-TETapt-tet MBs has stronger anti-interference ability and the detection signals in PBS buffer and fetal bovine serum were basically the same,however,the detection signal value of ROX-TETapt MBs in fetal bovine serum was only 55%of that in PBS buffer.Therefore,ROX-TETapt-tet MBs was selected to detect TET in subsequent experiments.Section 2,magnetic beads detection probes(ROX-TETapt-tet MBs)constructed based on aptamer-modified DNA tetrahedral nanostructures were used for the highly sensitive detection of TET in honey samples.When the detection system contains TET,TET would bind to the aptamer,causing the complementary strand ROX-c DNA to fall off the surface of the MBs and to release into the solution.Under the action of magnetic separation,the goal of quantitative detection of TET could be achieved by detecting the fluorescence of the supernatant.First,the ROX-TETapt-tet MBs probe was characterized by zeta potential and fluorescence spectra.Then,ROX-TETapt-tet MBs was used to detect the TET in the buffer.Under the optimal conditions,the concentration of TET had a good linear relationship with the fluorescence value of ROX-c DNA,the detection range was 0.1-50 ng m L^-1,the limit of detection and limit of quantification were as low as 6 and 20 pg m L^-1,respectively.ROX-TETapt-tet MBs could also be applied to the detection of TET in honey samples,and the recovery rate of standard addition was between 95.6%and 114.5%.Finally,a simple method will be established using TETapt-tet MBs for simultaneous detection of two antibiotics such as TET and kanamycin.Section 3,a fluorescence amplification detection method was established for the ultra-sensitive detection of TET in fish and honey based on DNA tetrahedron-immobilized magnetic beads detection probes(Primer-TETapt-tet MBs)combined with rolling circle amplification(RCA)reaction.Based on the experimental of the section 2,we designed a complementary DNA sequence of the aptamer into the primer that could initiate an RCA reaction(Primer-c DNA).When TET was contained in the detection system,the combination of TET and aptamer causes Primer-c DNA to be released into the solution and initiate RCA reaction to achieve the purpose of amplifying and detecting TET.Under optimal conditions,the linear range of was 0.001-10 ng m L^-1,and the detection limit was 0.724 pg m L^-1.At the same time,the method has been successfully applied to the detection of TET in fish and honey samples,the detection limits were 0.893 and 0.822 pg m L^-1,and the spiked recoveries were 96.1%-110.7%and 94.6%-107.5%,respectively.Those results were basically consistent with the results of the ELISA kit.Therefore,this method can achieve ultra-sensitive detection of TET in fish and honey.