Research On Sensitive Detection Of Kanamycin Based On Aptamer Recognition Combined With Signal Amplification Technology

Due to the abuse of antibiotics in the production of agriculture and animal husbandry,its negative impact on food has been widely concerned.Therefore,there is an urgent need to develop a fast,convenient,high sensitivity and high selectivity for the detection of antibiotics in complex foods.Kanamycin is a commonly used broad-spectrum aminoglycoside antibiotic,which is widely used in agriculture,animal husbandry and aquaculture.It is also often added to feed to promote the growth and development of animals.However,kanamycin residues in animal products will cause great harm to human health after long-term consumption,such as ototoxicity,nephrotoxicity and other side effects.Many countries and institutions,such as China,the European Union,the U.S.FDA and Japan,have made clear regulations on the residual limit of kanamycin in milk.Therefore,it is of great significance to design a simple and efficient aptamer sensor and establish a simple,feasible,rapid and accurate method for the detection of kanamycin in food to ensure food safety and human health.In this paper,the optical detection method based on aptamer sensor was established to detect kanamycin.The main research contents and results can be summarized into the following two aspects:1.Aptamer biorecognition-triggered hairpin switch and Nicking Enzyme assisted signal amplification for ultrasensitive colorimetric bioassay of kanamycin.A colorimetric aptasensing strategy for detection of kanamycin was designed based on aptamer biorecognition and signal amplification assisted by nicking enzyme.The aptamer of kanamycin was designed to be contained in the metastable state hairpin DNA.The target DNA as recycling DNA was located in the loop of hairpin DNA.The signal probe labeled with magnetic beads(MB)and platinum nanoparticles(PtNPs)was employed for colorimetric detection owing to its carrying the recognition sequence and cleavage site for the nicking enzyme.In the presence of kanamycin,the formation of hairpin probe was induced from metastable hairpin shape to stretch form,thus prompting the hybridization between the target DNA and signal probe.Under the function of the nicking enzyme,the signal probe was cleaved;the PtNPs modified in signal probe were released.After magnetic separation,a significant blue color was obtained due to the reaction of 3,3',5,5'-tetramethylbenzidine(TMB)and H2O2 catalyzed by numerous PtNPs accumulated through continuous enzyme cleavage.The aptasensor exhibited good selectivity and sensitivity for kanamycin in milk with a detection limit as low as 0.2 pg mL-1.In addition,the proposed assay is potentially to be extended for other antibiotics detection in foods by adapting the corresponding aptamer sequence.2.DNAzyme-powered DNA Walking Machine for Ultrasensitive fluorescence aptasensing of kanamycin.In this study,a fluorescence analysis method was developed to detect kanamycin by using aptamer's specific recognition of target and combined with DNAzyme powered DNA walker signal amplification method,which can be used to detect kanamycin in actual milk samples.In this experimental protocol,the complementary DNA(C-DNA)contained a rA cleavage point(RNA A bases)with FAM labelled at the 3' end and biotin modified 5' end,and was linked to magnetic beads modified streptavidin by the high affinity of biotin and streptavidin.Then,the free aptamer DNA in solution was captured on magnetic beads through hybridization between the C-DNA and the aptamer DNA.The aptamer left the magnetic beads in the presence of kanamycin because of specific binding between the target and the aptamer.Then the free walker DNA in the solution hybridized with the complementary so that it was immobilized to the magnetic beads.After adding Pb2+,the rA cleavage point of the C-DNA was identified and cut by Pb2+-specific DNAzymes,releasing a short DNA fragment labelled with FAM into the solution.The walker DNA then moved to the next C-DNA chain and repeated the above cleavage process.During this movement process,walker DNA acted as the enzyme and continuously released the short DNA fragment.Finally,the short DNA fragment labelled with FAM in the supernatant displayed a strong fluorescence signal after separating the magnetic beads.These results indicate that kanamycin can be quantitatively detected according to the change of the fluorescence intensity.Under optimized conditions,from 0.2 pg·mL-1 to 2?g ·mL-1,the logarithmic value of kanamycin concentration has a good linear relationship with the fluorescence intensity of the system.The LOD of detection is 0.14 pg mL-1(S/N=3).This method can also be extended to the analysis and detection of other analytes by changing the corresponding aptamer sequence of the target,thereby implementing food safety monitoring.