Study On The Construction Of An Aptamer-based Kanamycin Assay And Its Application

Kanamycin is an aminoglycoside antibiotic, which has been widely used as anantimicrobial drug. It shows particular inhibitory activity against gram-negative bacteria, andthe inhibition appears to be strong and durable. However, when it is overcommitted, residualkanamycin found in animal-derived foods may lead to the damage to human health. Therefore,to establish fast, sensitive and efficient methods for the detection of kanamycin residues infood is of significant importance in food safety control. Here we established two methods forkanamycin detection based on kanamycin-specific aptamer, one belongs to UV-visiblespectroscopy and the other is an electrochemical method.In UV-visible spectrophotometric analysis, two types of functionalized goldnanoparticles (AuNPs) were firstly synthesized by self-assembly of thiol-modifiedsingle-stranded DNAs (ssDNAs) which are complementary to the5’ terminus and3’ terminussequences of kanamycin-specific aptamer respectively. As the kanamycin aptamer is mixedwith these AuNPs, the aggregate of AuNPs forms due to the hybridization of aptamer with thecomplementary ssDNAs on the surface of AuNPs. However, in the presence of differentconcentrations of kanamycin, it binds with the aptamer specifically and competitively, whichleads to the disaggregation of AuNPs aggregate, and the change in UV-visible absorptionspectrum of AuNPs. The absorbance at527nm was utilized to detect the concentration ofkanamycin. The detection range of this method is within1-500nmol·L-1, with the detectionlimit of1nmol·L-1.An electrochemical method to detect kanamycin was also established. In this method, athio-modified kanamycin-specific aptamer covalently bound to the gold electrode surfacethrough self-assembly. The optimum concentration of the aptamer was1μmol·L-1. Then themodified electrode was immersed in kanamycin solution and the optimum incubation timewas30min. The conformational changed of the aptamer during its specifically binding withkanamycin leads to the change in electrochemical resistance of electrode surface. By using[Fe(CN6)3-/4-] as probe, the current changes in square wave voltammetry were employed todetect the concentration of kanamycin. This method can detect kanamycin in the range of0.2-2000nmol·L-1.The accuracy of the two methods was verified by comparing with widely used HPLCmethod. Different concentrations of kanamycin solution were prepared, and then analyzed bythe UV-visible spectroscopy, electrochemical method, and HPLC. To estimate the specificityof two mehods streptomycin, gentamicin, neomycin, tetracycline, and ultra pure water wereused as sample instead of kanamycin. The results showed that the two methods have highaccuracy and high specificity.Furthermore, the two methods mentioned above were utilized to detect kanamycin inmilk sample. The pretreatment conditions of the milk samples are as follows:20%trichoroacetic acid to adjust pH to4.6, precipitation at45oC for10min, centrifugation at10000r·min-1for25min and filtration with0.22μm filter membrane. The recovery rate ofkanamycin was94.43%. In electrochemical method, the milk samples were directly underwent detection after dilution for5times. The result showed the two methods canperform well in the detection of milk samples, therefore have great application prospects inthe detection of kanamycin residue in food samples.