Engineering Construction Of High Sensitivity Dexamethasone Aptamer Colorimetric Sensor Based On Truncation And Mutation Technology

Objective: Residual and illegal addition of Dexamethasone(DEX)in food has received widespread attention over the past few decades.Long-term intake of DEX will have a strong endocrine-disrupting effect on the human body,and the analytical method for determining the residual amount of dexamethasone is based on the traditional instrumental analysis method of liquid chromatography(HPLC)and mass spectrometry(MS)and immunoassay method.These methods are costly,time-consuming and require professional operators.Therefore,it is urgent to develop high sensitivity and fast on-site detection methods.Aptamer is a short oligonucleotide chain with high specificity,low cost and good biocompatibility.In this study:(1)A colorimetric sensor based on aptamers and gold nanoparticles(Au NPs)was designed to detect DEX in milk and glucosamine.(2)The binding mechanism of the aptamer and DEX was deeply studied,and the sequence of the aptamer was further optimized to improve the affinity of the aptamer to DEX.Methods:1.Through the synthesis and characterization of Au NPs and the optimization of various sensing conditions,a colorimetric sensor was constructed,and the detection limit,specificity and spiking recovery of the sensor were investigated.2.Molecular docking and molecular dynamics simulation are used to explore the intermolecular interaction and structure of the complex,and based on this,the original aptamer is truncated and mutated.Results: 1.The absorbance ratio of the established colorimetric sensor of Au NPs increased linearly with the concentration of DEX in the range of 10-350 nmol/m L(r2=0.997),the limit of detection(LOD)was 0.5 nmol/m L,and the spiked recovery was 93.6 to 117%.2.By truncating the redundant bases at both ends of the 59 nt long dexamethasone original aptamer sequence that are not involved in DEX-specific binding,and then introducing base mutations into the truncated sequence,the 33 nt long optimized aptamer Apt-M13 with Gquadruplex structure was obtained.The dissociation constant(Kd)of Apt-M13 was determined to be 200 n M by fluorescence method based on graphene oxide(GO).When the optimized Apt-M13 is used for the colorimetric aptamer sensor based on gold nanoparticles,the LOD is 3.2 times lower than the original aptamer colorimetric sensor described in the previous work,and the anti-interference ability of DEX analogues is further improved.Conclusion: The aptamer-based sensor established in this paper has the advantages of high-cost effectiveness and good selectivity,and has great potential in the detection of residual and illegal addition of DEX.In addition,the aptamer truncation strategy effectively improves the sensitivity of the aptamer to DEX,and the introduction of base mutation further improves the affinity and selectivity of the aptamer to DEX.The method of aptamer optimization proposed in this paper is expected to become a general strategy for optimizing various aptamer sequences.