| ObjectiveBeclomethasone(BEC)is an effective glucocorticoid that is predominantly used for the treatment of allergic asthma,eczema,allergic dermatitis,and various pruritus.However,the long-term use of BEC can cause a variety of side effects such as osteoporosis and skin induration,while BEC can be illegally added to some health products by some unscrupulous merchants,including Traditional Chinese Medicine(TCM)and cosmeticsy,which seriously damages the interests of consumers.Consequently,there is a significant need to develop methods for illegally added beclomethasone detection rapidly.Aptamers are short single-stranded nucleic acid that have high affinity and specificity for their targets which have a variety of range.Aptamers can form diverse structures for binding targets,and they can be engineered into aptasensors which are widely used in drug detection.In this study,the systematic evolution of exponential enrichment ligands(SELEX)technology was used to screen out aptamers with high affinity and specificity for beclomethasone,which can provide research materials for beclomethasonedetection;The screened beclomethasone aptamer was engineered into a fluorescence detection device based on quantum dots,which is used for beclomethasone detectionrapidly,to expand the application of aptamers in drug detection;The interaction between aptamer and beclomethasone was also studied by high resolution Fourier transform ion cyclotron resonance mass spectrometry(FT-MS)and molecular docking technology,which provide reliable research data for the application of beclomethasone aptamer.Methods1.Screening aptamers for beclomethasone:The aptamer of beclomethasone was screened out from random library by library immobilization.The enriched library was sequencing by high-throughput sequencing,and the sequences of aptamers were analysised through homology analysis andstructure prediction by ClustalW and M-fold respectively.The affinity and specificity of the aptamer candidates were characterized by fluorescence spectroscopy.2.An aptasensor for beclomethasonedetection:Using beclomethasone aptamer as recognition probe and quantum dots as output signal,amino-modified beclomethasone aptamer is fixed on the surface of carboxyl-modified magnetic beads through chemical bonds,and carboxyl-modified quantum dots are combined with single-chain binding protein(SSB)through chemical bonds.Based on the nonspecific binding between aptamer and SSB,the aptasensor was constructured.In theabsence of beclomethasone,beclomethasone competes with SSB for binding aptamer,and the complex of SSB and quantum dot was free in solution,and the signal intensity of quantum dots in solution was detected by fluorescence spectrometer.3.Study on the interaction between beclomethasone and aptamer:Under the optimized conditions of high-resolution mass spectrometry parameters,the affinity and the ratio of chemical binding stoichiometrybetween beclomethasone and aptamer were detected by the negative ion scanning mode with the electrospray ion source;Discovery Studio and Schrodinger molecular docking software were used to simulate the interaction between aptamer and beclomethasone.To verify their interaction,The traditional method of ultra-micro UV was also used to detect the change of absorption wavelength before and after binding of aptamer,and the ultraviolet absorption wavelength and dissociation constant(Kd)of the complex of aptamer and beclometasone were obtained.Results1.Screening aptamers for beclomethasone:After 14 rounds of SELEX screening,the enrichment library of 12th round was obtained,and 47 aptamer candidate sequences were obtained by sequence analysis.The affinity of 47 aptamer candidate sequences was initially screened by fluorescence spectrometry,and 6 aptamer candidate sequences with the highest affinity for beclomethasone were obtained.The 6 aptamer candidate sequences were characterized,and it was found that BEC-6 had the highest affinity and specificity for beclomethasone,and its Kd value was 0.15±0.02 μMwith a limit of detection(LOD)was 0.1μM.2.An aptasensor for beclomethasonedetection:Under the optimized conditions of the aptasensor,the detection linear range was 0.1～10 μM,and the LODwas 0.1 μM.The average recovery of beclomethasone in real samples was 81.72～91.84%.3.Study on the interaction between beclomethasone and aptamer:Under the optimized conditions of FT-MS,the complex of aptamer and beclomethasone was detected by the negative ion scanning mode with the electrospray ion source,and it was found that the most of the compounds were-8-valent ions.Before saturation,the ratio of chemical binding stoichiometry of the aptamer to beclomethasone was 1:1,and its Kd was 1.01±0.23μM.According to the calculation and simulation of molecular docking software,the ratio of chemical binding stoichiometry between the aptamer and beclomethasone was 1:1,and the binding free energy of two was-24 kcal/mol,and they were mainly combined by hydrogen bonding force.Moreover,Based on the results of the Ultra-micro UV,the Kd of the beclomethasone-aptamer complex was 1.67 ± 0.35 μM,which is similar to that of high resolution FT-MS.Conclusion1.The aptamer BEC-6 screened by SELEX technology has high affinity and specificity for beclomethasone.2.The established aptasensor has the advantages of simple operation,rapid detection,good specificity and low cost,which can realize qualitative and semi-quantitative detection of illegally added beclomethasone.Moreover,the established aptasensor can be used for detection of real samples of illegally added beclomethasone.3.High-resolution FT-MS detection and molecular docking simulation show that the aptamer and beclomethasone had the same ratio of chemical binding stoichiometry before saturation,as well as high affinity and stable binding.4.In this paper,the aptamers of beclomethasone were screened based on SELEX,and an aptasensor was established for beclomethasonedetection.The interaction between aptamer and beclomethasone was studied,which expanded the application of aptamer technology in the field of rapid detection of illegal drug addition.This study hasrealized the innovation of application of aptamer technology to a certain extent. |
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