New Antibiotic Optical-biosensing Strategies Based On The Functional Nucleic Acid Recognition And Signal Amplification

Recently,antibiotics,one category of important antibacterial drug,have been widely used in many fields such as agriculture,animal husbandry and human medical and health fields.However,the abuse of antibiotics and their residual enrichment in foods have brought serious threat to public health.At present,the traditional antibiotic detection methods often involve complicated sample pretreatment,skillful manipulation and expensive equipment requirement,which restricts their applications only suitable for laboratory analysis.In contrast,optical biosensors constructed on various functional nucleic acids such as aptamers have attracted considerable interest of analytical scientists because they can specifically convert the target biorecognition information into easily analytical and measurable optical signals.However,the content of antibiotics existing in real samples is usually very low.Therefore,how to construct the analytical method to realize the highly sensitive and on-site detection of target analytes with good reliability is of great significant.Fortunately,the programmability of functional nucleic acids enables their freely switching in three different states such as the free state,the target binding state and the double-stranded DNA state.This property can be easily combined with a large variety of isothermal nucleic acid signal amplification techniques to significantly improve the sensitivity of detection methods.Therefore,this thesis combines the functional nucleic acid recognition-based optical biosensing with several signal amplification strategies of nucleases,DNA nanostructure self-assembly and nanomaterials to design and construct two highly selective and sensitive optical biosensing methods for antibiotic assays in real samples,which are presented as follows:1.Dually Exo III-catalytic recycling amplification to construct a novel ratiometric fluorescence biosensing method for bleomycin assayHerein we report a novel ratiometric fluorescence biosensing method for BLM assays by utilizing two target-sensitive fluorescent probes to produce reverse signal response.One signal arose from the target recognition-triggered and exonuclease III（Exo III）-catalytic release of a G-quadruplex sequence.This could cause the recombination of the G-quadruplex with thioflavin T（Th T）to realize the“signal-on”fluorescence output.The other signal arose from the target recognition and Exo III-catalytic reaction-induced DNA hybridization of a ROX fluorophore-labeled strand with its quencher-labeled strand.This proximity quenching effect could realize the“signal-off”fluorescence output of the method.Meanwhile,the dually Exo III-catalytic recycling greatly amplified the Th T and ROX-based fluorescence response.Through the measurement of the ratio of the two reverse responses,a novel ratiometric fluorescence biosensing method was successfully constructed for the homogeneous assay of BLM in a very wide linear range from 50 p M to 5μM,with a very low detection limit of 15.8 p M.The excellent analytical performance,convenient manipulation,and low assay cost of the method determine its high potential for future applications.2.Amplified assembly of a hyperbranched DNA nanostructure with a dual cascade nucleic acid recycling strategy to develop a novel multicolor visual antibiotic biosensing methodHerein we designed a target-triggered dual cascade nucleic acid recycling strategy to amplify the assembly of a hyperbranched DNA nanostructure and developed a novel kanamycin colorimetric biosensing method.The first cycle arising from the aptamer recognition-triggered strand displacement reaction and another cascade cycle constructed on the catalytic reaction of two nucleases could release an output DNA to trigger the assembly of the DNA nanostructure.Based on the high capture of alkaline phosphatase by this DNA nanostructure to induce the localized surface plasmon resonance change of gold nanobipyramids（Au NBPs）,an ultrasensitive colorimetric signal transduction strategy was developed.Through the measurement of the shift of the characteristic absorption wavelength of Au NBPs,a very wide linear range from10 fg m L^-1 to 1 ng m L^-1 and a very low detection limit of 1.4 fg m L^-1 were obtained.Meanwhile,the obvious multicolor change of Au NBPs could be used for the visual semi-quantitative analysis of Kana residues.These determine the great potential of the method for future applications.