Study On The Nuclease-signal Amplification Based Homogeneous Fluorescence Biosensing For Antibiotic Detection

Recently,the residual pollution caused by abuse of antibiotics has brought serious negative effects for human health and ecological environment.Therefore,the accurate determination of low-abundance antibiotics in complex matrices is of great significance for food security and public health.Compared with conventional analytial methods,aptasensors often possess excellent analytical performances such as excellent selectivity,high sensitivity and rapid analysis speed.Meanwhile,the biorecognition reaction between aptamers and their target analytes can not only cause the conformation changes to related nucleic acids but also induce corresponding signal changes of the signal groups modified at their specific positions.Based on this and the combination of different nuclease-assisted signal amplification strategies,a variety of homogeneous biosensing methods can be developed to effectively avoid the complicated manipulation drawback involving in traditional heterogeneous bioassays.Therefore,this thesis combines the aptamer-based homogeneous bioassay with the nuclease-assisted nucleic acid signal amplification strategies to carry out two researches on the development of novel homogeneous fluorescent biosensing methods for ultrasensitive and accurate detection of antibiotic residues,which are liste as follows:1.Fe₃O₄@polydopamine and Exo Ⅲ-assisted homogeneous biosensing of kanamycinBased on the Fe₃O₄@polydopamine（Fe₃O₄@PDA）and exonuclease Ⅲ（Exo Ⅲ） assisted signal amplification technology,a novel homogeneous fluorescence biosensor has been successfully developed for ultrasensitive detection of kanamycin（Kana）.A hairpin DNA（HP）containing Kana aptamer sequence was first designed for the highly specific biorecognition of the target analyte.Due to the aptamer biorecognition-induced structure change of HP and the high-effectively catalyzed reaction of Exo Ⅲ,a large amount of FAM fluorophore labeled at the 3-terminus of the fluorescent DNA probe（S2）were also released.It realizes the successful fluorescence signal transduction of this method.During the homogeneous reaction process,the Exo Ⅲ assisted dual recycling significantly amplified the fluorescence signal output.Moreover,the excessive probes were easily adsorbed and separated by the Fe₃O₄@PDA nanocomposite,which decreased the background signal and increased the signal-to-noise ratio.These strategies result in the excellent analytical performance of the method,including a very low detection limit of 0.023 pg m L^-1 and a very wide linear range of six orders of magnitude.In addition,this method has convenient operation,excellent selectivity,repeatability and satisfactory reliability,and does not involve the design and utilization of complicated DNA sequences.Thus,it exhibits a promising prospect for practical applications.2.Nuclease-assisted DNA walking for homogeneous biosensing of kanamycinBy combination of the biorecognition reaction at a nanobiosensing platform with the nuclease and DNA Walker-assisted signal amplification,this work successfully develops a novel homogeneous fluorescence biosensing method for the ultrasensitive detection of Kana.The biosensing platform is prepared through the surface modification of gold nanoparticles（Au NPs）by the double-stranded DNA（ds DNA）formed by the hybridization of two FAM-labeled oligonucleotide chains of S1 and S2.In the presence of the target analyte of Kana,the highly specific biorecognition reaction between Kana and its aptamer chain of S1 can form a S1/Kana biocomplex and thus trigger the enzymatic reaction of Exo I to catalyze cleveage of S1.This can cause the release of Kana for target recycling and the same release of the FAM labels at the 5′end of S1.Meanwhile,the S2 chain exposed on the surface of Au NPs can be used to induce its hybridization reaction with the quadrupedal DNA walker prepared on the basis of streptavidin.This will trigger the restriction endonuclease of Nb.Bbv CI-assisted DNA walking to cleave the release of the FAM fluorescents labeled at the 5-terminus of S2.It thus realizes the successful fluorescence signal transduction of the method.Due to Exo I-assisted target recycling and the Nb.Bbv CI-assisted three-dimensional quadrupedal DNA walking for signal amplification,this method exhibits a very high analytical sensitivity.Under optimal conditions,this method can be used for the ultrasensitive and accurate detection of Kana at a range from 0.1 pg m L^-1 to 100 ng m L^-1 with a very low detection limit down to 15 fg m L^-1.Moreover,As Au NPs can well quench the fluorescence signal of the FAM labels of S1/S2 which are modified on the Au NP surfaces,all the reactions involved in this method can be one-step carried out in a homogeneous system,and no separation operation is required during the whole detection process.So this method has excellent operability and repeatability.Based on the outstanding performance advantages mentioned above,this method shows a promising application prospect in the practical analysis field.