DNA Nano-Assembly Mediated Multi-Signal Amplification For Ultrasensitive Biosensing Of Antibiotics

Antibiotic residues in animal-derived foods such as eggs,milk and meat can enter human bodies through the food chain.When the antibiotic residues accumulate to a certain level in human bodies,it will cause drug toxicity and even drug resistance,which will cause serious threats to the public health and safety.Therefore,it is of great significance to develop the analytical methods which can be used for the ultrasensitive,highly specific and accurate detection of antibiotic residues in animal-derived foods.Compared with traditional methods,the biosensors constructed on the nucleic acids with good chemical stability,easy synthesis,and high programmability often have the unique advantages such as high sensitivity,low sample consumption,and easy integration,miniaturization and intelligentialization.Thus,it has a good application prospect for the convenient and accurate detection of antibiotics in complex matrices.In addition,the nucleic acid structure changes caused by the highly specific biorecognition of aptamers toward targeting analytes also provide the possibility for the DNA nano-assembly and the highly efficient nuclease-catalytic reactions.These thereby provide more new ideas for the design and construction of various nucleic acid signal amplification-based ultrasensitive biosensing strategies.In recent years,how to combine various nucleic acid signal amplification technologies with highly sensitive optical/electrical signal transduction strategies to develop novel biosensing methods with excellent performances has become the core issue in the analytical science field.Therefore,by using the broad-spectrum antibiotic of kanamycin（Kana）as the model analyte,this thesis employs the highly specific aptamer-biorecognition and the nucleic acid signal amplification such as DNA nano-assembly mediated exonuclease-catalyzed reaction,hybrid chain reaction（HCR）,catalytic hairpin self-assembly（CHA）to design and construct two novel ultrasensitive optical and electrical biosensing strategies,which are listed as follows:1.Homogeneous biorecognition-induced assembly of DNA nanostructures for ultrasensitive electrochemical detection of kanamycin antibioticBy the employment of a homogeneous biorecognition reaction to induce the assembled formation of DNA nanostructures at an electrode,herein we develop a novel biosensing method for the ultrasensitive electrochemical detection of kanamycin（Kana）antibiotic.A DNA complex consisting of Kana-aptamer and a hairpin DNA with an exposed 3′-end was first designed for conducting the homogeneous reaction with Kana in the presence of exonuclease I（Exo I）.It resulted in the production of a hairpin DNA with a blunt terminus,which could be used for triggering the assembled formation of a layer of DNA nanostructures with orderly distribution and abundant biotin sites at a gold electrode.Then,high-content methylene blue and horseradish peroxidase（HRP）-functionalized gold nanotags would be captured onto the electrode to realize the electrocatalytic signal transduction.Due to the Exo I and HRP-assisted dual signal amplification,a very low detection limit of 9.1 fg m L^-1 was obtained for the Kana assay along with a very wide linear range over five-order of magnitude.Considering the excellent performance of the method,it exhibits a promising prospect for practical applications.2.Dual CHA-mediated assembly of a tripedal DNA walker for homogeneous dual-mode biosensing of kanamycinBy utilizing the specific single-target biorecognition-triggered dual CHA reaction to mediate the assembly of a Mg²⁺-dependent DNAzymes（MNAzymes）-functionalized tripod DNA walker,this work successfully develops a homogeneous biosensing method which can be used for the highly sensitive dual-mode detection of Kana.First,the specific biorecognition between Kana and its aptamer sequence designed in a hairpin DNA can trigger the cascade CHA reaction of three delicately designed hairpin DNAs to successfully form a large number of MNAzyme-functionalized tripedal DNA walkers.When this DNA walker is applied to the magnetic bead（MBs）platform modified by the probe hairpin DNA（HP）,the catalytic reaction of MNAzymes can produce a large number of triggering chains to initiate another cascade CHA reaction and thus form more tripedal DNA walkers.At the same time,the SP chain remaining on the MBs surface can trigger the HCR assembly to generate the double-stranded DNA structures composed of a large number of G4-DNAzymes with unique peroxidase-like catalytic activity.With the aid of the highly sensitive catalytic reaction of G4-DNAzymes,the colorimetric and photoelectrochemical dual-mode signal transduction can be thus realized for this method.Based on the dual CHA-mediated assembly of the tripedal DNA walker and the synergistic signal amplification of HCR,this method is not only very simple in operation but also has a very high analytical sensitivity.Based on the colorimetric signal transduction strategy,this method can be used for the rapid and accurate detection of Kana in a linear range from 0.1 pg m L^-1 to 10 ng m L^-1,with a detection limit of 9.4 fg m L^-1.By utilizing the photoelectrochemical signal transduction strategy,the linear range of this method is in the range from 0.01 pg m L^-1 to 1.0 ng m L^-1,and the detection limit can be low down to 0.55 fg m L^-1.As this method can not only realize the quick semi-quantitative analysis by the naked eye colorimetry but also be used for the accurate quantification by the photoelectrochemical method,it has a promising application value for the antibiotic screening and the low-abundance antibiotic residue detection in real samples.