Study On The Detection Method On DNA Nanomachine Of Enrofloxacin And Kanamycin Based On Split Aptamer

Enrofloxacin（ENR）and kanamycin（KANA）are broad-spectrum antibiotics widely used to treat infectious diseases in poultry.Their unregulated use can lead to their accumulation in the environment and food chain,which can cause adverse effects on the human digestive system or nervous system,which cause adverse effects on human digestive system or nervous system.Split aptamers are two or more fragments separated from a complete aptamer.Compared with traditional aptamers,the unique sandwich structure improves the sensitivity of detection and reduces non-specific binding.DNA nanomachine are used to detect specific target molecules through DNA self-assembly and molecular recognition technology.They can be designed,programmed and modified to achieve highly controllable and selective detection to improve accuracy and reproducibility.They can also be reused through simple washing and regeneration operations,greatly reducing cost and improving practicality.Based on the advantages of split aptamers and DNA nanomachine,a series of fluorescent aptasensor assays were constructed for the first time as follows:Firstly,an aptasensor based on split aptamer separated by magnetic nanoparticles（MNPs）and detecting ENR with the help of DNA tweezers’fluorescence signals was constructed.DNA tweezers use complementary base pairs to form a tweezer structure that captures small molecules precisely and keeps them in place.The tweezers use fluorescent labeling to convert the tweezers’conformational changes into fluorescent signals.SPA₁ is assembled on the surface of MNPs as a trapping probe.In the presence of ENR,SPA₁,ENR and split aptamer 2（SPA₂）combine to form a ternary complex（SPA₁/ENR/SPA₂）.The base pairs of SPA₂ are not paired with the DNA tweezers end,and the DNA tweezers structure"turn-on."A pair of fluorescence groups（FAM）and quenching groups（BHQ₁）were connected at both ends of the tweezers arm.In the absence of ENR,SPA₂ dissociates in the supernatant after magnetic separation and combines with DNA tweezers.Self-assembly results in the tweezers being"turn-off"and fluorescence decreasing.The fluorescence intensity had a good logarithmic relationship with ENR concentration in the range of 0.01 ng/m L-100 ng/m L,and limit of detection was 0.008ng/m L.The constructed DNA tweezers recognize the target SPA₂ sequence and alter its"on/off"structure,providing a significant fluorescence signal,which is highly specific and controllable.Secondly,based on the specific signal amplification performance of 3D-DNAwalkers and high reaction rate,a highly sensitive aptasensor based on magnetic separation split aptamer was constructed.DNA walkers utilize complementary hybridization and motility of DNA molecules to form specific structures to recognize and locate specific DNA sequences.The movement of DNA walkers is translated into measurable fluorescence by fluorescent labeling.In this work,DNA walkers consist of FAM fluorescence-labeled signal probes and locked DNAzyme that are silent in the presence of KANA.In the system without KANA,the fuel chain SPA₂ in the supernatant after magnetic separation replaces the target through the chain replacement reaction and hybridization with the locking strands,which provides power for the continuous migration of DNAzyme chain with the help of Mn²⁺and shear signal probes to release the FAM-labelled fragment.Under optimal conditions,the logarithm of the KANA concentration showed a good linear relationship with the fluorescence intensity,with a detection range of 0.05 ng/m L-500ng/m L and an LOD of 0.036 ng/m L.Finally,through rational design of DNA sequence and structure,a"turn-off"dual-DNA tweezers was constructed,which can detect ENR and KANA in food in one step.DNA nanomachine can be programmed through reasonable design of DNA sequence and structure to achieve different functions and applications.The two ends of the dual-DNA tweezers are cleverly connected with a specific split aptamer sequence.Using the split aptamer and the target to form a specific terpolymer complex,the tweezers identify and"clamp"the ENR and KANA at each end,effectively reducing false positive signals.The linear range of ENR was 0.2 ng/m L-1000 ng/m L,and the LOD was 0.18 ng/m L.The linear range of KANA was 0.5 ng/m L-1000ng/m L,and the LOD was 0.36 ng/m L.The dual-DNA tweezers not only solve the problem of single target recognition in the traditional single-DNA tweezers biosensor,but also provide a new idea for the construction of DNA nanomachines,which is also suitable for the recognition and detection of other small molecules.