| The advent of antibiotics has brought new solutions to the treatment of various human diseases.However,the abuse of antibiotics poses a great threat to human health.Therefore,in order to improve the detection limit of antibiotics in the environment,various methods such as high performance liquid chromatography(HPLC),capillary electrophoresis(CE),gas chromatography-mass spectrometry(GS-MS),and enzyme-linked immunosorbent assay(ELISA)have been developed for the identification of antibiotics.Although these technologies can quantitatively analyze kanamycin,they also have the disadvantages of cumbersome sample preparation steps and long analysis time.Therefore,it is very convenient to develop antibiotic detection strategies that are convenient to explore,short in detection cycle and high in sensitivity.Necessarily,biosensors have received extensive attention from scholars because of their high specificity,speed,and high sensitivity.In this topic,using the specific recognition between antibiotics and their aptamers,combined with DNA Walker and nucleic acid amplification technology,three fluorescent biosensors were constructed,and good feedback was obtained in the actual detection of residual antibiotics in milk samples.The main research content is as follows:Firstly,based on Endo IV-powered 3D DNA Walker and HCR signal amplification technology,a highly sensitive fluorescent biosensor for kanamycin detection was constructed.Fluorescence resonance energy transfer is generated by labeling a fluorescent group and a quenching group on the signal probe,which causes the fluorescence of the fluorescent group to be quenched.With the assistance of Endo IV,a trigger agent that triggers the HCR reaction is generated,and along with the formation of the cleavage conditions for the cleavage site on the FQP,the fluorescent group is far away from the quenching group,thereby generating a fluorescent signal.In this sensor,the detection limit of KANA is as low as 1.01 pM,and the accuracy is higher than the traditional detection method.This shows that the fluorescent biosensor constructed in this subject can be used to detect KANA in a liquid environment.Secondly,based on the DNA Walker and b-HCR amplification reaction induced by the target,a fluorescent biosensor containing G-quadruplex structure was constructed.Through the specific binding of ampicillin and aptamers,the DNA Walker on the gold nanoparticles is triggered to work on the surface of the gold nanoparticles.With the help of the nicking enzyme Nt.Bbvc1,b-HCR reaction triggered agent was produced.On the branch of the b-HCR product,ThT can form a stable G-quadruplex structure with two G-rich sequences,accompanied by the generation of fluorescent signals.In this sensor,the detection limit of AMP is as low as 3.68 pM,and the cost is relatively low,and it has good applicability.Thirdly,based on the target-induced bipedal DNA Walker and surface plasmon resonance,a fluorescent biosensor constructed by CHA signal amplification.The aptamer of kanamycin and the DNA Walker of the two feet are blocked with each other.The presence of KANA releases the DNA Walker so that it can be captured by the hairpin track probe.One end of the track probe is connected to the gold nanoparticles and a fluorescent group is labeled on the other one end,and after being combined with DNA Walker,the quenched fluorescent signal is restored.The addition of the hairpin HP1 forms a cyclic amplification reaction of CHA with the trajectory probe and Walker,which further expands the signal generation.In this sensor,the detection limit of KANA is as low as 2.77 pM,and it has been verified in actual milk,so it has high applicability in KANA detection. |
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