Preparation Of A Three-dimensional Functional Nucleic Acid-based Paper Device And Its Application In Antibiotic Detection

Antibiotics are regarded as emerging pollutants due to their extensive use in human and persistence in the environment.Different environmental matrix containing antibiotics residue has caused serious influence to the environment.As a representative aminoglycoside antibiotic,kanamycin can cause damage to many organs of human body,especially to kidney.And tetracycline,as a typical macrolide antibiotic,can damage the liver and affect the development of children’s bones.Therefore,detecting kanamycin and tetracycline is of great significance for human health and environment protection.The current detection methods mainly include liquid chromatography,mass spectrometry,capillary method,immunoassay and microbiological method.Among them,liquid chromatography and mass spectrometry requires complicated sample pretreatment procedures and thus having the disadvantages of time-consuming and expensive.And the immunoassay is a qualitative or semi-quantitative method with high false positive rate.Capillary method has low sensitivity and poor reproducibility.Concurretly,the microbiological method is also timeconsuming and the results need to be visually analyzed by trained professionals,thus maybe resuting in the incorrect results.To overcome these,it is so necessary to develop a fast,simple and sensitive method for the detection of antibiotic residues.Recently years,biosensor has been widely used as an effective alternative method for sensitive,fast and on-site detection of different antibiotics.However,most biosensing detection methods need to combine optical or electrochemical methods to detect antibiotics,which still has the problem of cumbersome operation and unsuitable for on-site detection.Therefore,it is necessary to develop a simple and suitable method for on-site detection of antibiotics.This study prepared a protein loaded three-dimensional functional nucleic acid(protein@3D RNA)for the construction of a fast response gating system to develop a paper-based device for the sensitive and simple detection of antibiotics.Here are the details:(1)A three-dimensional functional nucleic acid which can specifically detect antibiotics(kanamycin and tetracycline)was prepared.Firstly,the circular DNA containing the complementary sequence of kanamycin or tetracycline apatamer was used as a template.And then under the action of T7 RNA polymerase catalytic hoop transcription reaction(RCT),the template synthesized RNA long chain.It binded to the phosphatase precipitation produced by the reaction and formed 3D RNA.Concurrently,with the proceeding of roll ring transcription,the added protease can be directly incorporated into the 3D RNA.And kanamycin or tetracycline can recognize and bind to 3D RNA,triggering the release of protease and catalyzing the chromogenic reaction of TMB.(2)Kanamycin and tetracycline in the solution were detected by 3D RNA with the detection limits of 1 mM and 1 nM.Based on the HRP@3D DNA control system,a paperbased device was developed to detect kanamycin and tetracycline with high specificity within16 min through simple steps,and the sensitivity of the detection can reach 0.1 μM and 1 nM,respectively.The paper-based device is proved to be universal and can be used for specific detection of different antibiotics.When quantitative kanamycin and tetracycline were added to actual river water samples,the detection recovery rate reached more than 80% and relative standard deviation less than 6%,which proved the practicability and repeatability of the paperbased device.