Research On Electrochemical Biosensing Method Of Multi-target Foodborne Pathogenic Bacteria Based On Metal Organic Framework-bacons

The establishment of rapid detection methods for food-borne pathogens is of great significance for ensuring food safety.Salmonella typhimurium?S.ty?and Listeria monocytogenes?List.m?are the most common and lethal foodborne pathogens.And these two food-borne pathogens usually appear in contaminated food at the same time.The current detection of food-borne pathogens mainly relies on traditional methods that are tedious and time-consuming,far from meeting the needs of current social development and food safety.Therefore,there is an urgent need to develop a rapid,sensitive and convenient detection method that can simultaneously detect multiple food-borne pathogens in a sample.Electrochemical biosensor has been used in analysis and detection in various fields such as medical diagnosis,drug analysis,heavy metal analysis,and food analysis due to their advantages of easy operation,low cost,and high sensitivity.In this paper,the copper metal organic framework?CuMOF?and lead metal organic framework?PbMOF?prepared under mild conditions were used as nano-beacons.And the nucleic acid sandwich structure were used to construct a dual-channel electrochemical sensing platform based on signal amplification strategy for quickly detect the inv A gene of S.ty and the inl A gene of List.m.In order to improve the performance of nanostructures,magnetic core-shell structure metal organic framework nanoparticles were prepared as magnetic nanobeacons.The proposed electrochemical sensor had an ultra-sensitive electrochemical response to the target gene,with high selectivity and repeatability.At the same time,the analysis of real samples also showed good performance and has potential application prospects.The research content of this article mainly has the following three parts:1. The establishment of the correspondence between the inv A gene electrochemical biosensor based on signal amplification and the number of S.ty coloniesThe inv A gene electrochemical biosensing method established earlier was applied to the detection of actual samples.The S.ty colony count was determined by dilution coating method,and the total DNA was extracted by hydrothermal method.The inv A gene electrochemical sensor was used to directly analyze the DNA extract of S.ty.And the linear relationship between the number of S.ty colony-DNA concentration and the electrical signal mapping was established.S.ty inv A gene fragment polymerase chain reaction?PCR?and agarose electrophoresis were used as controls to evaluate the performance of the genosensor.The results showed that the inv A gene electrochemical sensor has good practical analysis and detection capabilities.In the range of S.ty concentration of 9.6–9.6×10⁴ CFU m L^-1,there was a good linear relationship between the current response and the logarithm of the bacterial concentration.The limit of detection?LOD?was 8.82 CFU m L^-1,and the sensitivity was 10³ times that of PCR.2. Preparation and characterization of nanostructures used to construct sensing interfaces and signal labelsA series of nanomaterials and structures with stable physical and chemical properties are prepared.1)According to the principles of non-toxic or low-toxicity,mild conditions,nanomaterials/structures with good size uniformity and controllable morphology were prepared:gold nanoparticles,three-dimensional metal organic framework?MOF?and magnetic core-shell structureMOF,etc.These nanomaterials and structures with good electronic properties were used to build the sensing interface.2)Biological functionalization of the nanomaterials/structures:through non-covalent and covalent assembly methods,the nanomaterials/structures were biofunctionalized to achieve more effective and reasonable molecular recognition and sensing purposes.3)Characterization of nanomaterials/structures:through the use of field emission transmission electron microscopy?FE-TEM?,ultraviolet-visible spectroscopy?UV-vis?,electrochemical and other technical means,the nanomaterials/structures involved were characterized and their effectiveness confirmed.3. Construction of dual-channel electrochemical genosensor based on metal organic framework nanoparticles for simultaneous detection of S.ty inv A gene and List.m inl A geneIn this part of the experiment,the prepared CuMOF and PbMOF nanomaterials were used to obtain two biofunctionalizedMOF nanobeacons through self-assembly adsorption signal DNA?s DNA?.The surface of the constructed dual-channel electrochemical sensor obtained gold-modified glassy carbon electrode by electrodeposition method.Then capture DNA?c DNA?was modified on the electrode surface by self-assembly covalent modification.In the presence of the target gene,half of the target gene's sequence was complementary to c DNA,and the remaining sequence was complementary to theMOF nanobeacon.The complementary hybridization of DNA formed a sandwich-type“signal-on”electrochemical biosensor.The twoMOF nanobeacons in acetate buffer solution will showed clear,separated electrochemical signals,which were proportional to the target gene.The detection limits?LOD?of the inv A and inl A genes of the two pathogens constructed by the two-channel electrochemical detection platform were 5.831×10^-16 mol L^-1 and 8.608×10^-15 mol L^-1,respectively.The proposed biosensor not only had simple construction process and low cost,but also had good sensitivity,selectivity,stability and broad application prospects.Reducing the background signal can also improve the performance of the sensor.This study further designed a class of magnetic nanobeacons that can reduce the background signal through the magnetic separation step.First,superparamagnetic Fe₃O₄nanoparticles were prepared.Then Fe₃O₄ nanoparticles were used as the inner core,andMOF was modified on the surface of the inner core to obtain metal-organic framework nanoparticles with magnetic core-shell structure?MOF@Fe₃O₄?.Finally,Au NP@MOF@Fe₃O₄ obtained by adsorbing Au NP on the surface ofMOF@Fe₃O₄ was used as the beacon to construct a dual-channel electrochemical sensor for simultaneously detecting S.ty inv A gene and List.m inl A gene.The background signal of the sensor using Au NP@MOF@Fe₃O₄ as a beacon was reduced to 40.33%and24.18%using Au NP@MOFNP.The prepared beacon not only maintained good electrochemical performance,but also could be separated and purified by applying an external magnetic field,which greatly improved the detection performance of the sensor.