Magnetism-resolved Separation And Fluorescence Quantification For Detection Of Multiplex Pathogens

Food safety and infectious diseases caused by pathogens are serious threats to human health.Moreover,multiple pathogens,eg,Salmonella typhimurium?S.typhimurium?and Escherichia coli O157:H7?E.coli O157:H7?,etc.may coexist in the same sample,usually with low infectious dosages.This calls for the development of a rapid,sensitive,and specific analytic method to simultaneously or near-simultaneously separate and detect multiple pathogens to achieve effective prevention and control of disease.Although conventional approaches based on culture and colony counting methods can simultaneously identify multiple pathogens,they cannot separate multiple pathogens with each other at the same time.Therefore,they have the problems of time-consuming,labor-intensive,low sensitivity and poor specificity.In recent years,various new methods,including polymerase chain reaction?PCR?techniques,immunoassays and other detection techniques have been extensively explored to rapidly and simultaneously detect multiplex pathogens.However,some of these methods have poor reproducibility,false-positive results,and high cost,respectively,and while others cannot simultaneously separate and collect different types of pure bacterium.Therefore,developing a new analytical strategy with near-simultaneous rapid,sensitive and high-specificity detection of multi-target bacteria has important basic research value and potential application for the diagnosis of infectious diseases caused by pathogens.This work developed a new multi-component analysis method for rapid separation and detection of multiple pathogens in complex samples using fluorescent-magnetic multifunctional nano-probes with different magnetic responses.Further,the target bacteria were subjected to antimicrobial susceptibility test.The strategy developed here is espected to provide technical support for clinical research such as diagnosis and treatment of infectious diseases cuased by pathogens.The main contents are as follows:Part 1:This part mainly constructed two kinds of fluorescent-magnetic multifunctional nanocomposites?W/S-FMNPs?via a layer-by-layer self-assembly approach based on the metal coordination between the metal elements from?-Fe₂O₃magnetic nanoparticles or the quantum dots and the sulfydryl from the surface of the?3-mercaptopropyl?trimethoxysilanesilica sphere.The W/S-FMNPs with different magnetic response intensity were achieved by controlled the assembly number of?-Fe₂O₃.The W/S-FMNPs with size of about 150 nm show strong fluorescence intensity.In addition,the W/S-FMNPs have obvious differences of magnetic gradient with the saturation magnetization values are 5.63 emu/g and 16.4 emu/g respectively,which laid the foundation for the separation of various target materials.Part 2:In this work,multifunctional nanoprobes targeting to E.coli O157:H7 and S.typhimurium respectively were fabricated by employing nucleic acid aptamer and W/S-FMNPs.Andthenbasedondifferentialmagneticresponseof pathogen@nanoprobes complex under the same external magnetic field,the model bacteria were fished out at different magnetic adsorption time points and subjected to fluorescence quantification with good linear ranges and detection limits within 1h.The enhanced fluorescence intensity was correlated with the concentration of S.typhimurium in the range of 63-10⁸ cfu/mL with a detection limit of 25 cfu/mL and correlated with E.coli O157:H7 in the range of 40-10⁸ cfu/mL with a detection limit of16 cfu/mL.Multiplex pathogens spiked in authentic samples?milk,serum and urine?were also effectively detected by the apt-FMNPs with good recovery and detection limit,and sequentially fished out for antimicrobial susceptibility testing?AST?,which showed similar results to that for pure pathogens.Therefore,this method has potential value in the clinical applications such as the diagnosis and treatment of infectious diseases caused by pathogens.