Construction And Application Of Integrated Multifunctional Sensing Platform For Pathogenic Bacterial Identification And Inactivation

The problem of food safety not only seriously threatens the health and safety of people in the whole society,but also adversely affects the economic development of food related industries.Microbial pollution dominated by pathogenic bacteria exacerbates the seriousness of the problem.Due to the strong infectious ability of pathogenic bacteria,they are very easy to spread to humans from contaminated food,drinking water or other media,causing serious diseases and infections.Considering that contaminated matrix and environment exist multiple bacteria,it is difficult to accurately control the pathogenic bacteria by detecting single bacteria.In particular,the outbreak and spread of coronavirus disease 2019（COVID-19）reminds people that such highly polluting pollutants should be eliminated right after the detection and identification of them,which can effectively avoid accessional contaminations.Therefore,developing a simple and effective integrated multifunctional platform for identifying and killing pathogenic bacteria simultaneously is of great significance for food quality control,clinical diagnosis and treatment.In this paper,the purpose of the constructed multifunctional sensing platform is to integrate the identification and inactivation of pathogenic bacteria,gradually simplify the construction of the sensing platform,greatly shorten the detection time and simplify the operation steps,which can improve the universality and convenience of the developed platform.Based on the binding ability to pathogenic bacteria and photothermal characteristics of poly（allylamine hydrochloride）（PAH）functionalized copper sulfide nanoparticles（CuS@PAH NPs）,a POC（Point of Care）detection platform was constructed for the detection and inactivation of Escherichia coli（E.coli）and Staphylococcus aureus（S.aureus）by using temperature as signal output.Cationic polymer PAH could be utilized as protective agent and capping agent to synthesis CuS@PAH NPs.Through electrostatic interaction,the positively charged CuS@PAH NPs can bind to the surface of the negatively charged pathogenic bacteria,and can be separated into the sediment by centrifugation.Due to the photothermal effect of CuS@PAH NPs,the precipitation can rapidly increase the temperature after being irradiated by 808 nm laser,which can be monitored by infrared thermal imager.Therefore,taking temperature as the output signal,the proposed method can detect E.coli and S.aureus with detection limits of 3.59 and 3.91 CFU m L^-1,respectively.Also,it can be applied to the detection of these two pathogens in milk samples.In addition,the rise of temperature during detection can realize the in-situ inactivation of pathogenic bacteria through photothermal therapy（PTT）,and the sterilization efficiency of E.coli and S.aureus are 99%and 100%,respectively.Based on the affinity with pathogenic bacteria and photothermal characteristics of 4-mercaptophenylboric acid（MPBA）functionalized copper selenide nanoparticles（CuSe@MPBA NPs）,a portable sensor array was constructed for the identification and inactivation of various pathogenic bacteria by using temperature as signal output.MPBA can bind to the homeopathic diol structure in the surface sugars of pathogenic bacteria,which can be regulated by p H.Due to the differences in the types and substances of sugars on the surface of different bacteria,a portable sensor array was constructed with CuSe@MPBA NPs as the probe,different p H as the sensing channel and temperature signal as the output.Based on LDA,ten kinds of bacteria and their gram-negative state were successfully identified with 100%accuracy.The sensor array has been proved to be for determining unknown bacteria in tap water samples with 93.3%accuracy.Moreover,depending on the photothermal characteristics of CuSe@MPBA NPs,the sensor array can effectively kill pathogenic bacteria,and the sterilization rate is close to 100%.3,3′,5,5′-Tetramethylbenzidine（TMB）can be oxidized into oxidized TMB（ox TMB）which shows diverse absorption wavelengths and also presents photothermal properties.Based on that,a multifunctional sensor array with TMB as a signal probe was constructed for identification of multiple bacteria and photothermal sterilization.The synthesized bimetallic nanozyme of palladium/platinum nanoparticles（Pd/Pt NPs）has outstanding oxidase-like property,and can directly oxidize TMB to ox TMB with four feature absorption peaks（370 nm,450 nm,650 nm and 880 nm）in the absence of H₂O₂.Bacteria have different ability on inhibiting the reaction between Pd/Pt NPs and TMB,resulting in absorbance intensity changes of the feature peaks.Taking the intensity change of four absorption peaks of ox TMB as the signal,nine kinds of bacteria were successfully identified by LDA with 100%accuracy.Moreover,the sensor array can distinguish drug-resistant and non drug-resistant strains of the same strain.The accuracy of identifying unknown pathogenic bacteria in tap water samples was 96.3%.Interestingly,ox TMB exhibits excellent photothermal property,which endows the sensor array with the capability of photothermal sterilization after identifying the bacteria.The antibacterial efficiency against KREC（Kanamycin resistant Escherichia coli）and MRSA（Methicillin resistant Staphylococcus aureus）were 99%and 99.4%respectively,and nearly 100%for other pathogens.In this study,the strategy of choosing TMB as a single probe greatly simplifies the experimental operation and reduces the detection time.Based on the colorimetric reaction system of HAu Cl₄ and TMB,a novel multifunctional colorimetric sensor array with TMB as single probe and HAu Cl₄ as oxidant and bactericide was constructed.TMB can be efficiently oxidized to generate ox TMB by HAu Cl₄ which displays four feature absorption peaks.The presence of different bacteria could inhibit the oxidation reaction and cause diverse change in the intensity of four feature peaks.Based on LDA,not only nine kinds of pathogenic bacteria were successfully identified with 100%accuracy,but also drug-resistant strains are distinguished from sensitive ones.The accuracy of determining unknown pathogenic bacteria in contaminated tap water samples was 97.3%.Moreover,HAu Cl₄ can effectively kill bacteria during the identification and inhibit their growth and reproduction,which can avoid the formation of bacterial biofilm by residual pathogenic bacteria and effectively reduce the risk of bacterial contaminations and transmissions.Therefore,the constructed multifunctional sensing platform skillfully integrates the detection,identification and inactivation of pathogenic bacteria,which can realize the inactivation of pathogenic bacteria when detecting and identifying them,and effectively prevent the pollution of pathogenic bacteria.The developed strategy provides a new and simple avenue for bacterial detection and identification in real samples with high accuracy.