Detection And Removal Of Salmonella Typhimurium Based On Hollow Carbon Nitride Nanosphere

With the continuous development of economy and society,food safety problems are frequent,especially food contamination caused by food-borne pathogenic bacteria,among which S.typhimurium is one of the most dangerous Gram-negative bacteria that can infect a range of hosts and cause a variety of diseases such as gastroenteritis and typhoid fever,and has received widespread attention.Therefore,the development of sensitive,accurate and rapid detection and removal methods for S.typhimurium is of great importance to ensure food and public health safety and to prevent and control foodborne disease outbreaks in a timely and effective manner.Graphitic-carbon nitride（g-C₃N₄）is a non-metallic conjugated semiconductor material with visible light response,good stability,low cost,good fluorescence performance and excellent peroxide-mimetic enzymes,which has been successfully applied in the detection of foodborne pathogenic bacteria;meanwhile,based on its large specific surface area and the ability to produce a large number of active species under visible light,it has been successfully used in the construction of drug-controlled release systems and it has shown good application prospects in the construction of drug-controlled release systems and photocatalytic antibacterial applications.Therefore,it is expected to construct a platform for rapid detection and removal of S.typhimurium based on g-C₃N₄.In this paper,g-C₃N₄ was used as the main material,and its functionalization was modified by morphological modulation to further enhance its fluorescence performance,and its specificity and peroxide-mimetic enzyme activity were improved by combining with aptamers to develop a ratiometric fluorescence and visualization detection method;meanwhile,by constructing a drug delivery system and modifying aptamers,an efficient synergistic antibacterial platform was developed to significantly improve the antibacterial performance.Based on this,the efficient detection and removal of S.typhimurium by g-C₃N₄ material was achieved.The specific studies are as follows.1.Detection of S.typhimurium based on aptamer recognition-HCNS fluorescenceHollow carbon nitride nanospheres（HCNS）were prepared by hard-template method,and an"off-on"ratio fluorescent biosensor,HCNS-Apt,was developed by binding a dye（Cy5）labeled aptamer（Cy5-Apt）with specific recognition function to HCNS by direct adsorption.Cy5 for the detection of S.typhimurium.In the absence of S.typhimurium,the fluorescence of Cy5-Apt was effectively burst by HCNS based on the PET principle after adsorption on the HCNS surface.In the presence of S.typhimurium,Cy5-Apt would preferentially bind to S.typhimurium,forming a S.typhimurium-Apt complex,which restored the fluorescence of Cy5,while the fluorescence of HCNS remained almost unchanged.The ratio of Cy5 fluorescence enhancement to the intrinsic fluorescence of HCNS was used to quantify the cell density of S.typhimurium.Under the optimized experimental conditions,the logarithm of the ratiometric fluorescence and the cell density of S.typhimurium showed good linearity between 30-3×10⁴CFU m L^-1（y=0.23x-0.33,R²=0.996）with a detection limit of 13 CFU m L^-1.The assay is specific for S.typhimurium and can be used to detect S.typhimurium in milk samples with good performance.The method is specific for S.typhimurium and can be used to detect S.typhimurium in milk samples with good accuracy and recovery.2.Visualization of S.typhimurium based on aptamer recognition-HCNS peroxide-mimetic enzymeDue to the property of peroxide-mimetic enzyme,HCNS,when combined with TMB and H₂O₂,can effectively catalyze H₂O₂ and oxidize TMB to ox TMB,so that the system shows a bright blue color with gradient.Based on this principle,an Apt recognition-HCNS peroxide-mimetic enzyme-based visual detection biosensor was constructed by interconnecting the Apt element with specific recognition function to HCNS by direct adsorption.In the absence of the presence of S.typhimurium,the absorbance atλ=652 nm increased with the increase of Apt concentration after the HCNS itself was connected to Apt.When S.typhimurium was present,the aptamer and S.typhimurium bound to each other,resulting in a decrease in absorbance atλ=652 nm after Apt detached from the HCNS surface.Under the optimized experimental conditions,the absorbance value at 652 nm showed a good linear relationship with the logarithm of S.typhimurium in the range of 3-10⁵ CFU m L^-1（y=-0.04x+0.81,R²=0.992）,and the detection limit was 15 CFU m L^-1.The assay was specific for S.typhimurium and could be used to detect S.typhimurium in milk samples.The method is specific for S.typhimurium and can be used to detect S.typhimurium in milk samples with good accuracy and recovery.3.Performance study of the aptamer-HCNS-based drug delivery system for the removal of S.typhimuriumChloramphenicol（Cap）was successfully loaded into the hollow structure of HCNS（HCNS-Cap）by electrostatic adsorption,and HCNS had 55%loading rate for Cap,while block g-C₃N₄（CN）had only 2%loading rate.Moreover,by adjusting the p H as well as the light conditions,HCNS-Cap could achieve 55%drug release rate and 302.5μg mg^-1 drug release within 24 h.A controlled drug delivery drug delivery system based on HCNS was constructed.Since HCNS has both drug delivery and photocatalytic antibacterial functions,it can achieve a synergistic antibacterial effect while slowing down the release of Cap.With the addition of light and p H adjustment,HCNS-Cap killed 99.2±0.1%of S.typhimurium cells within 12 h,while the bactericidal rates of HCNS or Cap alone were only 85.1%and 72.9%.Meanwhile,the aptamer-functionalized HCNS drug delivery system Apt-HCNS-Cap was constructed by combining HCNS-Cap and aptamer elements by direct adsorption.Since Apt can bind specifically to S.typhimurium,Apt-HCNS-Cap can preferentially recognize and rapidly kill S.typhimurium.Within 4 h,the killing rate of Apt-HCNS-Cap against S.typhimurium reached99.0±0.1%,while the killing rate against Gram-negative bacteria（e.g.Escherichia coli）and Gram-positive bacteria（e.g.Staphylococcus aureus）was only 48.2±0.2% and 13.3±0.1%.