Study On Electrochemical Sensing Method For Ultra-sensitive Detection Of Pseudomonas Aeruginosa 16R RRNA And Antibacterial Performance Of Novel TiO₂ Nanocomposites

Diseases caused by bacterial infections has became a serious threats to human health.The research of antibacterial material without drug resistance has attracted much attention.Pseudomonas aeruginosa can cause multiple infections,the accurate and rapid detection of it is great significance in medical diagnosis,food safety and other fields.Antibacterial nanomaterials represented by TiO₂have been gradually applied to various aspects of bacterial infection due to their stable properties,high biocompatibility and ability to act on most pathogenic bacteria.In this study,we developed an electrochemical DNA sensor for the ultra-sensitive detection of Pseudomonas aeruginosa based on the electrochemical biosensor with high sensitivity,good selectivity and low cost.Based on TiO₂nanomaterials,two kinds of TiO₂based photocatalytic antibacterial activity composites were developed.The main work is as follows.（1）Based on the rolling ring signal amplification（RCA）and DNA multi legged walking strategy,taking Pseudomonas aeruginosa 16S rRNA as the target sequence,a new electrochemical biosensor was established to realize the sensitive and specific detection of Pseudomonas aeruginosa.In this study,capture probe（CP）and methylene blue signal probe（SP）were assembled on the surface of gold electrode by sulfhydryl modification and Au-S bond.Subsequently,the addition of 6-mercaptohexanol（6-mch）inhibited the inactive sites,thus preventing nonspecific adsorption.If there is Pseudomonas aeruginosa 16S rRNA in the sample,the capture probe will hybridize with 16S rRNA to form a partial double stranded structure.After adding the auxiliary probe（AP）,it will hybridize with 16S rRNA and capture the ring lock probe,and then add the RCA reaction reagent（CPP,d NTPs,phi29 polymerase）to open the rolling ring amplification and generate a long ss DNA,which contains a large number of repeat units complementary to the walking probe（WP）,thus forming a lead ion(Pb²⁺)dependent DNase.With the addition of Pb²⁺,Pb²⁺-dependent DNase cleaves the signal probe,methylene blue away from the electrode surface,and then WP is released to trigger the next cutting reaction.With the progress of multiple cutting reactions,the electrochemical signal of methylene blue gradually decreases,so as to achieve the purpose of detecting Pseudomonas aeruginosa.The detection platform showed excellent analytical performance according to the change of methylene blue signal,and the detection limit for Pseudomonas aeruginosa reached 10 CFU/ml.Therefore,the electrochemical sensing platform can be used as a sensitive and rapid tool for the detection of Pseudomonas aeruginosa,and has broad application potential in the fields of food safety,environmental monitoring,disease diagnosis and so on.（2）The novel Cu_0.01@TiO₂nanocomposites with high energy band gap and high specific surface area were successfully prepared by sol-gel method.Compared with pure TiO₂,Cu@TiO₂exhibited stronger light capture efficiency in the visible region,inhibited the complexation between e^-and h⁺,produced·OH and reactive oxygen species under simulated natural light irradiation,and enhanced the photocatalytic bactericidal activity against E.coli.In this study,the photoelectrochemical response of Cu_0.01@TiO₂was sensitive and stable as shown by photocurrent density test.Further,due to the high specific surface area and high photoelectric properties of Cu_0.01@TiO₂,the nanocomposite achieved a high antibacterial efficiency of 98.7%against E.coli under simulated 60 min of natural light.（3）In this experiment,UCNPs@R-TiO₂nanocomposites were successfully prepared by coupling reduced TiO₂to the surface of Gd-enhancedβ-Na YF₄:Yb,Er,Gd fluorescent nanorods through an electrostatic assembly strategy.The material exhibited efficient photocatalytic bactericidal performance under a single near-infrared（NIR）light source（980 nm,1 W）.the UCNPs could adsorb NIR light and the emitted visible light could be effectively absorbed by R-TiO₂.According to the synergistic effect of UCNPs and R-TiO₂to generate strong reduced electron and oxidized hole pairs（e^-/h⁺）,they can react with H₂O and O₂to generate hydroxyl radicals（·OH）with extremely strong oxidizing properties to achieve sterilization effect.UCNPs@R-TiO₂nanocomposites with MIC（minimum inhibitory concentration）of 40μg/m L killed98.1%of E.coli in 20 min when irradiated with 980 nm NIR light（1 W）.The bactericidal performance was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF MS）,and the results showed that the UCNPs@R-TiO₂composites treated with UCNPs had efficient bactericidal ability.In addition,in vitro cytotoxicity experiments showed that the composites had low toxicity to mammals.Therefore,the low toxicity UCNPs@R-TiO₂nanocomposites prepared in this study have good prospects for application in the field of NIR-responsive photocatalysis.