A Novel Electrochemical Sensor For Rapid Detection Of Pseudomonas Aeruginosa Based On High Catalytic Cu-ZrMOF And Conductive Super P

Objective:P.aeruginosa is one of the most intractable multidrug-resistant bacteria to cause nosocomial infections.The conventional detection methods for P.aeruginosa are time-consuming or have lower detection sensitivity,which cannot meet the clinical requirements.Therefore,it is urgent to develop a more rapid and sensitive method for detection of P.aeruginosa.In present work,a novel electrochemical sensor is proposed to be constructed based on high catalytic Cu-ZrMOF and high conductive Super P to detect P.aeruginosa rapidly and sensitively in urine and serum,in order to provide a rapid and powerful laboratory support for the timely diagnosis of P.aeruginosa infection.Methods:1.Preparation of Super P and AuNPs:Super P was dispersed into chitosan solution to prepare homogeneous Super P dispersion,the crystal morphology of Super P was characterized by FESEM.AuNPs were synthesized with chloroauric acid and trisodium citrate.The appearance structure of AuNPs were characterized by TEM2.Synthesis of ZrMOF,Cu-ZrMOF,Cu-ZrMOF@Aptamer@DNA Zirconium(Ⅳ)chloride,2,2’-Bipyridine-5,5’-dicarboxylic acid and acetic acid were added into DMF,then the mixture was stirred continuously at 120℃ for 16 h to synthesize ZrMOF.Cu-ZrMOF was prepared by dispersing Copper(Ⅱ)chloride dihydrate and ZrMOF in THF and being stirred at room temperature for 2 h.Then,Cu-ZrMOF,Aptamer and DNA were added into DMF and stirred at 37℃ for 12 h to synthesize Cu-ZrMOF@Aptamer@DNA signal probe.The morphology of ZrMOF was characterized by HRSEM and HRTEM.The element composition of Cu-ZrMOF was characterized by EDS.The connective result of Aptamer and DNA was characterized by Zeta potential3.Fabrication of the electrochemical sensor:Super P,AuNPs,antibodies of P.aeruginosa and BSA were gradually modified on the gold electrode.When the P.aeruginosa is appeared,it was connected on the gold electrode by the specific recognition of the antibodies of P.aeruginosa CV and EIS were used to characterize the changes of current and resistance in the fabrication of electrochemical sensor.4.Optimization of the key parameters:the key parameters were optimized,such as the type of working solution,the pH of working solution,the concentration of the antibodies of P.aeruginosa,the volume ratio of Cu-ZrMOF to Aptamer,the concentration ratio of Aptamer to DNA,and the incubation time of the Cu-ZrMOF@Aptamer@DNA signal probe5.Analytical performance evaluation of the electrochemical sensor the analytical performance of new electrochemical sensor,such as repeatability,specificity,linear range,LOD and storage stability were evaluated by measuring different concentrations of P.aeruginosa6.Recovery test:different concentrations of P.aeruginosa were obtained by adding P.aeruginosa to the matrix of urine and serum of healthy people.The accuracy of the new constructed electrochemical sensor was evaluated by the recovery testResults:1.Characterization of Super P and AuNPs:FESEM showed that Super P was spherical and the particle size was about 70 nm.TEM revealed that the synthesized AuNPs were uniformly dispersed with an average diameter of 15 nm2.Characterization of ZrMOF,Cu-ZrMOF and Cu-ZrMOF@Aptamer@DNA:Both HRSEM and HRTEM indicated that ZrMOF had a typical octahedral structure with an average particle size of 120 nm.EDS confirmed the presence of Cu,Zr,Cl,C,O in Cu-ZrMOF.Zeta potential analysis showed that both Aptamer and DNA were successfully connected on the surface of Cu-ZrMOF3.Fabrication of the electrochemical sensor:CV results revealed that with the gradual modification of high conductive Super P and AuNPs,the current was increased gradually,and with the gradual modification of non-conductive substances(antibodies of P.aeruginosa and BSA),the current was decreased gradually.In the presence of P.aeruginosa,the current was further decreased.The results of EIS were same with that of CV.It indicated that the construction of electrochemical sensor was successful.4.Optimization results of the key parameters:the most suitable working solution was PBS.The optimal pH of PBS was 5.0.The optimum concentration of antibodies of P.aeruginosa was 50μg-mL-1.The optimal volume ratio of Cu-ZrMOF to Aptamer was 5:1.The optimum concentration ratio of Aptamer to DNA was 1:1.The optimal incubation time of Cu-ZrMOF@Aptamer@DNA signal probe was 50 min.5.Analytical performance evaluation results of the electrochemical sensor:the repeatability of the electrochemical sensor was studied by measuring different concentrations of P.aeruginosa(102,104,106 CFU·mL-1,n=10).The RSD was 4.0%,3.8%and 3.7%respectively.The specific experiments of many kinds of interferential bacteria showed that the method had good specificity to P.aeruginosa.The linear range of the method was 10-106 CFU·mL-1 and the LOD was 2 CFU·mL-1.The retained current signal was 98.1%and 83.9%after storing at 4℃ for 7 and 14 days respectively6.Recovery test:the recovery rate was calculated after detecting the different concentrations of P.aeruginosa in urine and serum(10,103,105 CFU·mL-1).The results were 96.9%,102.3%and 98.8%in urine respectively,and 106.4%,103.7%and 97.5%in serum respectivelyConclusion:Based on the synthesis of the high catalytic Cu-ZrMOF and the combination of the high conductive Super P,a novel electrochemical sensor was constructed in present work.The linear range of the electrochemical sensor was 10-106 CFU·mL-1 and LOD was 2 CFU·mL-1.Compared with the conventional method,this method had the advantages of high sensitivity and good specificity.It could finish the detection of P.aeruginosa in urine and serum within 120 min.