Electrochemical Sensor Based On CNMs/CS Composite For Detection Of Live Salmonella Typhimurium

Objective:In this study,Salmonella typhimurium was used as the research object,and two composites of carbon nanomaterials(CNMs)and chitosan,multi-walled carbon nanotubes/chitosan(MWCNT/CS)and graphene/chitosan(GR/CS)were used as signal amplification elements,two simple and sensitive electrochemical sensors were constructed to explore the electrochemical behavior of resazurin and realize the detection of live Salmonella typhimurium.Methods:(1)The MWCNT/CS and GR/CS composites were prepared by ultrasonic method,and the composites were analyzed by scanning electron microscopy.(2)Two kinds of electrochemical sensors were constructed using the composites as the signal amplification element.The single factor method was used for optimization to determine the best detection conditions.(3)Using the constructed electrochemical sensor,the electrochemical behavior of resazurin was explored.(4)By using the two constructed electrochemical sensing systems,resazurin was used as a redox probe to detect different concentrations of live Salmonella typhimurium,and the linear range,detection limit,repeatability,sensitivity and stability were determined,and the methodological evaluation was performed.(5)The two constructed electrochemical sensing detection systems were used to study the antibacterial effect of the ciprofloxacin on Salmonella typhimurium,monitor the metabolic level of live bacteria,and determine the minimum inhibitory concentration of ciprofloxacin.Methodological evaluation was carried out by comparing with the standard method of traditional drug susceptibility testing.Results:(1)The best conditions for building sensing platforms:(1)MWCNT/CS sensor:the feeding ratio of the modified material was 0.5:1;the amount of the composite material for the modified electrode was 8μL;the pH of the buffer solution was 7.4;The incubation time was 5 min.(2)GR/CS sensor:the dosage ratio of the modified material was 1:1;the amount of the composite material for the modified electrode was 6μL;the pH of the buffer solution was 7.4;the incubation time of the modified electrode and the test solution was 5 min.(2)Electrochemical behavior of resazurin:(1)Principle of electrochemical detection:On the electrode surface,the electrochemical reaction of resazurin was an electrochemical redox process controlled by diffusion.(2)Linear range and detection limit of resazurin:The linear range of the MWCNT/CS sensor was 10 nmol/L～50μmol/L,the detection limit was 5.8 nmol/L,and the standard curve equation was y=0.0015x+1.1289,R~2=0.9932;the linear range of the GR/CS sensor was 10 nmol/L～50μmol/L,the detection limit was 9.1nmol/L,and the standard curve equation was y=0.0008x+0.1432,R~2=0.9988.(3)Methodological evaluation of the detection of living Salmonella typhimurium:(1)MWCNT/CS sensor:the linear range was7.5×10~2CFU/mL～7.5×10~7 CFU/mL,the detection limit was 7.5×10~2CFU/mL,the standard curve equation was y=-0.2533x+4.937,R~2=0.9317.(2)GR/CS sensor:the linear range was 7.5×10~3CFU/mL～7.5×10~7CFU/mL,and the detection limit was 7.5×10~3 CFU/mL,the standard curve equation was y=-0.1826x+3.468 and R~2=0.9487.(3)The relative standard deviations of the repeatability of the two electrochemical sensors were 5.1%and 7.5%,both less than 10%,and the stability within 7 days remained above 74%.(4)Bacteriostatic effect of ciprofloxacin on Salmonella typhimurium:Both sensors detected that the inhibitory effect on bacteria was enhanced with the increase of antibiotic concentration.The changes of bacterial respiration activity under different antibiotic concentrations within 4 hours were observed.When the concentration reached 16μg/mL,there was a significant inhibitory effect,which was consistent with the results of the traditional method.Therefore,the minimum inhibitory concentration of ciprofloxacin against the Salmonella typhimurium strain in this experiment was determined to be 16μg/mL.Conclusions:(1)In this study,two electrochemical sensors based on MWCNT/CS and GR/CS were constructed,the electrochemical behavior of resazurin was explored,and a new method for simple and sensitive detection of living Salmonella typhimurium was established.(2)The detection limits of the two new electrochemical sensing methods are low,and they have good accuracy and stability.Comparing the two established methods,the detection limit of the MWCNT/CS sensor was relatively low and the sensitivity was relatively high,which is more conducive to the detection of a small amount of living bacteria.(3)Using the constructed MWCNT/CS and GR/CS electrochemical sensors,the respiration activity of bacteria was monitored in real time.With the increase in the concentration of ciprofloxacin,the inhibitory effect on the growth of the Salmonella typhimurium has gradually strengthened,and the minimum bacteriostatic concentration of ciprofloxacin was determined as 16μg/ml.