| Objective:Methicillin resistant Staphylococcus aureus(MRSA)is an important pathogen of hospital and community infections,and rapid and sensitive diagnostic methods for MRSA have important clinical significance.In this study,a high-performance electrochemical immunosensor was constructed for the detection of methicillin-resistant Staphylococcus aureus by preparing nanocomposites.The condition optimization and performance evaluation of the constructed electrochemical sensor were carried out after the construction.Methods:1.Cultivate the standard strains of methicillin-sensitive Staphylococcus aureus ATCC25923,methicillin-resistant Staphylococcus aureus USA300 and MRSA252,and obtain three bacterial samples to be tested after ultrasonic wall breaking treatment.Gram staining was performed on the bacteria and their morphology was observed under the microscope.The affinity between the selected PBP2a antibody and the sample to be tested was proved by enzyme linked immunosorbent assay(ELISA).2.MWCNT-PDA-AuNPs nanocomposites were prepared and characterized by scanning electron microscopy.3.The prepared MWCNT-PDA-AuNPs nanocomposite was modified to the surface of the gold electrode,the antibody of the MRSA marker protein PBP2a was connected to the surface of the nanocomposite,and bovine serum albumin was added to block the non-specific site,finally an electrochemical immunosensor for detecting MRSA was obtained.The electrochemical immunosensor was characterized by cyclic voltammetry and electrochemical impedance spectroscopy.4.The key conditions in the construction of electrochemical immunosensor were optimized by differential pulse voltammetry:material mixing ratio,material drying temperature,buffer system PH,antibody incubation time,sample incubation time.5.The linear range and detection limit of the electrochemical immunosensor for MRSA detection were investigated,and the specificity,repeatability,stability and recovery of the sensor were evaluated.Results:1.Antigen antibody specificity investigation:A bacterial sample was successfully prepared,and it was proved by ELISA that the PBP2a antibody used could specifically bind to the two types of methicillin resistant Staphylococcus aureus samples prepared:the USA300 standard strain and the MRSA252 strain,but could not bind to the prepared Staphylococcus aureus ATCC25923 and the control group PBS.The antibody has the highest affinity with USA300 standard strain,and the detection limit is 2×10~5CFU/m L。2.SEM results:MWCNT-PDA-AuNPs nanocomposite liquid was successfully prepared.When only MWCNT is used,the result of SEM characterization is smooth tubular shape.The characterization result of MWCNT-PDA is a rough pipe shape with increased diameter.The characterization result of MWCNT-PDA-AuNPs is that circular particles with smaller diameter are added on the basis of the characterization result of MWCNT-PDA.The scanning electron microscope results showed that the nanocomposites were successfully prepared and immobilized on the electrode surface.3.Construction of electrochemical sensor:The electrochemical sensor was characterized by cyclic voltammetry and electrical impedance spectroscopy,and the analysis results were consistent,which proved that the sensor was successfully constructed.4.The result of condition optimization:(1)The optimal mixing ratio of MWCNT-PDA:AuNPs is 1:2;(2)The optimum drying temperature of the material;(3)The optimum PH of buffer system is 6.4;(4)The optimal incubation time of antibody is 1h;(5)The best incubation time of the sample is 30 min.5.Performance evaluation results of electrochemical sensor:(1)Linear range and detection limit:the concentration logarithm of MRSA samples in the range of 20CFU/m L～2.0×10~7CFU/m L has a good linear relationship with the peak current value of differential pulse voltammetry,and the regression equation is I(μA)=-6.72lg C+84.26(R~2=0.9878),the detection limit is 1 CFU/m L;(2)Specific selectivity:Methicillin-sensitive Staphylococcus aureus,bovine serum albumin,ovalbumin and casein were added to MRSA samples as impurities for electrochemical detection,and the results were basically consistent with the electrochemical response of the control group;(3)Repeatability:The different concentrations of MRSA(2×10~2CFU/m L、2×10~4CFU/m L、2×10~6CFU/m L)were measured repeatedly with the proposed electrochemical biosensor,and the RSD values were 4.18%,3.59%,4.38%,respectively(n=8);(4)Stability:when the sensor is stored at 4℃for 3 days,5days,10 days and 20 days,the sensor electrical signal is 98.85%～99.15%,97.80%～98.98%,95.45%～96.29%,92.15%～93.58%of the initial electrical signal(n=3);(5)Recovery rate results:The recovery rate of the sensor for low,medium and high concentration MRSA samples is basically consistent with that of the plate counting method.Conclusion:1.In this study,a novel nanocomposite material was prepared and an electrochemical immunosensor was successfully constructed.2.The electrochemical immunosensor is simple to construct and can detect MRSA quickly,accurately and sensitively.3.The electrochemical immunosensor has good specificity,repeatability and stability,wide detection range,low detection limit and good recovery rate,and can be used as an alternative method for clinical detection of MRSA. |
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