Construction Of Electrochemical Biosensor And Application In Early Diagnosis Of Pathogens Infection

Objective1.A novel cascade sensitizing electrochemical biosensor for the detection of E.coli O157:H7 was constructed based on cascade sensitizing system,which was induced by DNA 3D walker and RCA@HCR nucleotide amplification reaction.The analytical performances of the cascade sensitizing electrochemical biosensor were studied,including the sensitivity,specificity,repeatability and stability.2.A dual-component magnetron electrochemical biosensor was constructed based on the magnetron electrochemical flow detection cell.In virtue of the excellent signal amplification efficiency of C₆₀@Au NPs and HCR reaction,the dual-component magnetron electrochemical biosensor with high efficiency and high sensitivity can be used to realize the parallel detection of E.coli O157:H7 and Vibrio cholera O1 pathogens.Methods1.Electrodeposition time of DepAu onto the glassy carbon electrode surface was optimized in the range from 5 s to 100 s.The morphology of electrodeposited DepAu was characterized by scanning electron microscope?SEM?JSM-4800.At the same time,the morphology of electrodeposited DepAu was also characterized by atomic force microscopy?AFM?.2.The effect of different signal amplification strategies were investigated by series experiments.The comparative studies included the following three groups:?1?hybridization chain reaction?HCR?;?2?rolling circle amplification?RCA?,in which the auxiliary sequences were hybridized with the single-stranded DNA sequence obtained by the reaction to form double-stranded DNA to immobilize the electrons medium Fc-Dox;?3?RCA@HCR,in which the generated double-stranded DNA structures were employed to immobilize the electrochemical media Fc-Dox.3.The cascade sensitizing electrochemical biosensor was established for the sensitive detection of E.coli O157:H7.The detection performances of the studied electrochemical biosensor,including the linear range,specificity and stability,were investigated based on the correspondence between the changes of current responses and the concentrations of E.coli O157:H7.4.In order to overcome the limitations of the conventional electrode,including the complexpreparation process,the high production cost and difficult regeneratable ability,a novel magnetron electrochemical flow detection cell was designed and developed independently,in which the detection was performed on the magnetic beads fixed on the electrode surface with the help of a magnet under the glassy carbon electrode.After every detection,the magnet could be removed,and the glassy carbon electrode could be regenerated by the washing buffer flowing through the magnetron electrochemical flow detection pool to remove the magnetic beads on the glassy carbon electrode surface.And then,another new detection process could be performed via the proposed the magnetron electrochemical flow detection cell.5.In order to investigate the amplification effect of different amplification strategies via nanotechnology and nucleic acid amplification,three different signal amplification strategies were investigated in this work,including?1?detection of antibodies directly with PbS or CdS nanoparticles as signal labels;?2?amplification strategy with the help of Au NPs to load nucleic acid and generate nucleic acid amplification strategy to load a large amount of PbS or CdS nanoparticles as signal labels;?3?amplification strategy with the help of C₆₀@AuNPs to load nucleic acid and generate nucleic acid amplification strategy to load amounts of Pb S or Cd S nanoparticles as signal labels.6.Based on the indigenous design for magnetron electrochemical flow detection cell,the dual-component electrochemical biosensor was constructed to detect E.coli O157:H7and Vibrio cholera O1 pathogens simultaneously.In this work,the C₆₀@AuNPs was employed as nanocarriers to adsorb a large number of initiators to generate HCR amplification for ultrasensitive detection of pathogenic bacteria.The concentration of E.coli O157:H7 and Vibrio cholera O1 pathogens were detected by dual-component electrochemical biosensor and the obtained results were compared with the plate culture counting technique.Results1.The morphology of electrodeposited gold nanoparticles?Dep Au?was characterized by scanning electron microscopy.The Dep Au was increased gradually with the increased electrodeposition time from 5 s to 100 s.When the electrodeposition time was on 30 s,the DepAu are basically covered on the glassy carbon electrodesurface.The larger flower-like DepAu are aggregated with the increased electrodeposited reaction time more than 30 s.Thus,30s was employed as the best electrodeposited reaction time to modify Dep Au onto the glassy carbon electrode surface for the construction of electrochemical biosensors.At the same time,the DepAu were characterized by atomic force microscopy.The results show that the Dep Au were homogeneous and evenly distributed on the surface of the glassy carbon electrode.2.The different signal amplification strategies were investigated with three different amplification strategies including?1?hybridization chain reaction?HCR?;?2?rolling circle amplification?RCA?,in which the auxiliary sequence is hybridized with the single-stranded DNA sequence obtained by the reaction to form double-stranded DNA to immobilize the electrons medium Fc-Dox;?3?RCA@HCR,in which the generated double-stranded DNA structures were employed to immobilize the electrochemical media Fc-Dox.Under the optimum experimental conditions,the electrochemical response signal was only 2.95?A based on HCR.The electrochemical response signal was 3.14?A only based on the RCA.The electrochemical response signal was significantly enhanced,up to 5.25?A,via RCA@HCR indicating the good amplification effect of the proposed RCA@HCR amplification strategy,which could effectively improve the immobilization of electrochemical electronic media,and then improve the electrochemical signals to improve the detection performances of electrochemical biosensor.3.The electrochemical signals of the cascade sensitizing electrochemical biosensor showed a good linear relationship to the concentrations of the target nucleic acid in the range of 1.0×10¹¹.0×10⁴ CFU/m L with the linear equation is y=0.9803 lgc _{E.Coli O157:H7}+5.73 and the detection limit of 7 CFU/m L.Compared with the detection atrategies in some previous literatures,the cascade sensitizing electrochemical biosensor showed good linear range and sensitivity.It could be expected that the proposed electrochemical biosensors with good analytical performances could be further applied to the actual sample analysis.4.The analysis of the different nucleic acid sequences,including single base and multi-base differential nucleic acid,showed that there was no significant interference between the identified nucleic acid sequences,indicating that the cascade sensitizing electrochemical biosensor was well specific.At the same time,the stability study showed that the standard deviation of inter-and intra-assay,and the standard deviation of the long-term stability test were less than 5%,which indicated the good stability of the cascade sensitizing electrochemical biosensor.5.The main components of the magnetron electrochemical flow detection cell included organ glass body cavity,limit plate,limit plate fixing screw,electrode piece,permanent magnet,permanent magnet fixing screw,sealing ring,counter electrode,reference electrode and so on.6.The results showed that the detection current obtained by direct detection of PbS or CdS nanoparticles was 4.29?A and 4.64?A.The detection current was significantly increased,to 9.82?A and 11.13?A based on the nucleic acidamplification strategy,indicating its good amplification effect to improve the electrochemical detection signals.Based on the amplification strategy with the help of C₆₀@Au NPs to load nucleic acid and generate nucleic acid amplification strategy to load amounts of PbS or Cd S nanoparticles as signal labels,the current signal was 19.19?A and 22.11?A,showing good signal amplification effect.7.The performance of the dual-component magnetron electrochemical biosensor was evaluated for simultaneous determination of different concentrations of E.coli O157:H7 and Vibrio cholerae O1 in standard solution.The current signals were increased according to the concentrations of E.coli O157:H7 and Vibrio cholerae O1 in the range from 5×10¹ to 1×10⁶CFU/m L.There is a good linear relationship with the logarithm concentrations of E.coli O157:H7 and Vibrio cholerae O1 with egression equation of?I??A?=3.43logc_O157:H7-5.05and?I??A?=3.51logc_O1-4.54,and detection limit of 39 CFU/m L and 32 CFU/m L,respectively,in which the equation was defined as LOD=3 SB/m,where SB was the standard deviation of the blank and m was the analytical sensitivity.These results indicated that the E.coli O157:H7 and Vibrio cholerae O1 concentrations could be quantitatively measured by the electrochemical signal using the proposed electrochemical biosensor.Furthermore,the performance of the dual-component magnetron electrochemical biosensor was compared with that of the previously reported studies as shown in Table 1.The results indicated the superior sensitivity of the biosensor for the detection of bacteria with a low detection limit and wide linear range.At the same time,the dual-component magnetron electrochemical biosensor showed good repeatability and stability.8.The concentration of E.coli O157:H7 and Vibrio cholera O1 pathogens were detected by the dual-component magnetron electrochemical biosensor and plate culture counting technique.Both the correlation coefficient and slope were close to 1,indicating a good agreement between the study results of the proposed biosensor and those from the plate count method.Conclusion1.Cascade amplification technology based on RCA@HCR amplification initiated by DNA 3D Walker,can realize the triple amplification of the nucleotide sequence,construct the nucleotide tree molecular sequence to efficiently supporte the electronic medium Fc-Dox,improve the responding signals of the electrochemical biosensor,and improve the analytical performance of electrochemical biosensor.2.A novel high-sensitive electrochemical biosensor for the rapid detection of E.coli O157:H7 was constructed based on cascade sensitizing system,which was induced by DNA 3D walker and RCA@HCR nucleotide amplification reaction.The cascade sensitizing electrochemical biosensor has the rapid detection time and the high sensitivity,which can provide a new method for the early diagnosis of pathogenic microorganisms.3.In this paper,a novel magnetron electrochemical flow detection cell was designed and developed,and the electrochemical sensing analysis is carried out on the surface of the electrode by moving the magnet to fix the magnetic beads onto the glassy carbon electrode surgace to perform the electrochemical assay.After the every detection,the magnetic beads,which were adsorbed on the surface of the electrode,could be washed away and the glassy carbon electrode could be regenerated for the next detection.In addition,the flow cell showed the advantages of simple structure,easy preparation and good versatility,and could effectively realize the coordinated application of various target detection systems.4.Based on the magnetron electrochemical flow detection cell,a dual-component magnetron electrochemical biosensor with high efficiency and high sensitivity was constructed based on the excellent signal amplification efficiency of C₆₀@AuNPs and HCR reaction.5.The parallel detection of E.coli O157:H7 and Vibrio cholera O1 pathogens can be realized by using the dual-component magnetron electrochemical biosensor,which has excellent detection performance.At the same time,the dual-component magnetron electrochemical biosensor has good specificity,repeatability and stability.The proposed biosensor provides a new method of immunoassay for early clinical diagnosis of pathogens infection.