Study On A Novel Electrochemical Immunosensor For Serous Myeloperoxidase

Objective:Myeloperoxidase (MPO) is an abundant mammalian phagocytehemoprotein that is in culprit lesions of individuals with sudden cardiacdeath, such as those found in human atheroma thought to be a potentialparticipant in multiple phases of the atherosclerotic process. Biochemicaland genetic studies in humans demonstrate that blood-MPO levels areindependently associated with the prevalence of cardiovascular diseases. Incontrast to cTnI, Myo and CK-MB, MPO can identify patients at risk forcardiac events in the absence of myocardial necrosis, as well as the risk ofmajor adverse cardiac events in the ensuing30-day and6-month periods.Therefore, determination of MPO plays an important role in early clinicaldiagnosis of ACS. Electrochemical immunosensors based on the specificityof immunoreaction and the sensitivity of electrochemical methods have beenwidely applied in the field of clinical diagnosis. In this study, we are aimingto construct a novel electrochemical immunosensor for MPO fast andsensitive detection to make up for the shortcomings of conventionalmethods. Methods:1. The composite film PoPD-MWCNTs-IL on the surface of indium tinoxide electrode electrode (ITO) was prepared by in situelectropolymerization using the ionic liquid as a supporting electrolyte.2. Negatively charged AuNPs were then adsorbed on the modifiedelectrode via amine-gold affinity and to immobilize MPO antibody.3. Bovine serum albumin was employed to block possible remainingactive sites on the AuNPs. The novel electrochemical immunosensor forMPO detection was completed fabricated.4. The modification of the electrode was characterized by cyclicvoltammetry and scanning electron microscopy.5. The factors affecting the performance of the immunosensor such asconcentration of oPD, pH, incubation temperature and time were studied indetail.6. Under the optimized experimental condition, the immunosensor wasincubated in different concentrations of the MPO standard solution. Thestandard curve was prepared according to the relationship between MPOconcentration and current change. The selectivity, stability, repeatability andaccuracy of the immunosensor were also analyzed in detail.7. The serum samples were measured by both the method and ELISA inorder to investigate whether the new immunosensor can be applied to humanserum samples analysis. Results:1. The SEM images show that the modification process and theformation of anti-MPO layer on AuNPs/PoPD-MWCNTs-IL/ITO electrodewere successfully. The CV of anti-MPO/AuNPs/PoPD-MWCNTs-IL/ITOobtained in PBS (pH7.0) containing1mmol/L [Fe(CN)3-/4-6]and0.1mol/LKCl showed high peak current. The anodic and cathodic peak currents wereproportional to the square root of the scan rates, suggesting a diffusioncontrolled redox process.2. When the concentration of oPD was0.05mol/L, pH value was7.0,incubation temperature was35℃and incubation time was20min, theimmunosensor had superior behavior. Under the optimized experimentalcondition, the immunosensor show good response to MPO in the range from0.2to23.4ng/mL (I=﹣0.4793C+93.460，R2=0.995) and23.4to300ng/mL (I=﹣0.0739C+84.7023，R2=0.998). The detection limit wascalculated to be0.07ng/mL at a signal-to-noise ratio of3(S/D=3).3. The peak current responses in the MPO solutions with and withoutinterference showed a difference of less than5.7%. When the immunosensorwas stored in the refrigerator at4℃for30days, there was no obviousdecline (about8%) in current response. The results of intra-and inter-assaywere3.4%and4.7%respectively, and the results of recovery test rangedfrom92.44%-103.2%.Conclusion: 1. The AuNPs/PoPD-MWCNTs-IL composite prepared by in situelectropolymerization could increase the amount of MPO loading and keeptheir bioactivity. At the same time, the composite improved theelectroactivity of PoPD, greatly enhanced current response and increased thedetection limit.2. The performance of the immunosensor in this study has beenimproved compared with that of previous work attributing to its wide linearrange, high selectivity and stability and low detection limit.