Construction Of ZnO Sol-gel Electrochemical Biosensor And Accurate Discrimination For RhEPO/EPO Of Peptide Stimulants

Objectives:The aim of this study was to construct an ultrasensitive electrochemical biosensordetection system to rapidly detect the rhEPO/EPO, and to examine the superiority of thesystem by detecting the rhEPO/EPO.Methods:1. Zn （CH₃COO）22H₂O was weighed and added into absolute alcohol. Then,LiOH H₂O was weighed and added into absolute alcohol, followed by sonication at roomtemperature to facilitate dissolution.2. To prepare the recognition element of the novel sensor, the surface of a glassycarbon electrode （GCE） was modified with erythropoietin receptor （EPOR） using ZnOsol-gel as matrix.3. The rhEPO/EPO electrochemical biosensor system was made up of an EPORmodified GCE as working electrode, a saturated calomel electrode as reference electrode, aplatinum electrode as counter electrode.4.2mmol/L K₃[Fe（CN）₆]/K₄[Fe（CN）₆] PBS solution （pH6.2⁹.0,0.05mol/L） as testbase solution. The electrochemical properties of EPOR modified electrodes werecharacterized by cyclic voltammetry. The electric potential scanned ranged between-0.9Vand0.7V, and the electric potential scanning speed was10¹00mV s-1.5. The sample solution’s erythropoietin concentration is calculated according to thepeak current at the potential of0.14V⁰.17V and the erythropoietin standard curve, and/or the sample solution’s concentration of recombinant human erythropoietin is calculatedaccording to the peak current at the potential of0.06V⁰.09V and the recombinant humanerythropoietin standard curve, and according to the correspondence between the current change and rhEPO/EPO to determine the detection sensitivity and detection range of theelectrochemical biosensor.6. To explore the sensitivity, specificity, reproducibility and stability of the the ZnO sol-gel electrochemical biosensor to detects rhEPO/EPO through a series of experiments.7. sandwich-type nano-Au/ZnO sol-gel/nano-Au signal amplification to improve thedetection sensitivity of the biosensor, optimize time optimization for nano-Auelectrodeposition and concentration.Results:1. Optimum the dilution ratio of ZnO sol gel and absolute alcohol is1:2, the dilutionratio of ZnO sol-gel and receptor is1:1, when ZnO sol-gel pH is9.0, there caused thechanges in the value of the maximum peak current response.2. The most preferred pH is7.4, incubation time is20minutes, scanning potentialwithin-0.3V⁰.7V, EPOR concentration is1μg/L affects the sensor’s current responsemarkedly.3. The sol-gel facilitates maintenance of protein bioactivity, and it is also highlyporous, thermally stable, chemically inert and biocompatible. Because rhEPO and EPOhave different isoelectric points, rhEPO-EPOR complexes and EPO-EPOR complexesexhibit different working potentials. So, EPO and rhEPO can be discriminated accurately.4. The current response firstly enhanced and then tended gently for hybridization withrhEPO/EPO, when concentration of rhEPO/EPO varied from5pg/L to5μg/L, and500n g/Lwas the saturation point. The rhEPO statistic linear regression equation wasy=1.5737x+14.765among the range of rhEPO concentration from5pg/L to500ng/L and thecorrelating coefficient was0.9935. The EPO statistic linear regression equation wasy=2.1674x+17.691among the range of target concentration from5pg/L to500ng/L and thecorrelating coefficient was0.9966.5. The intraassay and interassay CV for the ZnO sol-gel electrochemical biosensorwere3.99%and7.21%for0.10μg/L,3.29%and5.15%for1.00μg/L,6.76%and8.44%for10.00μg/L respectively. The intraassay mean CV was4.39%and the interassay mean CVwas6.13%. Both the CVs were lower than10%.6. The response current of the EPOR modified biosensor was95%,82%, and77%of its original value after20d,40d, and50d of storage in the dark at4°C. Meanwhile, theEPOR modified biosensor with a coefficient of variability of2.79%,7.59%, and10.50%.After60d of storage, the current response curve of the EPOR modified sensor was verysimilar to that of the unmodified electrode.7. Shows the results of biosensor detection of EPO, rhEPO, and interfering substances,respectively.The response currents of the electrode remained largely unchanged before andafter incubation in solution containing interfering substances only. In contrast, afterincubation in solution containing EPO/rhEPO, the electrode’s response current changevalue （I） was8.2μA and9.7μA, respectively.8. The first round of nano-Au electrodeposition is60s, the second round of nano-Auelectrodeposition is30s, nano-Au concentration is10mmol/L, and the sensor’s currentreached its maximum.9. Under the optimal experimental conditions, various concentrations of rhEPO/EPOwere detected. The rhEPO statistic linear regression equation was y=3.7068x+31.796among the range of rhEPO concentration from5pg/L to500ng/L and the correlatingcoefficient was0.9912. The EPO statistic linear regression equation was y=3.4389x+29.685among the range of target concentration from5pg/L to500ng/L and the correlatingcoefficient was0.9925.10. The response current of the nano-Au-modified biosensor was97.45%,94.33%,83.29%and75.07%of its original value after10d,30d,50d,70d of storage in the dark at4°C. Meanwhile, the EPOR modified biosensor with a coefficient of variability of1.83%,2.57%,6.38%and10.48%. After80d of storage, the current response curve of the EPORmodified sensor was very similar to that of the unmodified electrode.11. The intraassay and interassay CV for nano-Au ZnO sol-gel electrochemicalbiosensor were2.22%and4.32%for0.10μg/L,2.25%and4.17%for1.00μg/L,2.40%and3.68%for10.00μg/L respectively.12. Shows the results of nano-Au-modified biosensor detection of EPO, rhEPO, andinterfering substances, respectively.The response currents of the electrode remained largelyunchanged before and after incubation in solution containing interfering substances only. Incontrast, after incubation in solution containing EPO/rhEPO, the electrode’s response current change value （I） was29.50μA and27.31μA, respectively. After incubation insolution containing rhEPO and interfering substances, the electrode’s response currentchange value （I） was28.63μA. After incubation in solution containing EPO andinterfering substances, the electrode’s response current change value （I） was26.42μA.13. According to the standard curves of biosensor determined serum rhEPO and EPOconcentrations in a patient treated with rhEPO and a healthy volunteer. Then, serumsamples were diluted, generating analyte solutions with various concentrations of rhEPOand EPO. The rhEPO statistic linear regression equation was y=3.647x+13.555amongthe range of rhEPO concentration from0.125ng/L to125ng/L and the correlatingcoefficient was0.9893. The EPO statistic linear regression equation was y=3.180x+12.380among the range of target concentration from0.073ng/L to73ng/L and thecorrelating coefficient was0.9925.Conclusions:1. We prepared an electrochemical biosensor which allows fast and accuratediscrimination of rhEPO/EPO by utilizing the minor difference in isoelectric point betweenrhEPO and EPO.2. The detectability of the electrochemical biosensor was5pg/L. It was greatly moresensitive than other detection. It could quickly accurate discrimination rhEPO/EPO.3. ZnO sol-gel is highly absorbable and persistently maintains biological activity ofEPOR on the electrode surface.4. The intraassay CV showed very low SD（mean CV＝3.89%） and the interassay CVhad a slightly higher SD（mean CV＝6.16%）. However, they were both lower than10%.Therefore, our study indicated that the reproducibility of the ZnO sol-gel electrochemicalbiosensor system was satisfactory.5. The biosensor exhibited strong resistance to interference and was highly selectivefor rhEPO and EPO.6. rhEPO/EPO from clinical samples could be directly detected with ZnO sol-gelelectrochemical biosensor. Biosensor was highly selective for rhEPO and EPO, and lesstime was consumed.7. The sensitivity was effectively improved and the detection time was significantly shortened by applying nano-Au complex to the ZnO sol-gel. The electrochemical biosensorhas a wider linear range, and have good sensitive, stability, reproducibility and specificity.