ZnO溶胶—凝胶电化学生物传感器的构建及精准甄别肽类兴奋剂rhEPO/EPO的实验研究
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摘要
目的：
构建超灵敏ZnO溶胶-凝胶电化学生物传感器，实现肽类兴奋剂rhEPO/EPO的快速精准甄别，并通过系列实验验证传感器甄别检测rhEPO/EPO的有效性和优越性。
方法：
1.将乙酸锌溶于无水乙醇中，在超声波作用下加入氢氧化锂，制备ZnO溶胶-凝胶溶液。
2.利用电极表面化学修饰技术，将促红细胞生成素受体通过ZnO溶胶-凝胶固定于玻碳电极表面，制备促红细胞生成素受体修饰电极，作为传感器的识别元件。
3．以促红细胞生成素受体修饰电极为工作电极、铂电极为对电极、饱和甘汞电极为参比电极，构建成促红细胞生成素和/或重组人促红细胞生成素电化学生物传感器。
4.以含有2mmol/L K_3Fe(CN)_6/K_4Fe(CN)_6的磷酸盐缓冲液(6.2~9.0，0.05mol/L)为测试底液，采用循环伏安法进行扫描测定，电位扫描范围为-0.9V～0.7V，电位扫描速率为10~100mv/s，进修样品检测。
5.根据电位0.14~0.17V处的峰电流和促红细胞生成素标准曲线计算样品溶液中促红细胞生成素的浓度，和/或根据电位0.06~0.09V处的峰电流和重组人促红细胞生成素标准曲线计算样品溶液中rhEPO/EPO的浓度。根据电流的变化和rhEPO/EPO浓度之间对应的关系来确定生物传感器的检测灵敏度及检测范围。
6.通过系列实验研究，探索ZnO溶胶-凝胶电化学生物传感器检测rhEPO/EPO灵敏度、重复性、稳定性及特异性等传感器的性能参数，并对rhEPO治疗患者的临床血清样本进行检测，并进行方法学比较。
7.用三明治型“纳米金-ZnO溶胶凝胶-纳米金”为生物信号放大系统提高传感器的检测灵敏度，探索并优化两次纳米金电沉积的最佳时间和纳米金的反应浓度。
结果：
1.当ZnO溶胶-凝胶的贮备液与无水乙醇最佳稀释比为1：2, ZnO溶胶-凝胶与EPOR体积比为1：1,电极的溶胶-凝胶的最优pH值为9.0时修饰的ZnO溶胶-凝胶EPOR电极在杂交反应前后引起的峰电流响应变化值最大。
2.自主创建的ZnO溶胶-凝胶电化学生物传感器在电解液的最优pH值为7.4,工作电位为-0.3V-0.7V, EPOR浓度为1μg/L,孵育时间为20min时,传感器杂交反应前后引起的电流响应变化值最大。
3.利用ZnO溶胶-凝胶具有多孔性、高的热稳定性和化学惰性及良好的生物兼容性等优点,采用循环伏安法进行扫描测定，由于EPO与rhEPO有等电点的差异,导致EPOR-rhEPO复合物与EPOR-EPO复合物的工作电位略有差异，从而将EPO和rhEPO精确甄别。
4.随着EPO/rhEPO浓度从5pg/L到5μg/L变化时，孵育前后所引起的电流响应变化值先上升后趋于饱和，以500ng/L为饱和点；在5pg/L到500ng/L的EPO/rhEPO浓度范围内，rhEPO的线性回归方程为：y=1.5737x+14.765，相关系数为0.9935; EPO的线性回归方程为：y=2.1674x+17.691，相关系数0.9966。
5. EPOR浓度分别是0.10、1.00及10.00μg/L时，天内重复性和天间重复性的实验CV值分别为3.99%和7.21%，3.29%和5.15%，6.76%和8.44%。上述三种不同EPOR浓度的天内和天间重复性实验平均CV值分别是4.39%和6.13%，均小于10%。
6. EPOR修饰传感器4℃避光放置20天、40天、50天后其响应电流分别为初始值的95%、82%、77%，变异系数分别为是2.79%、7.59%、10.50%。传感器放置60天后的电流响应曲线与裸电极的电流响应极其相似，传感器响应电流的变化较小。
7.传感器分别检测EPO、rhEPO和干扰物质的实验结果表明,在仅含有干扰物质的溶液中孵育的电极在孵育前后响应电流基本保持不变，而在含有EPO和rhEPO溶液中孵育的电极在孵育前后响应电流变化值(I)分别为8.2μA和9.7μA。
8. HAuCl4溶液第一次电沉积时间为60s，第二次电沉积时间为30s，HAuCl4溶液浓度为10mmol/L时，传感器的响应电流变化值最大(I)。
9.通过实验的优化，自主创建的纳米金溶胶-凝胶电化学生物传感器的检测rhEPO/EPO线性范围是5pg/L到500n g/L，rhEPO的线性回归方程为：y=3.7068x+31.796，相关系数为0.9912，EPO的线性回归方程为：y=3.4389x+29.685，相关系数0.9925。
10.纳米金/凝胶受体/纳米金修饰电极4℃避光放置10天、30天、50天、70天后其响应电流分别为初始值的97.45%、94.33%、83.29%、75.07%，变异系数分别为是1.83%、2.57%、6.38%、10.48%。传感器放置80天后的峰电流变化较小，电流响应曲线与阴性电极的电流响应极其相似。
11.纳米金/凝胶受体/纳米金生物传感器的EPOR浓度为0.1.00μg/L时，其天内和天间重复性实验的CV值分别为2.22%和4.32%；EPOR浓度是1.00μg/L时，天内及天间的重复性实验CV值分别是2.25%和4.17%；当EPOR浓度是100.00μg/L时，其天内和天间重复性实验的CV值分别为2.40%和3.68%%。
12.纳米金/凝胶受体/纳米金修饰电化学生物传感器分别检测EPO、rhEPO和干扰物质的实验结果表，而在含有rhEPO和EPO溶液中孵育的电极在孵育前后响应电流变化值(I)分别为29.50μA和27.31μA，在含有rhEPO及干扰物质和EPO及干扰物质溶液中中孵育的电极在孵育前后响应电流变化值(I)分别为28.63μA和26.42μA，在仅含有干扰物质的溶液中孵育的电极在孵育前后响应电流基本保持不变。
10.根据传感器的线性曲线计算rhEPO治疗的患者及健康志愿者血清样本中浓度，将血清样品浓度倍比稀释，rhEPO浓度在0.125ng/L~125ng/L的范围内时，其线性回归方程为：y=3.647x+13.555，相关系数为0.9893； EPO浓度在0.073ng/L~73ng/L的范围内时，其线性回归方程为：y=3.180x+12.380，相关系数0.9891。
结论：
1.自主创建了rhEPO/EPO精确甄别的电化学生物传感器，利用二者只有等电点的细微差别进行检测，实现rhEPO和EPO的快速精确区分。
2. ZnO溶胶-凝胶电化学生物传感器可达到pg/L级的检测限灵敏度，与其他检测方法相比，其检测灵敏度有了很大提高，并实现了rhEPO/EPO的快速甄别检测。
3. ZnO sol-gel具有很强的吸附能力，并可使电极表面修饰的EPOR保持良好的生物活性，因而确保了传感器的稳定性。
4.三种不同EPOR浓度的天间精密性实验的平均CV值为6.16%，略高于天内精密性实验的平均CV值3.89%，但二者均小于10%。因此，ZnO溶胶-凝胶电化学传感器具有良好的重复性，从而保证了检测结果的可靠性。
5.自主构建的电化学生物传感器抗干扰能力强，对EPO和rhEPO具有良好的选择性，并能对EPO和rhEPO进行精确甄别。
6. ZnO溶胶-凝胶电化学生物传感器实现了对临床标本中rhEPO/EPO的直接检测。与其他定量方法相比，ZnO溶胶-凝胶电化学生物传感器法不但对rhEPO/EPO进行了定量检测，而且还精确甄别，所用检测时间更短。
7.纳米金颗粒与ZnO溶胶-凝胶相结合可以有效提高传感器的检测灵敏度，线性范围更宽，并且灵敏度、稳定性、重复性、特异性均较好。
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 (CH_3COO)22H_2O was weighed and added into absolute alcohol. Then,LiOH H_2O 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 K_3[Fe(CN)_6]/K_4[Fe(CN)_6] PBS solution (pH6.2~9.0,0.05mol/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 was10~100mV s-1.
5. The sample solution's erythropoietin concentration is calculated according to thepeak current at the potential of0.14V~0.17V 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 of0.06V~0.09V 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-0.3V~0.7V, 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.
构建超灵敏ZnO溶胶-凝胶电化学生物传感器，实现肽类兴奋剂rhEPO/EPO的快速精准甄别，并通过系列实验验证传感器甄别检测rhEPO/EPO的有效性和优越性。
方法：
1.将乙酸锌溶于无水乙醇中，在超声波作用下加入氢氧化锂，制备ZnO溶胶-凝胶溶液。
2.利用电极表面化学修饰技术，将促红细胞生成素受体通过ZnO溶胶-凝胶固定于玻碳电极表面，制备促红细胞生成素受体修饰电极，作为传感器的识别元件。
3．以促红细胞生成素受体修饰电极为工作电极、铂电极为对电极、饱和甘汞电极为参比电极，构建成促红细胞生成素和/或重组人促红细胞生成素电化学生物传感器。
4.以含有2mmol/L K_3Fe(CN)_6/K_4Fe(CN)_6的磷酸盐缓冲液(6.2~9.0，0.05mol/L)为测试底液，采用循环伏安法进行扫描测定，电位扫描范围为-0.9V～0.7V，电位扫描速率为10~100mv/s，进修样品检测。
5.根据电位0.14~0.17V处的峰电流和促红细胞生成素标准曲线计算样品溶液中促红细胞生成素的浓度，和/或根据电位0.06~0.09V处的峰电流和重组人促红细胞生成素标准曲线计算样品溶液中rhEPO/EPO的浓度。根据电流的变化和rhEPO/EPO浓度之间对应的关系来确定生物传感器的检测灵敏度及检测范围。
6.通过系列实验研究，探索ZnO溶胶-凝胶电化学生物传感器检测rhEPO/EPO灵敏度、重复性、稳定性及特异性等传感器的性能参数，并对rhEPO治疗患者的临床血清样本进行检测，并进行方法学比较。
7.用三明治型“纳米金-ZnO溶胶凝胶-纳米金”为生物信号放大系统提高传感器的检测灵敏度，探索并优化两次纳米金电沉积的最佳时间和纳米金的反应浓度。
结果：
1.当ZnO溶胶-凝胶的贮备液与无水乙醇最佳稀释比为1：2, ZnO溶胶-凝胶与EPOR体积比为1：1,电极的溶胶-凝胶的最优pH值为9.0时修饰的ZnO溶胶-凝胶EPOR电极在杂交反应前后引起的峰电流响应变化值最大。
2.自主创建的ZnO溶胶-凝胶电化学生物传感器在电解液的最优pH值为7.4,工作电位为-0.3V-0.7V, EPOR浓度为1μg/L,孵育时间为20min时,传感器杂交反应前后引起的电流响应变化值最大。
3.利用ZnO溶胶-凝胶具有多孔性、高的热稳定性和化学惰性及良好的生物兼容性等优点,采用循环伏安法进行扫描测定，由于EPO与rhEPO有等电点的差异,导致EPOR-rhEPO复合物与EPOR-EPO复合物的工作电位略有差异，从而将EPO和rhEPO精确甄别。
4.随着EPO/rhEPO浓度从5pg/L到5μg/L变化时，孵育前后所引起的电流响应变化值先上升后趋于饱和，以500ng/L为饱和点；在5pg/L到500ng/L的EPO/rhEPO浓度范围内，rhEPO的线性回归方程为：y=1.5737x+14.765，相关系数为0.9935; EPO的线性回归方程为：y=2.1674x+17.691，相关系数0.9966。
5. EPOR浓度分别是0.10、1.00及10.00μg/L时，天内重复性和天间重复性的实验CV值分别为3.99%和7.21%，3.29%和5.15%，6.76%和8.44%。上述三种不同EPOR浓度的天内和天间重复性实验平均CV值分别是4.39%和6.13%，均小于10%。
6. EPOR修饰传感器4℃避光放置20天、40天、50天后其响应电流分别为初始值的95%、82%、77%，变异系数分别为是2.79%、7.59%、10.50%。传感器放置60天后的电流响应曲线与裸电极的电流响应极其相似，传感器响应电流的变化较小。
7.传感器分别检测EPO、rhEPO和干扰物质的实验结果表明,在仅含有干扰物质的溶液中孵育的电极在孵育前后响应电流基本保持不变，而在含有EPO和rhEPO溶液中孵育的电极在孵育前后响应电流变化值(I)分别为8.2μA和9.7μA。
8. HAuCl4溶液第一次电沉积时间为60s，第二次电沉积时间为30s，HAuCl4溶液浓度为10mmol/L时，传感器的响应电流变化值最大(I)。
9.通过实验的优化，自主创建的纳米金溶胶-凝胶电化学生物传感器的检测rhEPO/EPO线性范围是5pg/L到500n g/L，rhEPO的线性回归方程为：y=3.7068x+31.796，相关系数为0.9912，EPO的线性回归方程为：y=3.4389x+29.685，相关系数0.9925。
10.纳米金/凝胶受体/纳米金修饰电极4℃避光放置10天、30天、50天、70天后其响应电流分别为初始值的97.45%、94.33%、83.29%、75.07%，变异系数分别为是1.83%、2.57%、6.38%、10.48%。传感器放置80天后的峰电流变化较小，电流响应曲线与阴性电极的电流响应极其相似。
11.纳米金/凝胶受体/纳米金生物传感器的EPOR浓度为0.1.00μg/L时，其天内和天间重复性实验的CV值分别为2.22%和4.32%；EPOR浓度是1.00μg/L时，天内及天间的重复性实验CV值分别是2.25%和4.17%；当EPOR浓度是100.00μg/L时，其天内和天间重复性实验的CV值分别为2.40%和3.68%%。
12.纳米金/凝胶受体/纳米金修饰电化学生物传感器分别检测EPO、rhEPO和干扰物质的实验结果表，而在含有rhEPO和EPO溶液中孵育的电极在孵育前后响应电流变化值(I)分别为29.50μA和27.31μA，在含有rhEPO及干扰物质和EPO及干扰物质溶液中中孵育的电极在孵育前后响应电流变化值(I)分别为28.63μA和26.42μA，在仅含有干扰物质的溶液中孵育的电极在孵育前后响应电流基本保持不变。
10.根据传感器的线性曲线计算rhEPO治疗的患者及健康志愿者血清样本中浓度，将血清样品浓度倍比稀释，rhEPO浓度在0.125ng/L~125ng/L的范围内时，其线性回归方程为：y=3.647x+13.555，相关系数为0.9893； EPO浓度在0.073ng/L~73ng/L的范围内时，其线性回归方程为：y=3.180x+12.380，相关系数0.9891。
结论：
1.自主创建了rhEPO/EPO精确甄别的电化学生物传感器，利用二者只有等电点的细微差别进行检测，实现rhEPO和EPO的快速精确区分。
2. ZnO溶胶-凝胶电化学生物传感器可达到pg/L级的检测限灵敏度，与其他检测方法相比，其检测灵敏度有了很大提高，并实现了rhEPO/EPO的快速甄别检测。
3. ZnO sol-gel具有很强的吸附能力，并可使电极表面修饰的EPOR保持良好的生物活性，因而确保了传感器的稳定性。
4.三种不同EPOR浓度的天间精密性实验的平均CV值为6.16%，略高于天内精密性实验的平均CV值3.89%，但二者均小于10%。因此，ZnO溶胶-凝胶电化学传感器具有良好的重复性，从而保证了检测结果的可靠性。
5.自主构建的电化学生物传感器抗干扰能力强，对EPO和rhEPO具有良好的选择性，并能对EPO和rhEPO进行精确甄别。
6. ZnO溶胶-凝胶电化学生物传感器实现了对临床标本中rhEPO/EPO的直接检测。与其他定量方法相比，ZnO溶胶-凝胶电化学生物传感器法不但对rhEPO/EPO进行了定量检测，而且还精确甄别，所用检测时间更短。
7.纳米金颗粒与ZnO溶胶-凝胶相结合可以有效提高传感器的检测灵敏度，线性范围更宽，并且灵敏度、稳定性、重复性、特异性均较好。
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 (CH_3COO)22H_2O was weighed and added into absolute alcohol. Then,LiOH H_2O 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 K_3[Fe(CN)_6]/K_4[Fe(CN)_6] PBS solution (pH6.2~9.0,0.05mol/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 was10~100mV s-1.
5. The sample solution's erythropoietin concentration is calculated according to thepeak current at the potential of0.14V~0.17V 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 of0.06V~0.09V 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-0.3V~0.7V, 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.
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