WO2014036772A1 - 促红细胞生成素受体修饰电极及其制备方法和应用 - Google Patents

促红细胞生成素受体修饰电极及其制备方法和应用 Download PDF

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WO2014036772A1
WO2014036772A1 PCT/CN2012/082621 CN2012082621W WO2014036772A1 WO 2014036772 A1 WO2014036772 A1 WO 2014036772A1 CN 2012082621 W CN2012082621 W CN 2012082621W WO 2014036772 A1 WO2014036772 A1 WO 2014036772A1
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electrode
erythropoietin
solution
modified electrode
gel
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PCT/CN2012/082621
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French (fr)
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王云霞
张立群
府伟灵
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中国人民解放军第三军医大学第一附属医院
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Priority to CA2809647A priority Critical patent/CA2809647C/en
Priority to US14/007,350 priority patent/US8986535B2/en
Priority to CH00900/13A priority patent/CH707252B1/de
Publication of WO2014036772A1 publication Critical patent/WO2014036772A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D3/00—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials
    • B05D3/002—Pretreatement
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48714—Physical analysis of biological material of liquid biological material by electrical means for determining substances foreign to the organism, e.g. drugs or heavy metals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/746—Erythropoetin

Definitions

  • the invention belongs to the technical field of electrochemical detection, relates to a modified electrode and a preparation method thereof, and relates to an electrochemical biosensor formed by using the modified electrode as a working electrode and a detection method thereof.
  • Erythropoietin is a glycoprotein hormone, a hematopoietic growth factor produced by the kidneys in the human body. Its physiological function is to promote the production and release of bone marrow red blood cells.
  • humans first synthesized recombinant human erythropoietin (recombinant) using genetic engineering techniques.
  • Human Erythopoietin (rhEPO) due to its dual function of mitogen and differentiation, can produce blood transfusion without blood transfusion, so that patients can avoid the risk of viral infection and excessive blood transfusion, and have played an important role in the treatment of renal anemia. effect.
  • rhEPO has become a new stimulant in sports because of its ability to improve the oxygen carrying capacity of the body and enhance exercise endurance. Since 2005, in the banned list of the International Olympic Committee and the World Anti-Doping Agency, rhEPO has been listed as the first peptide substance banned in sports competition.
  • EPO and rhEPO have the same biological activity and basically the same molecular structure, the only difference is that the isoelectric point is different, the isoelectric point of EPO is 3.7 ⁇ 4.7, and the isoelectric point of rhEPO is 4.4 ⁇ 5.1, so the EPO is accurately distinguished.
  • one of the objects of the present invention is to provide a modified electrode, and the second object thereof is to provide a method for preparing the modified electrode, and the third objective is to provide an electrochemical method using the modified electrode as a working electrode.
  • the fourth object of the invention is to provide a method for detecting EPO and/or rhEPO by using the electrochemical biosensor, which is simple in preparation and stable in performance, and the electrochemical biosensor formed by using the working electrode can be fast, Specific and sensitive detection of EPO and / or rhEPO, especially for rapid and accurate screening of EPO and rhEPO.
  • the present invention provides the following technical solutions:
  • An erythropoietin receptor (EPOR) modified electrode is a glassy carbon electrode in which an EPOR is immobilized as a recognition element by a ZnO sol-gel on the surface of the electrode.
  • the preparation method of the EPOR modified electrode comprises the following steps:
  • Pretreatment of glassy carbon electrode polishing the surface of the glassy carbon electrode, cleaning, drying, and standby;
  • EPOR solution is mixed with the ZnO sol-gel solution prepared in step b, added dropwise to the surface of the glassy carbon electrode pretreated by the step a, dried to form a film, and washed to prepare an EPOR modified electrode.
  • the step a is to sequentially use the glassy carbon electrode
  • the 0.3 ⁇ m and 0.05 ⁇ m aluminum oxide powders were polished and polished, washed with water each time after grinding, and then ultrasonically washed with nitric acid, acetone and water, and air-dried.
  • step b zinc acetate is dissolved in absolute ethanol to prepare a solution having a concentration of 0.1 mol/L, and lithium hydroxide is added under ultrasonic wave to a final concentration of 0.067 mol/L to prepare a ZnO sol.
  • a gel stock solution is prepared by diluting with anhydrous ethanol at a volume ratio of 2:1 to 1:3 before use to prepare a ZnO sol-gel solution.
  • the step b is to dissolve zinc acetate in anhydrous ethanol to prepare a solution having a concentration of 0.1 mol/L, and then add lithium hydroxide to a final concentration of 0.067 mol/L under ultrasonic wave to obtain ZnO.
  • the sol-gel stock solution was diluted with anhydrous ethanol at a volume ratio of 1:2 before use to prepare a ZnO sol-gel solution.
  • the ZnO sol-gel solution prepared in the step b and the EPOR solution having a concentration of 10 ng/L to 100 ⁇ g/L are uniformly mixed according to a volume ratio of 4:1 to 1:1.15, and then mixed.
  • the droplets were applied to the surface of the glassy carbon electrode pretreated in the step a, dried in the air, and thoroughly washed with a phosphate buffer to prepare an EPOR-modified electrode.
  • the step c is that the ZnO sol-gel solution prepared in the step b is uniformly mixed with the EPOR solution having a concentration of 1 ⁇ g/L in a volume ratio of 1:1, and then the mixed liquid droplet is added in the step a.
  • the surface of the pretreated glassy carbon electrode was dried in air and thoroughly washed with phosphate buffer (PBS) to prepare an EPOR modified electrode.
  • PBS phosphate buffer
  • the EPO and rhEPO electrochemical biosensor comprises a working electrode, a counter electrode, a reference electrode and a test bottom liquid, the working electrode is the aforementioned EPOR modified electrode, the counter electrode is a platinum electrode, and the reference electrode is a saturated calomel electrode;
  • the test bottom solution contains 2 mmol/L K 3 [Fe(CN) 6 ] and 2 mmol/L K 4 [Fe(CN) 6 ] (subsequently abbreviated as 2 mmol/L K 3 [Fe(CN) 6 ]-K 4 [Fe(CN) 6 )) Phosphate buffer with a pH of 6.2 to 9.0.
  • the test bottom liquid is a phosphate buffer solution containing 2 mmol/L K 3 [Fe(CN) 6 ]-K 4 [Fe(CN) 6 ] and having a pH of 7.4.
  • the method for detecting EPO and/or rhEPO by using the aforementioned EPO and rhEPO electrochemical biosensor is to incubate the EPOR modified electrode with the sample solution for more than 20 minutes, and then use the EPOR modified electrode as the working electrode, the platinum electrode as the counter electrode, and the saturated calomel.
  • the electrode was a reference electrode, a phosphate buffer containing 2 mmol/L K 3 [Fe(CN) 6 ]-K 4 [Fe(CN) 6 ] and a pH of 6.2-9.0 was used as a test liquid to construct an electrochemical biosensor. Cyclic voltammetry was used for scanning measurement.
  • the potential scanning range was -0.3V ⁇ 0.7V, the potential scanning rate was 10 ⁇ 100mv/s, and the EPO concentration in the sample solution was calculated according to the peak current at the potential of 0.14 ⁇ 0.17V and the EPO standard curve. And/or calculate the concentration of rhEPO in the sample solution based on the peak current at a potential of 0.06 to 0.09 V and the rhEPO standard curve.
  • the EPOR modified electrode is co-incubated with the sample solution for 20 minutes, and the potential scanning rate is 50 mv/s.
  • the invention has the advantages that the preparation method of the EPOR modified electrode of the invention is simple and the performance is stable, and the response current value after being kept away from the light for 4 days at 4 ° C is still maintained at about 77% of the initial value, and the three electrodes constructed as the working electrode are used.
  • the system electrochemical biosensor can detect EPO and/or rhEPO quickly, specifically and sensitively.
  • the linear range is 5pg/L ⁇ 500ng/L, and the detection limit is as low as 0.5pg/L, especially according to the peak potential EPO.
  • Quick and accurate screening with rhEPO not only for the detection of low concentration EPO or rhEPO, but also for the detection of stimulant rhEPO in sports competition.
  • Figure 1 shows the effect of the dilution ratio of ZnO sol-gel stock solution and absolute ethanol on the current response of the EPOR modified electrode.
  • Figure 2 shows the effect of the volume ratio of ZnO sol-gel solution to EPOR solution on the current response of the EPOR modified electrode.
  • Figure 3 shows the effect of EPOR solution concentration on the current response of the EPOR modified electrode.
  • Figure 4 shows the effect of the pH of the test solution on the current response of EPO and rhEPO electrochemical biosensors.
  • Figure 5 shows the effect of incubation time of the working electrode in the sample solution on the current response of the EPO and rhEPO electrochemical biosensors.
  • Figure 6 shows the effect of cyclic voltammetric scanning potential on the current response of EPO and rhEPO electrochemical biosensors.
  • Figure 7 shows the electrochemical response and sensor specific experimental results of an electrochemical biosensor constructed using an EPOR modified electrode as a working electrode.
  • a is the cyclic voltammetry curve of a simple ZnO sol-gel modified electrode in PBS solution
  • b is a bare glassy carbon electrode containing 2 mmol/L K 3 [Fe(CN) 6 ]-K 4 [Fe(CN) 6 ] Cyclic voltammetry curve in PBS solution
  • c is the cyclic volt of pure ZnO sol-gel modified electrode in PBS solution containing 2mmol/L K 3 [Fe(CN) 6 ]-K 4 [Fe(CN) 6 ]
  • d is the cyclic voltammetry curve of the EPOR modified electrode in PBS solution containing 2mmol/L K 3 [Fe(CN) 6 ]-K 4 [Fe(CN) 6 ]
  • e is the EPOR modified electrode in the interfering substance solution Cyclic voltammetry curve
  • Figure 8 shows the EPO standard curve and rhEPO standard curve obtained by EPO and rhEPO electrochemical biosensors under optimal conditions.
  • Figure 9 shows the change in current response of EPO and rhEPO electrochemical biosensors after storage for different times.
  • the main reagents and instruments used in the examples are as follows: Lithium hydroxide monohydrate LiOH•H 2 O, zinc acetate dihydrate Zn(Ac) 2 •2H 2 O was purchased from Shanghai Shenggong Bioengineering Co., Ltd., K 3 [Fe (CN) 6 ], K 4 [Fe(CN) 6 ] was purchased from Chongqing Oriental Reagent Factory, glassy carbon electrode, saturated calomel electrode, platinum electrode, 0.3 ⁇ m and 0.05 ⁇ m Al 2 O 3 powder were purchased from Tianjin Aida Hengyi Technology Development Co., Ltd., PBS powder was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., EPOR was purchased from Novus Biologicals, USA, and EPO and rhEPO standards were purchased from Abnova, USA.
  • KQ-5200B ultrasonic cleaner is the product of Kunshan Ultrasonic Instrument Co., Ltd. of Jiangsu Province
  • CHI660C electrochemical workstation is the product of Shanghai Chenhua Instrument Co., Ltd.
  • ZD-2 automatic potentiometric titrator is the product of Shanghai Jingke Lei Magnetic Co., Ltd.
  • the preparation method of the EPOR modified electrode specifically includes the following steps:
  • Pretreatment of glassy carbon electrode take a glassy carbon electrode with a diameter of 3mm, and polish it with 0.3 ⁇ m and 0.05 ⁇ m Al 2 O 3 powder in turn. After each grinding, wash it with ultrapure water, then sequentially with nitric acid. Ultrasonic washing in acetone and ultrapure water for 5 minutes, air drying;
  • ZnO sol-gel solution 2.20 g (0.01 mol) of Zn(Ac) 2 •2H 2 O was dissolved in 100 ml of absolute ethanol, and then slowly added LiOH•H 2 O 0.28 g (6.7 mmol) under ultrasonication.
  • the ZnO sol-gel stock solution is prepared, stored at 4 ° C for use, and diluted with absolute ethanol in a volume ratio of 1:2 before use to prepare a ZnO sol-gel solution;
  • an EPOR modified electrode prepared with different parameters was used as a working electrode, a saturated calomel electrode was used as a reference electrode, and a platinum electrode was used as a counter electrode to construct an electrochemical biosensor containing 2 mmol/L K 3 [Fe(CN) 6 ]-K 4 [ Fe(CN) 6 ] in PBS (pH 7.4, 0.05 mol / L) as a test solution, scanning at room temperature by cyclic voltammetry, potential scan range of -0.3V ⁇ 0.7V, potential scan rate It is 50mv/s.
  • ZnO sol-gel stock The dilution ratio of the liquid to the absolute ethanol is preferably 2:1 to 1:3, more preferably 1:2 (Fig. 1), and the volume ratio of the ZnO sol-gel solution to the EPOR solution is preferably 4:1 to 1:1.15. More preferably, it is 1:1 (Fig. 2), and the concentration of the EPOR solution is preferably from 10 ng/L to 100 ⁇ g/L, more preferably 1 ⁇ g/L (Fig. 3).
  • the EPOR modified electrode was incubated with the sample solution for 20 minutes, and then the EPO and rhEPO electrochemical biosensor were constructed with the EPOR modified electrode as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum electrode as the counter electrode to contain 2 mmol/L K 3 [ Fe(CN) 6 ]-K 4 [Fe(CN) 6 ] in PBS (pH 7.4, 0.05 mol/L) was used as the test solution, and was measured by cyclic voltammetry at room temperature.
  • the potential scanning range was -0.3V ⁇ 0.7V, the potential scanning rate is 50mv/s.
  • the main parameters affecting the current response of EPO and rhEPO electrochemical biosensors are also optimized in the research process.
  • the results show that the peak current of the sensor is larger when the pH of the test solution is 6.2 to 9.0, and the peak current of the sensor is the highest when the pH is 7.4. Therefore, the pH of the test solution is preferably 6.2 to 9.0, more preferably 7.4. (Fig.
  • the incubation time of the EPOR modified electrode with 500ng/L EPO or rhEPO standard solution increased from 5 minutes to 20 minutes, the peak current of the sensor gradually decreased and reached the minimum value, and then continued to increase the incubation time to 40 minutes, the peak The current remained substantially unchanged, indicating that the bound EPO or rhEPO on the EPOR modified electrode had reached saturation at 20 minutes, so the incubation time of the EPOR modified electrode with the sample solution was preferably 20 minutes or more, more preferably 20 minutes (Fig. 5).
  • the change of the scanning potential has little effect on the peak potential of the redox peak of K 3 [Fe(CN) 6 ]-K 4 [Fe(CN) 6 ], but the influence on the current response of the sensor is more obvious.
  • the best current response is between 0.3V and 0.7V ( Figure 6).
  • the potential scanning rate affects the shape of the cyclic voltammogram. The study found that the potential scanning rate is feasible in the range of 10 ⁇ 100 mv/s. When it is 50 mv/s, the cyclic voltammetry curve is the smoothest.
  • the EPOR modified electrode was incubated with the sample solution for 20 minutes, and then the EPOR modified electrode and the platinum electrode and the saturated reference electrode were combined to form an electrochemical biosensor containing 2 mmol/L K 3 [Fe(CN) 6 ]-K 4 [Fe(CN) 6 ) PBS solution (pH 7.4, 0.05mol/L) was used as the test solution, and was measured by cyclic voltammetry at room temperature.
  • the potential scanning range was -0.3V ⁇ 0.7V, and the potential scanning rate was 50mv/ s.
  • the specific experimental results of the sensor are shown in Fig. 7.
  • the curve a is the cyclic voltammetry curve of the pure ZnO sol-gel modified electrode in PBS solution, only the background current can be observed;
  • the curve b is the bare glass carbon electrode containing 2 mmol.
  • EPOR Since EPOR is a biomacromolecule, it is adsorbed on the surface of the electrode and hinders electron transport.
  • the peak current of the redox peak Further lower than curve c; curve e is the cyclic voltammetry curve of the EPOR modified electrode after incubation for 20 minutes in the interfering substance solution (500 ng/L IgA, 500 ng/L IgG and 500 ng/L IgM), compared with curve d, basic It remained unchanged, indicating that interfering substances such as IgA, IgG, and IgM did not affect the detection of EPO and rhEPO; curve f was the cyclic voltammetry curve of the EPOR modified electrode after incubation for 20 minutes in 500 ng/L EPO standard solution, and the response before and after incubation.
  • the current change value ( ⁇ I) is 8.2 ⁇ A, and the peak current appears at the potential of 0.16V.
  • the EPO-EPOR complex formed by the specific combination of EPO in the solution and EPOR on the electrode covers more electrode surface, further hindering. Electron transport, so the peak current of the redox peak is significantly lower than curve d; curve g is the cyclic voltammetry curve of the EPOR modified electrode after incubation for 20 minutes in 500ng/L rhEPO standard solution, and the response current change value ( ⁇ I) before and after incubation It is 9.7 ⁇ A, and the rhEPO-EPOR complex formed by the specific combination of rhEPO and EPOR hinders electron transport.
  • the peak current of redox peak is significantly lower than curve d, but the isoelectric point of rhEPO and EPO Similarly, the operating potential of the rhEPO-EPOR complex is different from that of the EPO-EPOR complex.
  • the redox peak of the curve g moves toward the negative potential, and the peak current appears at the potential of 0.08 V, thereby undergoing oxidation reduction.
  • the peak potential of the peak can accurately distinguish EPO from rhEPO.
  • the above experimental results show that the EPOR modified electrode constructed by the invention has strong anti-interference ability, good selectivity to EPO and rhEPO, and accurate screening detection of EPO and rhEPO.
  • the EPOR modified electrode was incubated with different concentrations of EPO standard solution and rhEPO standard solution for 20 minutes, and then an electrochemical biosensor was constructed with a platinum electrode and a saturated reference electrode to contain 2 mmol/L K 3 [Fe(CN) 6 ].
  • the potential scanning range is -0.3V ⁇ 0.7V, and the potential scanning rate is 50mv/s. The results are shown in Fig. 8.
  • the newly prepared EPOR modified electrode was placed in the dark at 4 ° C for 10, 20, 30, 40, 50, 60 days, and then an electrochemical biosensor was constructed with a platinum electrode and a saturated reference electrode to contain 2 mmol/L K 3 [Fe (CN) 6 ]-K 4 [Fe(CN) 6 ] in PBS (pH 7.4, 0.05 mol/L) as the test solution, and the cyclic voltammetry was used for scanning at room temperature to examine the EPOR modified electrode. Stability, potential sweep range is -0.3V ⁇ 0.7V, potential scan rate is 50mv / s. The results are shown in Fig. 9.
  • the response current value of the EPOR modified electrode was about 95% of the initial value; after 40 days of storage, the response current value decreased to 82% of the initial value; after 50 days of storage, the response current value remained at the initial value.
  • the value of about 77% indicates that the EPOR modified electrode of the present invention has good stability and a long service life.

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Abstract

本发明公开了一种促红细胞生成素受体修饰电极，是在电极表面通过ZnO溶胶-凝胶固定有促红细胞生成素受体作为识别元件的玻碳电极，该修饰电极制备方法简单、性能稳定，4℃避光放置50天后响应电流值仍维持在初始值的77%左右；以该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、含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的磷酸盐缓冲液为测试底液组建电化学生物传感器，可以快速、特异、灵敏地检测促红细胞生成素（EPO）和/或重组人促红细胞生成素（rhEPO），线性范围为5pg/L~500ng/L，检出限低至0.5pg/L，特别是能根据峰电位对EPO和rhEPO进行精确甄别，不但适用于低浓度EPO或rhEPO的检测，而且适用于体育竞技中对兴奋剂rhEPO的检测。

Description

促红细胞生成素受体修饰电极及其制备方法和应用 技术领域
本发明属于电化学检测技术领域，涉及一种修饰电极及其制备方法，还涉及以该修饰电极作为工作电极组建的电化学生物传感器及其检测方法。
 背景技术
促红细胞生成素（erythropoietin，EPO）是一种糖蛋白激素，在人体内主要是由肾脏生成的一种造血生长因子，其生理功能是促进骨髓红细胞的生成和释放。1985年人类首次利用基因工程技术合成了重组人促红细胞生成素（recombinant human erythopoietin，rhEPO），由于其具有分裂原和分化原的双重功能，能在不输血的情况下产生输血效应，使病人避免病毒感染和输血过量的危险，已在肾性贫血的治疗方面发挥出重要作用。同时，因其具有提高机体携氧能力、增强运动耐力的特性，rhEPO在体育运动中成为新的兴奋剂。2005年起，国际奥委会和世界反兴奋剂机构的禁用清单中，rhEPO被列为体育竞技中禁用的首个肽类物质。
由于EPO与rhEPO具有完全相同的生物活性和基本一致的分子结构，唯一差别仅在于等电点不同，EPO的等电点为3.7~4.7，rhEPO的等电点为4.4~5.1，因此精确区分EPO与rhEPO具有诸多困难。长期以来，EPO与rhEPO的甄别检测大多采用质谱、等电聚焦、凝胶电泳等方法相结合，但这些检测方法存在实验分离时间长、检测效率低、特异性差等缺点，不能满足对EPO和rhEPO进行快速、精确甄别的要求。因此，研究一种特异性好、灵敏度高、能够快速、精确甄别EPO和rhEPO的检测方法及工具势在必行。
 发明内容
有鉴于此，本发明的目的之一在于提供一种修饰电极，目的之二在于提供一种所述修饰电极的制备方法，目的之三在于提供一种以该修饰电极作为工作电极组建的电化学生物传感器，目的之四在于提供一种利用所述电化学生物传感器检测EPO和/或rhEPO的方法，所述修饰电极制备简单、性能稳定，以其为工作电极组建的电化学生物传感器可以快速、特异、灵敏地检测EPO和/或rhEPO，特别是能对EPO和rhEPO进行快速、精确甄别。
为达到上述目的，本发明提供如下技术方案：
1．促红细胞生成素受体（EPOR）修饰电极，所述修饰电极是在电极表面通过ZnO溶胶-凝胶固定有EPOR作为识别元件的玻碳电极。
2．EPOR修饰电极的制备方法，包括以下步骤：
a．玻碳电极的预处理：将玻碳电极表面抛光，清洁，干燥，备用；
b．ZnO溶胶-凝胶的制备：将乙酸锌溶于无水乙醇中，再在超声波作用下加入氢氧化锂，制得ZnO溶胶-凝胶溶液，备用；
c．EPOR的固定：将EPOR溶液与步骤b制得的ZnO溶胶-凝胶溶液混匀，滴加至经步骤a预处理的玻碳电极表面，干燥成膜，洗涤，即制得EPOR修饰电极。
优选的，所述步骤a是将玻碳电极依次用 0.3μm和0.05μm的三氧化二铝粉末抛光打磨，每次打磨后先用水洗净，再依次用硝酸、丙酮和水超声洗涤，空气中晾干。
优选的，所述步骤b是将乙酸锌溶于无水乙醇中制成浓度为0.1mol/L的溶液，再在超声波作用下加入氢氧化锂至终浓度为0.067mol/L，制得ZnO溶胶-凝胶贮备液，临用前用无水乙醇按体积比为2:1~1:3进行稀释，制得ZnO溶胶-凝胶溶液。
更优选的，所述步骤b是将乙酸锌溶于无水乙醇中制成浓度为0.1mol/L的溶液，再在超声波作用下加入氢氧化锂至终浓度为0.067mol/L，制得ZnO溶胶-凝胶贮备液，临用前用无水乙醇按体积比为1:2进行稀释，制得ZnO溶胶-凝胶溶液。
优选的，所述步骤c是将步骤b制得的ZnO溶胶-凝胶溶液与浓度为10ng/L~100μg/L的EPOR溶液按照体积比为4:1~1:1.15混合均匀，再将混合液滴加在经步骤a预处理的玻碳电极表面，空气中干燥，用磷酸盐缓冲液充分洗涤，即制得EPOR修饰电极。
更优选的，所述步骤c是将步骤b制得的ZnO溶胶-凝胶溶液与浓度为1μg/L的EPOR溶液按照体积比为1:1混合均匀，再将混合液滴加在经步骤a预处理的玻碳电极表面，空气中干燥，用磷酸盐缓冲液（PBS）充分洗涤，即制得EPOR修饰电极。
3．EPO和rhEPO电化学生物传感器，包括工作电极、对电极、参比电极和测试底液，所述工作电极为前述EPOR修饰电极，对电极为铂电极，参比电极为饱和甘汞电极；所述测试底液为含有2mmol/L K3[Fe(CN)6]和2mmol/L K4[Fe(CN)6]（后续简写为2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]）且pH为6.2~9.0的磷酸盐缓冲液。
优选的，所述测试底液为含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]且pH为7.4的磷酸盐缓冲液。
4．利用前述EPO和rhEPO电化学生物传感器检测EPO和/或rhEPO的方法，是将EPOR修饰电极与样品溶液共孵育20分钟以上，然后以EPOR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、含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]且pH为6.2~9.0的磷酸盐缓冲液为测试底液构建电化学生物传感器，采用循环伏安法进行扫描测定，电位扫描范围为-0.3V~0.7V，电位扫描速率为10~100mv/s，根据电位0.14~0.17V处的峰电流和EPO标准曲线计算样品溶液中EPO的浓度，和/或根据电位0.06~0.09V处的峰电流和rhEPO标准曲线计算样品溶液中rhEPO的浓度。
优选的，所述EPOR修饰电极与样品溶液的共孵育时间为20分钟，所述电位扫描速率为50mv/s。
 有益效果
本发明的有益效果在于：本发明的EPOR修饰电极制备方法简单，性能稳定，4℃避光放置50天后的响应电流值仍然维持在初始值的77%左右，以其为工作电极构建的三电极体系电化学生物传感器可以快速、特异、灵敏地检测EPO和/或rhEPO，线性范围均为5pg/L~500ng/L，检出限均低至0.5pg/L，特别是能根据峰电位对EPO和rhEPO进行快速、精确甄别，不但适用于低浓度EPO或rhEPO的检测，而且适用于体育竞技中对兴奋剂rhEPO的检测。
附图说明
图1为ZnO溶胶-凝胶贮备液和无水乙醇的稀释比对EPOR修饰电极电流响应的影响。
图2为ZnO溶胶-凝胶溶液与EPOR溶液的体积比对EPOR修饰电极电流响应的影响。
图3为EPOR溶液浓度对EPOR修饰电极电流响应的影响。
图4为测试底液的pH值对EPO和rhEPO电化学生物传感器电流响应的影响。
图5为工作电极在样品溶液中的孵育时间对EPO和rhEPO电化学生物传感器电流响应的影响。
图6为循环伏安法扫描电位对EPO和rhEPO电化学生物传感器电流响应的影响。
图7为以EPOR修饰电极为工作电极构建的电化学生物传感器的电化学响应及传感器特异性实验结果。其中a为单纯ZnO溶胶-凝胶修饰电极在PBS溶液中的循环伏安曲线；b为裸玻碳电极在含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液中的循环伏安曲线；c为单纯ZnO溶胶-凝胶修饰电极在含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液中的循环伏安曲线；d为EPOR修饰电极在含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液中的循环伏安曲线；e为EPOR修饰电极在干扰物质溶液（500ng/L IgA、500ng/L IgG和500ng/L IgM）中孵育20分钟后的循环伏安曲线；f为EPOR修饰电极在500ng/L EPO标准品溶液中孵育20分钟后的循环伏安曲线；g为EPOR修饰电极在500ng/L rhEPO标准品溶液中孵育20分钟后的循环伏安曲线。
图8为EPO和rhEPO电化学生物传感器在最佳条件下检测获得的EPO标准曲线和rhEPO标准曲线。
图9为EPO和rhEPO电化学生物传感器在贮存不同时间后的电流响应值变化。
 本发明的实施方式
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明的优选实施例进行详细的描述。
实施例中所用的主要试剂和仪器来源如下：一水氢氧化锂LiOH•H2O、二水乙酸锌Zn(Ac)2•2H2O购自上海生工生物工程有限公司，K3[Fe(CN)6]、K4[Fe(CN)6]购自重庆东方试剂厂，玻碳电极、饱和甘汞电极、铂电极、0.3μm和0.05μm 的Al2O3粉末购自天津艾达恒晟科技发展有限公司，PBS粉末购自北京中杉金桥生物技术有限公司，EPOR购自美国Novus Biologicals公司，EPO和rhEPO标准品购自美国Abnova公司。KQ-5200B型超声波清洗器为江苏省昆山市超声仪器有限公司产品，CHI660C型电化学工作站为上海辰华仪器有限公司产品，ZD-2自动电位滴定仪为上海精科雷磁有限公司产品。
一、EPOR修饰电极的制备及参数优化
EPOR修饰电极的制备方法具体包括以下步骤：
a．玻碳电极的预处理：取直径为3mm的玻碳电极，依次用0.3μm和0.05μm的Al2O3粉末进行抛光打磨，每次打磨后先用超纯水洗净，再依次于硝酸、丙酮和超纯水中超声洗涤5分钟，空气中晾干；
b．ZnO溶胶-凝胶溶液的制备：将Zn(Ac)2•2H2O 2.20g（0.01mol）溶于100ml无水乙醇中，再在超声波作用下缓慢加入LiOH•H2O 0.28g（6.7mmol），制得ZnO溶胶-凝胶贮备液，4℃保存备用，临用前按照体积比为1:2加入无水乙醇进行稀释，制得ZnO溶胶-凝胶溶液；
c. EPOR的固定：将浓度为1μg/L的EPOR溶液与步骤b制得的ZnO溶胶-凝胶溶液按照体积比1:1混匀，取混合液10μl滴在经步骤a预处理的玻碳电极表面，室温干燥16小时使电极表面的胶体形成凝胶，最后用PBS溶液（pH7.4，0.05mol/L）充分洗涤，即制得EPOR修饰电极，不用时置4℃避光保存。
本发明在研究过程中对影响EPOR修饰电极电流响应的主要参数进行了优化。将以不同参数制备的EPOR修饰电极作为工作电极、饱和甘汞电极作为参比电极、铂电极作为对电极组建电化学生物传感器，以含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液（pH7.4，0.05mol/L）作为测试底液，在室温下采用循环伏安法进行扫描测定，电位扫描范围为-0.3V~0.7V，电位扫描速率为50mv/s。结果显示，ZnO溶胶-凝胶贮备液与无水乙醇的稀释比、ZnO溶胶-凝胶溶液与EPOR溶液的体积比、EPOR溶液的浓度都会影响EPOR修饰电极的电流响应，ZnO溶胶-凝胶贮备液与无水乙醇的稀释比优选为2:1~1:3，更优选为1:2（图1），ZnO溶胶-凝胶溶液与EPOR溶液的体积比优选为4:1~1:1.15，更优选为1:1（图2），EPOR溶液的浓度优选为10ng/L~100μg/L，更优选为1μg/L（图3）。
二、EPO和rhEPO电化学生物传感器的构建及参数优化
将EPOR修饰电极与样品溶液孵育20分钟，然后以EPOR修饰电极为工作电极、饱和甘汞电极为参比电极、铂电极为对电极构建EPO和rhEPO电化学生物传感器，以含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液（pH7.4，0.05mol/L）为测试底液，在室温下采用循环伏安法进行扫描测定，电位扫描范围为-0.3V~0.7V，电位扫描速率为50mv/s。
本发明在研究过程中对影响EPO和rhEPO电化学生物传感器电流响应的主要参数也进行了优化。结果显示，测试底液的pH值为6.2~9.0时传感器的峰电流较大，pH值为7.4时传感器的峰电流最大，因此，测试底液的pH值优选为6.2~9.0，更优选为7.4（图4）；EPOR修饰电极与500ng/L EPO或rhEPO标准品溶液的孵育时间从5分钟增加至20分钟，传感器的峰电流逐渐降低并达到最小值，之后继续增加孵育时间至40分钟，峰电流基本保持不变，说明20分钟时EPOR修饰电极上结合的EPO或rhEPO已达到饱和，因此EPOR修饰电极与样品溶液的孵育时间优选为20分钟以上，更优选为20分钟（图5）。此外，扫描电位的变化对K3[Fe(CN)6]-K4[Fe(CN)6]氧化还原峰的峰电位的影响不大，但对传感器的电流响应的影响较为明显，在-0.3V~0.7V之间有最好的电流响应（图6）。电位扫描速率影响循环伏安曲线的形状，本发明研究发现，电位扫描速率在10~100 mv/s范围内都可行，当其为50 mv/s时循环伏安曲线最光滑。
三、EPO和rhEPO电化学生物传感器的检测性能
1、特异性
将EPOR修饰电极与样品溶液孵育20分钟，然后将EPOR修饰电极与铂电极、饱和参比电极组成电化学生物传感器，以含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液（pH7.4，0.05mol/L）为测试底液，在室温下采用循环伏安法进行扫描测定，电位扫描范围为-0.3V~0.7V，电位扫描速率为50mv/s。
传感器的特异性实验结果如图7所示，曲线a为单纯ZnO溶胶-凝胶修饰电极在PBS溶液中的循环伏安曲线，只能观测到背景电流；曲线b为裸玻碳电极在含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液中的循环伏安曲线，由于在PBS溶液中加入了氧化还原探针K3[Fe(CN)6]-K4[Fe(CN)6]，循环伏安曲线有明显改变，出现了一对准可逆的氧化还原峰；曲线c为单纯ZnO溶胶-凝胶修饰电极在含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液中的循环伏安曲线，由于ZnO溶胶-凝胶薄膜在一定程度上阻碍了溶液中导电离子在电极上的电子传递，氧化还原峰的峰电流降低；曲线d为EPOR修饰电极在含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液中的循环伏安曲线，与曲线c有明显不同，说明EPOR已成功修饰到电极表面，由于EPOR是生物大分子，被吸附在电极表面后阻碍电子传输，氧化还原峰的峰电流较曲线c进一步降低；曲线e为EPOR修饰电极在干扰物质溶液（500ng/L IgA、500ng/L IgG和500ng/L IgM）中孵育20分钟后的循环伏安曲线，与曲线d相比，基本保持不变，说明IgA、IgG、IgM等干扰物质不会影响EPO和rhEPO的检测；曲线f为EPOR修饰电极在500ng/L EPO标准品溶液中孵育20分钟后的循环伏安曲线，孵育前后响应电流变化值（ΔI）为8.2μA，峰电流出现在电位0.16V处，由于溶液中的EPO与电极上的EPOR特异性结合形成的EPO-EPOR复合物覆盖了更多的电极表面，进一步阻碍了电子传输，因此氧化还原峰的峰电流较曲线d明显降低；曲线g为EPOR修饰电极在500ng/L rhEPO标准品溶液中孵育20分钟后的循环伏安曲线，孵育前后响应电流变化值（ΔI）为9.7μA，同样由于rhEPO与EPOR特异性结合形成的rhEPO-EPOR复合物阻碍了电子传输，氧化还原峰的峰电流较曲线d明显降低，但由于rhEPO与EPO的等电点不同，导致rhEPO-EPOR复合物与EPO-EPOR复合物的工作电位不同，与曲线f相比，曲线g的氧化还原峰向负电位方向移动，峰电流出现在电位0.08V处，从而通过氧化还原峰的峰电位可以将EPO和rhEPO精确区分开来。以上实验结果表明，本发明构建的EPOR修饰电极抗干扰能力强，对EPO和rhEPO具有良好的选择性，并能对EPO和rhEPO进行精确甄别检测。
2、线性范围和检出限
将EPOR修饰电极分别与不同浓度的EPO标准品溶液、rhEPO标准品溶液孵育20分钟，然后与铂电极、饱和参比电极构建电化学生物传感器，以含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液（pH7.4，0.05mol/L）为测试底液，在室温下采用循环伏安法进行扫描测定，考察峰电流与EPO、rhEPO浓度的关系，电位扫描范围为-0.3V~0.7V，电位扫描速率为50mv/s。结果见图8，当EPO的浓度为5pg/L~500ng/L时，EPO浓度的对数值与峰电流呈良好的线性关系，线性回归方程为：y=2.1674x+17.691，相关系数为0.9966，检出限为0.5pg/L；当rhEPO的浓度为5pg/L~500ng/L时，rhEPO浓度的对数值与峰电流呈良好的线性关系，线性回归方程为：y=1.5737x+14.765，相关系数为0.9935，检出限为0.5pg/L。以上结果表明本发明构建的EPO和rhEPO电化学生物传感器具有较宽的线性范围和较低的检出限。
3、稳定性
将新制备的EPOR修饰电极分别于4℃避光放置10、20、30、40、50、60天，然后与铂电极、饱和参比电极构建电化学生物传感器，以含有2mmol/L K3[Fe(CN)6]-K4[Fe(CN)6]的PBS溶液（pH7.4，0.05mol/L）为测试底液，在室温下采用循环伏安法进行扫描测定，考察EPOR修饰电极的稳定性，电位扫描范围为-0.3V~0.7V，电位扫描速率为50mv/s。结果见图9，EPOR修饰电极在放置20天后，响应电流值约为初始值的95%；放置40天后，响应电流值下降至初始值的82%；放置50天后，响应电流值仍然维持在初始值的77%左右，说明本发明的EPOR修饰电极具有良好的稳定性，使用寿命较长。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管通过参照本发明的优选实施例已经对本发明进行了描述，但本领域的普通技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离所附权利要求书所限定的本发明的精神和范围。

Claims (10)

  1. 促红细胞生成素受体修饰电极，其特征在于，所述修饰电极是在电极表面通过ZnO溶胶-凝胶固定有促红细胞生成素受体作为识别元件的玻碳电极。
  2. 权利要求1所述的促红细胞生成素受体修饰电极的制备方法，其特征在于，包括以下步骤：
    a．玻碳电极的预处理：将玻碳电极表面抛光，清洁，干燥，备用；
    b．ZnO溶胶-凝胶的制备：将乙酸锌溶于无水乙醇中，再在超声波作用下加入氢氧化锂，制得ZnO溶胶-凝胶溶液，备用；
    c．促红细胞生成素受体的固定：将步骤b制得的ZnO溶胶-凝胶溶液与促红细胞生成素受体溶液混匀，滴加至经步骤a预处理的玻碳电极表面，干燥成膜，洗涤，即制得促红细胞生成素受体修饰电极。
  3. 根据权利要求2所述的促红细胞生成素受体修饰电极的制备方法，其特征在于，所述步骤a是将玻碳电极依次用0.3μm和0.05μm的三氧化二铝粉末抛光打磨，每次打磨后先用水洗净，再依次用硝酸、丙酮和水超声洗涤，空气中晾干。
  4. 根据权利要求2所述的促红细胞生成素受体修饰电极的制备方法，其特征在于，所述步骤b是将乙酸锌溶于无水乙醇中制成浓度为0.1mol/L的溶液，再在超声波作用下加入氢氧化锂至终浓度为0.067mol/L，制得ZnO溶胶-凝胶贮备液，临用前用无水乙醇按体积比为2:1~1:3进行稀释，制得ZnO溶胶-凝胶溶液。
  5. 根据权利要求4所述的促红细胞生成素受体修饰电极的制备方法，其特征在于，所述步骤b是将乙酸锌溶于无水乙醇中制成浓度为0.1mol/L的溶液，再在超声波作用下加入氢氧化锂至终浓度为0.067mol/L，制得ZnO溶胶-凝胶贮备液，临用前用无水乙醇按体积比为1:2进行稀释，制得ZnO溶胶-凝胶溶液。
  6. 根据权利要求2所述的促红细胞生成素受体修饰电极的制备方法，其特征在于，所述步骤c是将步骤b制得的ZnO溶胶-凝胶溶液与浓度为10ng/L~100μg/L的促红细胞生成素受体溶液按照体积比为4:1~1:1.15混合均匀，再将混合液滴加在经步骤a预处理的玻碳电极表面，空气中干燥，用磷酸盐缓冲液充分洗涤，即制得促红细胞生成素受体修饰电极。
  7. 根据权利要求6所述的促红细胞生成素受体修饰电极的制备方法，其特征在于，所述步骤c是将步骤b制得的ZnO溶胶-凝胶溶液与浓度为1μg/L的促红细胞生成素受体溶液按照体积比为1:1混合均匀，再将混合液滴加在经步骤a预处理的玻碳电极表面，空气中干燥，用磷酸盐缓冲液充分洗涤，即制得促红细胞生成素受体修饰电极。
  8. 促红细胞生成素和重组人促红细胞生成素电化学生物传感器，其特征在于，包括工作电极、对电极、参比电极和测试底液；所述工作电极为权利要求1所述的促红细胞生成素受体修饰电极，对电极为铂电极，参比电极为饱和甘汞电极；所述测试底液为含有2mmol/L K3[Fe(CN)6]和2mmol/L K4[Fe(CN)6]且pH为6.2~9.0的磷酸盐缓冲液。
  9. 根据权利要求8所述的促红细胞生成素和重组人促红细胞生成素电化学生物传感器，其特征在于，所述测试底液为含有2mmol/L K3[Fe(CN)6]和2mmol/L K4[Fe(CN)6]且pH为7.4的磷酸盐缓冲液。
  10. 利用权利要求8所述电化学生物传感器检测促红细胞生成素和/或重组人促红细胞生成素的方法，其特征在于，将促红细胞生成素受体修饰电极与样品溶液共孵育20分钟以上，然后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、含有2mmol/L K3[Fe(CN)6]和2mmol/L K4[Fe(CN)6]且pH为6.2~9.0的磷酸盐缓冲液为测试底液构建电化学生物传感器，采用循环伏安法进行扫描测定，电位扫描范围为-0.3V~0.7V，电位扫描速率为10~100mv/s，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重组人促红细胞生成素的浓度。
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