• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The invention discloses an erythropoietin receptor-modified electrode formed of a glassy carbon electrode having an erythropoietin receptor fixed on the electrode surface via a ZnO sol gel as a recognition element. The modified electrode can be easily manufactured and its performance is stable. After storage for over 50 days in the dark at 4 ° C, its response current remains at approximately 77% of the original value. An electrochemical biosensor using this modified electrode as a working electrode, a platinum electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and a phosphate buffer containing 2 mmol / LK 3 [Fe (CN) 6] -K 4 [Fe (CN) 6] as a test base solution Detect erythropoietin (EPO) and / or recombinant human erythropoietin (rhEPO) in a fast, specific and sensitive manner with a linear range of 5 pg / L-500 ng / L and a detection limit of 0.5 pg / L. In particular, the biosensor allows a precise distinction of EPO and rhEPO due to the different potential maxima. It can be used not only for the detection of low concentrations of EPO or rhEPO, but also for the detection of the stimulant rhEPO in sports competitions.
    公开号:CH707252B1
    申请号:CH00900/13
    申请日:2012-10-09
    公开日:2016-12-15
    发明作者:Zhang Liqun；Fu Weiling；Wang Yunxia
    申请人:First Affiliated Hospital Third Military Medical University Chinese People's Liberation Army P R Of；
    IPC主号:
    专利说明:

    Field of the invention
    The invention belongs to the technical field of electrochemical detection. It relates to a modified electrode and its manufacturing method, and it also relates to an electrochemical biosensor comprising the modified electrode as a working electrode, as well as associated detection methods.
    background
    Erythropoietin (EPO) is a glycoprotein hormone and a hematopoietic factor that is mainly produced in the human kidneys. EPO favors the formation and release of red blood cells in the bone marrow. In 1985, recombinant human erythropoietin (rhEPO) was synthesized by genetic engineering. Due to its mitogenic and differentiation-promoting effects, rhEPO can induce the effect of a blood transfusion without exposing the patient to the risk of viral infection or excessive transfusion. Accordingly, it plays an important role in the treatment of renal anemia. Meanwhile rhEPO is a new stimulant in sports competitions because of its effect of increasing oxygen storage capacity and exercise tolerance. In 2005, rhEPO was listed by the International Olympic Committee (IOC) and the World Anti-Doping Agency (WADA) as the first peptide substance banned in sports competitions.
    EPO and rhEPO have the same biological activities and a very similar molecular structure, and their only difference is the isoelectric point. EPO has an isoelectric point of 3.7-4.7, and rhEPO has an isoelectric point of 4.4-5.1. Accordingly, it is difficult to distinguish EPO from rhEPO. The distinction between EPO and rhEPO has long depended on the combination of mass spectrometry, isoelectric focusing, and gel electrophoresis. However, these detection methods have some disadvantages such as long separation time, low detection efficiency and low specificity. Accordingly, they are not suitable for rapid, accurate discrimination of EPO and rhEPO. It is imperative to develop a highly specific, sensitive, fast and accurate method for distinguishing EPO from rhEPO.
    Zusammenfssung
    An object to be solved by the invention disclosed herein is to provide a modified electrode. Another object to be achieved is to provide a manufacturing method for the said modified electrode. Yet another object to be achieved is to provide an electrochemical biosensor using the modified electrode as a working electrode. Yet another object to be achieved is to provide an EPO and / or rhEPO detection method using said electrochemical biosensor. The said modified electrode can be easily manufactured and its performance is stable. The electrochemical biosensor using the modified electrode as the working electrode is capable of detecting EPO and / or rhEPO in a fast, specific and sensitive manner. In particular, it allows a fast, accurate distinction between EPO and rhEPO.
    The objects are achieved by providing the following technical protocols:
    1. erythropoietin receptor (EPOR) modified electrode. Said modified electrode is a glassy carbon electrode having an EPOR fixed to the electrode surface via a ZnO sol gel as a recognition element.
    2. Production method for the EPOR-modified electrode, comprising the following steps:<tb> a. <SEP> Pre-treatment of glassy carbon electrode: The surface of the glassy carbon electrode is polished, cleaned and dried for later use;<tb> b. <SEP> Preparation of a ZnO sol gel: Zinc acetate is dissolved in absolute alcohol; while the mixture is subjected to ultrasonic stirring, lithium hydroxide is added to give a ZnO sol-gel solution for later use;<EP> Fixing: The ZnO sol-gel solution prepared in step b and an EPOR solution are thoroughly mixed, and the resulting solution is dropped on the surface of a pretreated glassy carbon as described in step a followed by drying and washing; thus the erythropoietin receptor-modified electrode is made.
    Preferably, the glassy carbon electrode is polished in said step a first with 0.3 microns and then with 0.05 micron alumina powder. Between the polishing steps, the electrode is first washed with water and then in an ultrasonic bath with nitric acid, acetone and water. After each washing step, the electrode is air-dried.
    Preferably, the zinc acetate is dissolved in said step b in absolute alcohol, resulting in a 0.1 mol / L solution. While the mixture is subjected to ultrasonic stirring, lithium hydroxide is added, resulting in a ZnO sol-gel stock solution having a final concentration of 0.067 mol / L. Immediately prior to use, ZnO sol-gel solution is prepared by diluting the stock solution with absolute alcohol in a vol / vol ratio of 2: 1 ~ 1: 3.
    More preferably, the zinc acetate is dissolved in said step b in absolute alcohol, resulting in a 0.1 mol / L solution. While the mixture is subjected to ultrasonic stirring, lithium hydroxide is added, resulting in a ZnO sol-gel stock solution having a final concentration of 0.067 mol / L. Immediately prior to use, ZnO sol-gel solution is prepared by diluting the stock solution with absolute alcohol in a vol / vol ratio of 1: 2.
    Preferably, the ZnO sol-gel solution prepared in step b and 10 ng / L ~ 100 μg / L erythropoietin receptor solution in said step c in a vol / vol ratio of 4: 1 ~ 1: 1.15 thoroughly and the resulting solution is dropped onto the surface of a pretreated vitreous carbon electrode as described in step a, followed by air drying and thorough washing in a phosphate buffer. Thus, the erythropoietin receptor-modified electrode is made.
    More preferably, the ZnO sol-gel solution prepared in step b and 1 μg / L erythropoietin receptor solution are thoroughly mixed in said step c in a vol / vol ratio of 1: 1, and the resulting solution is dissolved the surface of a pre-treated glassy carbon electrode as described in step a, followed by air drying and thorough washing in a phosphate buffer. Thus, the erythropoietin receptor-modified electrode is made.
    3. The electrochemical biosensor for EPO and rhEPO comprises a working electrode, a counter electrode, a reference electrode and the test base solution. The said working electrode is said erythropoietin receptor-modified electrode according to claim 1, the counter electrode is a platinum electrode, and the reference electrode is a saturated calomel electrode. Said test base solution is a phosphate buffer (pH = 6.2 ~ 9.0) containing 2 mmol / L K3 [Fe (CN) 6] and 2 mmol / L K4 [Fe (CN) 6].
    Preferably, said test base solution is a phosphate buffer (pH = 7.4) containing 2 mmol / L K3 [Fe (CN) 6] and 2 mmol / L K4 [Fe (CN) 6].
    4. EPO and / or rhEPO is detected using said electrochemical biosensor for EPO and rhEPO as follows: the erythropoietin receptor-modified electrode and sample solution are co-incubated for more than 20 minutes, then using the electrochemical biosensor, which contains the erythropoietin receptor-modified electrode as a working electrode, a platinum electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and a phosphate buffer (pH: 2) containing 2 mmol / L K3 [Fe (CN) 6] and 2 mmol / L K4 [Fe (CN) 6] = 6.2 ~ 9.0) as the test base solution, a cyclic voltammetry scan was performed with a potential scan range of -0.3V ~ 0.7V and a potential scan rate of 10 mV / s ~ 100 mV / s. The erythropoietin concentration of the sample solution is calculated from the peak current at the potential of 0.14 V ~ 0.17 V and the erythropoietin standard curve, and / or the concentration of the sample solution of recombinant human erythropoietin becomes 0.06 V ~ 0.09 V and the standard curve of the peak current due to peak current recombinant human erythropoietin.
    Preferably, said EPOR-modified electrode and sample solution are co-incubated for 20 minutes and said potential scan rate is 50 mV / s.
    The advantages of the invention are as follows: The inventive EPOR-modified electrode can be easily prepared and their performance is stable. After storage for over 50 days in the dark at 4 ° C, its response current remained at approximately 77% of the original value. An electrochemical biosensor using this modified electrode as a working electrode can be erythropoietin (EPO) and / or recombinant human erythropoietin (rhEPO) in a fast, specific and sensitive manner with a linear range of 5 pg / L-500 ng / L and a detection limit of 0.5 prove pg / L. In particular, the biosensor allows a precise distinction between EPO and rhEPO due to the different potential maxima. It can be used not only for the detection of low concentrations of EPO or rhEPO, but also for the detection of the stimulant rhEPO in sports competitions.
    Brief description of the drawings
    [0018]<Tb> FIG. 1 <SEP> shows the effect of the dilution ratio of the ZnO sol-gel stock solution and absolute alcohol on the current response of the EPOR-modified electrode.<Tb> FIG. 2 <SEP> shows the effect of the vol / vol ratio of ZnO sol-gel solution and EPOR solution on the current response of the EPOR-modified electrode.<Tb> FIG. 3 <SEP> shows the effect of the concentration of the EPOR solution on the current response of the EPOR-modified electrode.<Tb> FIG. 4 <SEP> shows the effect of the pH of the test base solution on the current response of the electrochemical biosensor for EPO and rhEPO.<Tb> FIG. 5 <SEP> shows the effect of the incubation time of the working electrode in the sample solution on the current response of the electrochemical biosensor for EPO and rhEPO.<Tb> FIG. 6 <SEP> shows the effect of the potential of the cyclic voltammetry scan on the electrochemical biosensor current response for EPO and rhEPO.<Tb> FIG. 7 <SEP> shows the results of the electrochemical response and specificity of the electrochemical biosensor using the EPOR-modified electrode as a working electrode. a: cyclic voltammogram of a simple ZnO sol-gel modified electrode in a PBS solution; b: cyclic voltammogram of a non-modified glassy carbon electrode in a 2 mmol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] -containing PBS solution; c: cyclic voltammogram of a simple ZnO sol-gel modified electrode in a 2 mmol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] -containing PBS solution; d: cyclic voltammogram of an EPOR-modified electrode in a 2 mmol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] -containing PBS solution; e: cyclic voltammogram of an EPOR-modified electrode after incubation for 20 minutes in an interferent-containing solution (500 ng / L IgA, 500 ng / L IgG and 500 ng / L IgM); f: cyclic voltammogram of an EPOR-modified electrode after incubation for 20 minutes in a 500 ng / L EPO standard preparation containing solution; g: cyclic voltammogram of an EPOR-modified electrode after 20 minutes incubation in a 500 ng / L rhEPO standard preparation containing solution.<Tb> FIG. 8 <SEP> Shows EPO and rhEPO standard curves obtained using an electronic biosensor for EPO and rhEPO under optimal conditions.<Tb> FIG. 9 <SEP> shows the change in the current response of an electrochemical biosensor for EPO and rhEPO after various periods of storage.
    Detailed description
    In order to clarify the objects, technical protocols and advantages of the invention, the preferred embodiments of the invention are described in detail below with reference to the drawings.
    The reagents and instruments used in the embodiments are listed below: lithium hydroxide (LiOH⋅H2O), zinc acetate [Zn (Ac) 2⋅2H2O] from Shanghai Sangon Bioengineering Co., Ltd. (Shanghai, China); K3 [Fe (CN) 6], K4 [Fe (CN) 6] from Chongqing Dongfang Reagents Factory (Chongqing, China); Glassy Carbon Electrode, Saturated Calom Electrode, Platinum Electrode, 0.3 μm and 0.05 μm Al2O3 Powder from Tianjin Aidahengsheng Tech Co., Ltd. (Tianjin, China); Beijing Zhong Shan PBS Powder Golden Bridge Biotech Co., Ltd. (Beijing, China); EPOR from Novus Biologicals (USA); EPO and rhEPO standard preparation from Abnova (USA); electronic workstation model CHI660C of Shanghai Chenhua Instruments Co., Ltd., China; Ultrasonic Bath Model KQ-5200B from Kunshan Ultrasound Instruments Co., Ltd. (Jiangsu, China), and ZD-2 Automatic Electric Potential Indicator from Shanghai Jingke Leici Co., Ltd. (Shanghai, China).
    I. Making the EPOR-modified electrode and parameter optimization
    The manufacturing method for an EPOR-modified electrode comprises the following steps:<tb> a. <SEP> Pre-treatment of glassy carbon electrode: Glassy carbon electrodes (3 mm in diameter) are first polished with 0.3 μm and then with 0.05 μm Al2O3 powder. Between the polishing steps, the electrodes are first washed with ultrapure water and then each for 5 minutes in an ultrasonic bath with nitric acid, acetone and ultrapure water. After washing, the electrodes are air dried.<tb> b. <SEP> Preparation of a ZnO sol-gel solution: Dissolve 2.20 g (0.01 mol) of Zn (Ac) 2⋅2H2O in 100 mL of absolute alcohol. Subsequently, 0.28 g (6.7 mmol) of LiOH⋅H2O are slowly added under sonication to produce ZnO sol-gel stock which is stored for later use at 4 ° C. Immediately prior to use, ZnO sol-gel solution is prepared by diluting the stock solution with absolute alcohol in a vol / vol ratio of 1: 2.<tb> c. <EP> Fixation: The ZnO sol-gel solution prepared in step b and 1 μg / L EPOR solution are thoroughly mixed in a vol / vol ratio of 1: 1, and 10 μl of the obtained Solution is dropped onto the surface of a pretreated glassy carbon as described in step a, followed by drying at room temperature for 16 hours, allowing the formation of gel on the electrode surface. Finally, the electrode is washed thoroughly in PBS solution (pH 7.4, 0.05 mol / L). The manufactured EPOR-modified electrode is stored in the dark until use at 4 ° C.
    The invention includes the optimization of important parameters that influence the current response of EPOR-modified electrodes. An electrochemical biosensor comprising an EPOR-modified electrode with different parameters as working electrode, saturated calomel electrode as reference electrode, platinum electrode as counterelectrode and 2 mmol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] (pH 7.4, 0.05 mol / L) PBS solution is prepared as a test base solution, is for a cyclic voltammetry scan at room temperature within the potential scan range of -0.3V ~ 0.7V and with the potential scan speed of 50 mV / s used. The results show that the dilution ratio of ZnO sol-gel stock solution and absolute alcohol, the volume / volume ratio of ZnO sol-gel solution and EPOR solution, and the EPOR concentration influence the current response of the EPOR-modified electrode and that the preferred dilution ratio is in the range of 2: 1 ~ 1: 3 and the most preferred ratio is 1: 2 for ZnO sol-gel stock solution and absolute alcohol (Figure 1). The preferred vol / vol ratio of ZnO sol-gel solution and EPOR solution is in the range of 4: 1 ~ 1: 1.15, and the most preferred ratio is 1: 1 (Figure 2). The preferred EPOR concentration is in the range of 10 ng / L ~ 100 μg / L, and the most preferred concentration is 1 μg / L (Figure 3).
    II. Preparation of an Electrochemical Biosensor for EPO and rhEPO and Parameter Optimization
    The EPOR-modified electrode and the sample solution are co-incubated for 20 minutes, and the electrochemical biosensor for EPO and rhEPO, which is an EPOR-modified electrode as a working electrode, a saturated calomel electrode as a reference electrode, a platinum electrode as a counter electrode, and 2 mmol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] (pH 7.4, 0.05 mol / L) containing PBS solution as the test base solution is used for a cyclic voltammetry scan at room temperature within the potential Scanning range of -0.3V ~ 0.7V and with the potential scanning speed of 50 mV / s used.
    The invention includes the optimization of important parameters that influence the current response of the electrochemical biosensor for EPO and rhEPO. The results show that the peak current of the sensor at pH of the test base solution is high within 6.2 ~ 9.0 and highest at a pH of 7.4. Accordingly, the preferred pH of the test base solution is in the range 6.2 ~ 9.0, and the most preferred pH is 7.4 (Figure 4). If the incubation time of the EPOR-modified electrode and 500 ng / L EPO or rhEPO standard preparation solution is increased from 5 minutes to 20 minutes, the peak current of the sensor gradually decreases to a minimum, while further increasing the incubation time to 40 minutes Peak current remains unchanged. It is concluded that after 20 minutes of incubation EPO or rhEPO binding to the EPOR-modified electrode is saturated. Accordingly, the preferred incubation time of the EPOR-modified electrode and the sample solution is 20 minutes or more, and the most preferred incubation time is 20 minutes (Figure 5). In addition, the change in scan potential only marginally affects the K3 [Fe (CN) 6] -K4 [Fe (CN) 6] redox tip potential, but it has a pronounced effect on the current response of the sensor, especially within -0.3V ~ 0.7V (Figure 6). The potential scan rate affects the shape of the cyclic voltammogram. Within the scope of the invention, the allowable potential scan rate has been found to be in the range of 10 mV / s ~ 100 mV / s, but at 50 mV / s, the cyclic voltammogram is the smoothest.
    III. Performance of electrochemical biosensor for EPO and rhEPO detection
    1. Specificity
    The EPOR-modified electrode and the sample solution are co-incubated for 20 minutes, and the electrochemical biosensor comprising an EPOR-modified electrode as a working electrode, a saturated calomel electrode as a reference electrode, a platinum electrode as a counter electrode, and a 2 mmol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] (pH 7.4, 0.05 mol / L) containing PBS solution as the test base solution is used for a cyclic voltammetry scan at room temperature within the potential scan range of -. 0.3V ~ 0.7V and with the potential scan speed of 50 mV / s.
    The experimental results on the specificity of the sensor are shown in FIG. 7. Curve a is the cyclic voltammogram of a simple ZnO sol-gel modified electrode in PBS solution showing only the background current; Curve b is the cyclic voltammogram of a non-modified glassy carbon electrode in a 2 mmole / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] -containing PBS solution. Since the PBS solution containing the redox sample K3 [Fe (CN) 6] -K4 [Fe (CN) 6] is added, the cyclic voltammogram changes markedly, showing a pair of quasi-reversible redox peaks; Curve c is the cyclic voltammogram of a simple ZnO sol-gel modified electrode in a 2 mmol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] -containing PBS solution. Since the ZnO sol-gel film prevents the electron transfer of electroconductive ions from the solution to the electrode, the redox peak currents decrease. Curve d, which represents the cyclic voltammogram of the EPOR-modified electrode in a 2 mmol / L K3 [Fe (CN) 6] / K4 [Fe (CN) 6] -containing PBS solution, differs significantly from curve c indicates that EPOR successfully modifies the electrode surface. As a biological macromolecule, EPOR prevents electron transfer once it has been absorbed on the electrode surface, resulting in a further decrease in redox peak current compared to curve c. Curve e represents the cyclic voltammogram of the EPOR-modified electrode after 20 minutes incubation in a solution containing interfering substances (500 ng / L IgA, 500 ng / L IgG and 500 ng / L IgM), and curve d remains largely unchanged, indicating that interfering substances, eg IgA, IgG, IgM do not affect the detection of EPO and rhEPO. Curve f is the cyclic voltammogram of the EPOR-modified electrode after 20 minutes incubation in 500 ng / L EPO standard preparation solution with the response current changing by 8.2 μA between before and after incubation (ΔI), and peak current at the potential of 0.16 V appears. EPO-EPOR complexes, which result from the specific binding of EPO in the solution to EPOR on the electrode surface, cover a larger area of the electrode surface, which further complicates the electron transfer. As a result, the redox peak current decreases significantly compared to the curve d. Curve g is the cyclic voltammogram of the EPOR-modified electrode after 20 minutes incubation in 500 ng / L rhEPO standard preparation solution. The response currents change by 9.7 μA (ΔI) before or after the incubation. Similarly, because the rhEPO-EPOR complexes, which result from the specific binding of rhEPO and EPOR, make electron transfer more difficult, the redox peak current decreases significantly in comparison with curve d. Nevertheless, the rhEPO-EPOR complexes and EPO-EPOR complexes have different work potentials, since rhEPO and EPO have different isoelectric points. Compared to curve f, the redox peak shifts towards the negative potential in curve g, where the peak current appears at the potential of 0.08V. EPO and rhEPO can be distinguished precisely due to the redox peak potential. These experimental results show that the EPOR-modified electrode of the present invention has strong resistance to interference and high selectivity for EPO and rhEPO, and that it allows detection of EPO and rhEPO with good distinctness.
    2. Linear range and detection limits
    The EPOR-modified electrode and the sample solution are co-incubated for 20 minutes, and the electrochemical biosensor for EPO and rhEPO, which has an EPOR-modified electrode as a working electrode, a saturated calomel electrode as a reference electrode, a platinum electrode as a counter electrode, and a 2 mMol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] (pH 7.4, 0.05 mol / L) containing PBS solution as the test base solution becomes within potential for a cyclic voltammetry scan at room temperature Scanning range of -0.3V ~ 0.7V and used with the potential scanning speed of 50 mV / s. The results are shown in FIG. When the EPO concentration is in the range between 5 pg / L and 500 ng / L, the logarithmic value of the EPO concentration and the peak current have a good linear correlation. For EPO the linear regression equation was: y = 2.1674x + 17.691, the correlation coefficient is 0.9966 and the detection limit is 0.5 pg / L. When the rhEPO concentration is in the range between 5 pg / L and 500 ng / L, the logarithmic value of the rhEPO concentration and the peak current have a good linear correlation. For rhEPO the linear regression equation resulted: y = 1.5737x + 14.765, where the correlation coefficient is 0.9935 and the detection limit is 0.5 pg / L. These results show that the electrochemical biosensor for EPO and rhEPO is characterized by a broad linear range and a low detection limit.
    3. Stability
    After storage of the freshly prepared EPOR-modified electrode at 4 ° C in the dark for 10, 20, 30, 40, 50, 60 days, the electrochemical biosensor, which is the modified electrode, a platinum electrode and a saturated reference electrode comprises, for a cyclic voltammetry scan in the test base solution containing a 2 mMol / L K3 [Fe (CN) 6] -K4 [Fe (CN) 6] -containing PBS solution (pH 7.4, 0.05 mol / L), at room temperature with the potential scan range of -0.3V ~ 0.7V and at the potential scan rate of 50 mV / s used to investigate the stability of the EPOR-modified electrode. The results are shown in FIG. After 20 days of storage, the response current of the EPOR modified electrode is approximately 95% of the original value; after 40 days of storage, the response current is about 82% of the original value; after 50 days of storage, the response current is approximately 77% of the original value. These results show that the EPOR-modified electrode of the present invention has good stability and long life.
    The above embodiments are intended to explain the technical protocol of the invention and are not limiting. While the invention has been described in terms of the preferred embodiments of the invention, those skilled in the art should understand that various modifications in form and detail may be implemented without departing from the spirit and scope of the invention as defined by the appended claims.
    权利要求:
    Claims (10)
    [1]
    An erythropoietin receptor-modified electrode, characterized in that said modified electrode is a glassy carbon electrode provided with a erythropoietin receptor as a recognition element fixed to the electrode surface via a ZnO sol gel.
    [2]
    2. An erythropoietin receptor-modified electrode manufacturing method according to claim 1, characterized by comprising the steps of:a. Pre-treating the glassy carbon electrode: The surface of the glassy carbon electrode is polished, cleaned and dried for later use;b. Preparation of a ZnO sol gel: zinc acetate is dissolved in absolute alcohol; while the mixture is subjected to ultrasonic stirring, lithium hydroxide is added to give a ZnO sol-gel solution for later use;c. Erythropoietin receptor fixation: The ZnO sol-gel solution prepared in step b and an erythropoietin receptor solution are mixed, and the resulting solution is dropped onto the surface of a pretreated glassy carbon as described in step a, followed by drying and washing.
    [3]
    3. The manufacturing method for the erythropoietin receptor-modified electrode according to claim 2, characterized in that the step a is as follows: The glassy carbon electrode is polished first with 0.3 μm and then with 0.05 μm alumina powder; between the polishes, the electrode is first washed with water and then in an ultrasonic bath with nitric acid, acetone and water, followed by air drying.
    [4]
    4. The manufacturing method for the erythropoietin receptor-modified electrode according to claim 2, characterized in that the step b is as follows: zinc acetate is dissolved in absolute alcohol, resulting in a 0.1 mol / L solution; while the mixture is subjected to ultrasonic stirring, lithium hydroxide is added, resulting in a ZnO sol-gel stock solution having a final concentration of 0.067 mol / L ZnO; Immediately prior to use, ZnO sol-gel solution is prepared by diluting the stock solution with absolute alcohol in a vol / vol ratio of 2: 1 to 1: 3.
    [5]
    The manufacturing method for the erythropoietin receptor-modified electrode according to claim 4, characterized in that the step b is as follows: zinc acetate is dissolved in absolute alcohol, resulting in a 0.1 mol / L solution; while the mixture is subjected to ultrasonic stirring, lithium hydroxide is added, resulting in a ZnO sol-gel stock solution having a final concentration of 0.067 mol / L ZnO; Immediately prior to use, ZnO sol-gel solution is prepared by diluting the stock solution with absolute alcohol in a vol / vol ratio of 1: 2.
    [6]
    The manufacturing method for the erythropoietin receptor-modified electrode according to claim 2, characterized in that the step c is as follows: The ZnO sol-gel solution prepared in the step b and 10 ng / L to 100 μg / L erythropoietin receptor solution at a vol / vol ratio of 4: 1 to 1: 1.15, and the resulting solution is dropped on the surface of a pretreated glassy carbon as described in step a, followed by air drying and washing in a phosphate buffer.
    [7]
    The production method for the erythropoietin receptor-modified electrode according to claim 6, characterized in that the step c is as follows: The ZnO sol-gel solution prepared in the step b and 1 μg / L erythropoietin receptor solution are prepared at a vol / vol 1: 1 ratio, and the resulting solution is dropped onto the surface of a pretreated glassy carbon as described in step a, followed by air drying and washing in a phosphate buffer.
    [8]
    8. An electrochemical biosensor for erythropoietin and recombinant human erythropoietin, characterized by comprising a working electrode, a counter electrode, a reference electrode and the test base solution; the working electrode is the erythropoietin receptor-modified electrode of claim 1, the counter electrode is a platinum electrode, and the reference electrode is a saturated calomel electrode; the test base solution is a phosphate buffer (pH = 6.2 to 9.0) containing 2 mmol / L K3 [Fe (CN) 6] and 2 mmol / L K4 [Fe (CN) 6].
    [9]
    9. An electrochemical biosensor for erythropoietin and recombinant human erythropoietin according to claim 8, characterized in that the test base solution comprises a 2 mMol / L K3 [Fe (CN) 6] and 2 mMol / L K4 [Fe (CN) 6] -containing phosphate buffer (pH = 7.4).
    [10]
    10. A method for the detection of erythropoietin and / or recombinant human erythropoietin using the electrochemical biosensor according to claim 8, characterized in that the following procedure is carried out: The erythropoietin receptor-modified electrode and sample solution are co-incubated for more than 20 minutes, then is carried out using the electrochemical biosensor using the erythropoietin receptor-modified electrode as a working electrode, a platinum electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and a 2 mmol / L K3 [Fe (CN) 6] and 2 mmol / L K4 [Fe (CN 6) containing phosphate buffer (pH = 6.2 to 9.0) as the test base solution, a cyclic voltammetry scan with a potential scan range of -0.3 V to 0.7 V and with a potential scan speed of 10 mV / s to 100 mV / s ; the erythropoietin concentration of the sample solution is calculated from the peak current at the potential of 0.14V to 0.17V and the erythropoietin standard curve, and / or the concentration of the sample solution of recombinant human erythropoietin becomes 0.06V to 0.09V due to the peak current and the standard curve of the recombinant human erythropoietin.
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    CN102854231A|2013-01-02|
    WO2014036772A1|2014-03-13|
    CN102854231B|2014-05-14|
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    AU1203699A|1997-10-29|1999-05-17|Ana R. Guadalupe|Electroanalytical applications of screen-printable surfactant-induced sol-gel graphite composites|
    WO2001059096A1|2000-02-08|2001-08-16|Ssp Co., Ltd.|Method of detecting ligand or ligand-like low-molecular weight compound|
    WO2002088732A1|2001-04-27|2002-11-07|Laboratoire National De Depistage Du Dopage|Improved qualitative and/or quantitative immunoassay method by immunoblot, kit and device therefor|
    CN1209622C|2003-07-17|2005-07-06|南京大学|Method for preparing titania dioxide gel film electrochemical biological sensor|
    US7469076B2|2003-09-03|2008-12-23|Receptors Llc|Sensors employing combinatorial artificial receptors|
    TWI336782B|2007-07-05|2011-02-01|Apex Biotechnology Corp|Composite modified electrode trip|
    CN101960300B|2008-05-28|2013-05-29|松下电器产业株式会社|Method for detecting or quantitating target substance by using electrochemical measurement device, electrochemical measurement device, and electrode plate for electrochemical measurement|
    CN101625334B|2009-08-05|2012-09-05|西北师范大学|Enzyme modified electrode based on sol gel layer and preparation method thereof|
    CN101672814B|2009-09-27|2012-11-07|上海大学|Electrochemical receptor biosensor and application thereof|CN103175879B|2013-03-12|2014-09-24|中国人民解放军第三军医大学第一附属医院|Electrode based on modification of nano-gold and erythropoietin receptor as well as preparation method and application thereof|
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    CN110702749B|2019-11-04|2021-11-09|太原理工大学|Method for constructing electrochemical immunosensing interface based on conductive gel with active sites|
    CN111122685B|2020-01-15|2022-03-01|杭州电子科技大学|Method for detecting organic carbon content of mangrove forest sediment|
    CN112858411B|2021-01-27|2021-12-28|山东农业大学|Method for detecting 5fdC by using photoelectrochemical biosensor based on silver sulfide @ disulfide-copper oxide ternary heterojunction|
    法律状态:
    2016-04-15| AZW| Rejection (application)|
    2016-09-15| PK| Correction|Free format text: BERICHTIGUNG ERFINDER |
    2020-05-29| AEN| Modification of the scope of the patent|Free format text: :DIE PATENTANMELDUNG IST AUFGRUND DES WEITERBEHANDLUNGSANTRAGS VOM 06. JUNI 2016 REAKTIVIERT WORDEN. |
    优先权:
    申请号 | 申请日 | 专利标题
    CN201210328850.5A|CN102854231B|2012-09-07|2012-09-07|Erythropoietin receptor modified electrode, preparation method and applications thereof|
    PCT/CN2012/082621|WO2014036772A1|2012-09-07|2012-10-09|Erythropoietin receptor modified electrode, preparation method and use thereof|
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