Label-free self-enhanced ECL sensor and rapid detection method for recombinant human erythropoietin

Abstract

The present invention relates to the technical field of doping detection, and more specifically, to a label-free self-enhanced ECL sensor and a rapid detection method for recombinant human erythropoietin. The working electrode of the sensor is coated with a self-enhanced MOF solid-phase luminescent material containing luminol. This solid-phase luminescent material uses a copper-based MOF material to encapsulate the luminol small molecule, effectively increasing the luminescence intensity. The rapid detection method utilizes the inherent properties of EPO and combines the advantages of electrochemiluminescence detection, making the detection principle simpler and more direct, enabling rapid detection of EPO. It also has high detection accuracy, a low detection limit, and high sensitivity. In the future, it may be applicable to the detection of dried blood samples, thus expanding its application range and making the detection process more convenient.

Description

Technical Field

[0001] The present invention relates to the technical field of doping detection, and in particular to a label-free self-enhanced ECL sensor and a rapid detection method for recombinant human erythropoietin. Background Art

[0002] The physiological function of erythropoietin (EPO) is to increase the number of red blood cells in the human blood, improve the blood's oxygen-carrying capacity, and thus improve the body's aerobic level. In the field of doping detection, it is an important detection indicator.

[0003] However, traditional testing methods are limited in speed. Sampling and testing all athletes would be extremely time-consuming and expensive. Furthermore, traditional testing methods are cumbersome, time-consuming, and expensive, making them difficult to promote.

[0004] Furthermore, to stimulate EPO production, athletes regularly train in low-pressure, low-oxygen environments at high altitudes. EPO levels are a key indicator of training performance. However, traditional methods have limited rapid, accurate, and cost-effective EPO testing, severely hindering the development of technology for evaluating athlete training performance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a label-free self-enhanced ECL sensor and a rapid detection method for recombinant human erythropoietin.

[0006] The technical solution of the present invention to solve the above technical problems is as follows

[0007] The present invention provides a method for preparing a copper metal organic framework material. Luminol and gallic acid are dissolved in a sodium hydroxide solution to obtain a solution A. 2+ The solution of is mixed with the solution A, heated and reacted to obtain the copper metal organic framework material.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, luminol, sodium hydroxide, gallic acid and Cu 2+ The mass ratio is: 1:3:3:6.67.

[0010] Furthermore, the heating temperature is 85° C. to 95° C., the heating time is 1.5 to 3 hours, and stirring is performed during the heating process.

[0011] The present invention also provides a copper metal organic framework material, which is prepared by the above method.

[0012] The present invention also provides a label-free self-enhanced ECL sensor, comprising a working electrode layer, a luminescent material layer coated on the working electrode layer, and a film layer coated on the luminescent material layer; wherein the component of the luminescent material layer is the luminol-functionalized self-enhanced MOF solid-phase luminescent material as described above.

[0013] Furthermore, the working electrode layer is an ITO electrode, and the film layer is a perfluorosulfonic acid polymer.

[0014] The present invention also provides a method for rapid detection of recombinant human erythropoietin, which uses the above-mentioned label-free self-enhanced ECL sensor for detection.

[0015] Further, the following steps are included:

[0016] S1, connecting an Ag / AgCl electrode as a reference electrode, a spiral platinum wire electrode as a counter electrode, and the electrochemiluminescence sensor as a working electrode to a chemiluminescence detection device;

[0017] S2. Using a phosphate buffer solution containing hydrogen peroxide, the electrochemiluminescence signal intensity generated by different concentrations of erythropoietin standards is detected by electrochemiluminescence;

[0018] S3. drawing a working curve based on the obtained linear relationship between the electrochemiluminescence signal intensity value and the corresponding erythropoietin standard concentration;

[0019] S4. Detect the erythropoietin sample to be tested using the method of steps S1 and S2 to obtain an electrochemiluminescence intensity value of the sample, and compare the electrochemiluminescence intensity value of the sample with the working curve to obtain the concentration of erythropoietin in the erythropoietin sample to be tested.

[0020] Furthermore, the detection parameters of the chemiluminescence detection device are: the high voltage of the photomultiplier tube is 500V, the scanning rate is 0.2V / s; and the scanning range is -1.0 to 1.2V.

[0021] Furthermore, in step S2, the content of hydrogen peroxide in the phosphate buffer solution is 200 mM, the content of phosphate is 0.1 M, and the pH value of the phosphate buffer solution is 7.4.

[0022] The beneficial effects of the present invention are:

[0023] (1) The preparation method of the copper metal organic framework material of the present invention uses copper-based MOF materials to encapsulate luminol small molecules, effectively improving the luminescence intensity;

[0024] (2) The copper metal organic framework material of the present invention is a self-enhanced nanoluminescent material that can not only achieve high-throughput detection of human recombinant erythropoietin, but also effectively improve the efficiency and accuracy of detection;

[0025] (3) The label-free self-enhanced ECL sensor of the present invention can not only achieve high-throughput detection of human erythropoietin, but also effectively improve the efficiency and accuracy of detection;

[0026] (4) The label-free self-enhanced ECL sensor of the present invention also has the advantages of being simple, portable, and easy to prepare;

[0027] (5) The rapid detection method of recombinant human erythropoietin of the present invention adopts the label-free self-enhanced ECL sensor of the present invention, takes advantage of the properties of recombinant human erythropoietin itself and combines the advantages of sensitive and fast electrochemiluminescence detection, making the detection principle simpler and more direct, and can achieve rapid detection of recombinant human erythropoietin. It also has the advantages of high detection accuracy, low detection limit, high sensitivity, and applicability to various sample forms;

[0028] (6) The rapid detection method of recombinant human erythropoietin of the present invention is applicable to the detection of various forms of samples, which makes its application range wider and the detection process more convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The copper metal organic framework material of the present invention is a SEM image of Luminol@CuGAMOF and CuGAMOF prepared in Example 1; Figure 1 The middle a is the SEM image of CuGAMOF with a scale of 200 nm. Figure 1 Middle b is the SEM image of CuGAMOF with a scale of 500 nm. Figure 1 Middle c is the SEM image of Luminol@CuGAMOF with a scale of 200 nm. Figure 1 Middle d is the SEM image of Luminol@CuGAMOF with a scale of 500 nm;

[0030] Figure 2 The copper metal organic framework material of the present invention, in Example 1, is the EDAX spectrum of CuGAMOF; Figure 2 a is the SEM image of CuGAMOF with a scale bar of 2 μm. Figure 2 b is the overall element distribution diagram of CuGAMOF, Figure 2 c~f are the distribution diagrams of C, N, O, and Cu elements in CuGAMOF, respectively;

[0031] Figure 3The copper metal organic framework material of the present invention, in Example 1, is the EDAX spectrum of Luminol@CuGAMOF; Figure 3 The scale bar in a is 5 μm for the SEM image of Luminol@CuGAMOF. Figure 3 b is the overall element distribution diagram of Luminol@CuGAMOF. Figure 3 c~f are the element distribution diagrams of C, N, O, and Cu in Luminol@CuGAMOF respectively;

[0032] Figure 4 The copper metal organic framework material of the present invention, in Example 1, FT-IR spectra of GA, Luminol@CuGAMOF, CuGAMOF and Luminol;

[0033] Figure 5 The copper metal organic framework material of the present invention, in Example 1, has XRD patterns of Luminol@CuGAMOF, CuGAMOF and Luminol;

[0034] Figure 6 The label-free self-enhanced ECL sensor of the present invention, in Example 2, uses the working electrodes GCE, GE, and ITO respectively to measure the effect of rHu EPO on the "luminol-hydrogen peroxide" ECL;

[0035] Figure 7 This is the label-free self-enhanced ECL sensor of the present invention, and the ECL intensity-potential diagram of each modified electrode in Example 3;

[0036] Figure 8 The label-free self-enhanced ECL sensor of the present invention is a cyclic voltammetry characterization curve of each step in the assembly process of the label-free self-enhanced ECL detection cell in Example 4;

[0037] Figure 9 For the label-free self-enhanced ECL sensor of the present invention, in Example 4, the standard curve of ECL signal (I) and rHu EPO concentration;

[0038] Figure 10 This is a graph showing the ECL signal stability of the label-free self-enhanced ECL sensor of the present invention, in Example 4, under continuous cyclic potential scanning. DETAILED DESCRIPTION

[0039] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0040] The preparation method of the copper metal organic framework material of the present invention comprises dissolving luminol (C8H7N3O2) and gallic acid (GA, C7H6O5) in sodium hydroxide solution to obtain a solution A, and then adding Cu 2+ The solution of is mixed with the solution A, heated and reacted to obtain a copper metal organic framework material. The copper metal organic framework material is a luminol functionalized self-enhanced MOF solid phase luminescent material, denoted as Luminol@CuGAMOF.

[0041] The preparation method of the copper metal organic framework material of the present invention is based on the effect of different concentrations of recombinant human erythropoietin (rHuEPO) on the enhanced chemiluminescence intensity of "luminol-hydrogen peroxide". The copper-based MOF material is used to encapsulate the luminol small molecule, thereby effectively improving the luminescence intensity. The luminol functionalized self-enhanced MOF solid-phase luminescent material prepared by this method is a self-enhanced nanoluminescent body that can not only realize high-throughput detection of human erythropoietin, but also effectively improve the efficiency and accuracy of detection.

[0042] Preferably, luminol, sodium hydroxide, GA and Cu 2+ The mass ratio is 1:3:3:6.67.

[0043] Preferably, the heating temperature is 85° C. to 95° C., the heating time is 1.5 to 3 hours, and stirring is performed during the heating process.

[0044] More preferably, the heating temperature is 85°C, 88°C, 90°C, 92°C, 95°C, etc., and the heating time is 1.5, 1.8, 2, 2.5, 2.8, 3 hours, etc.

[0045] Preferably, the preparation method of the luminol-functionalized self-enhanced MOF solid-phase luminescent material of the present invention comprises the following specific steps:

[0046] (1) Prepare a 30 mM Luminol solution by dissolving it in 0.4 M NaOH solution;

[0047] (2) Weigh 0.155 g of GA and dissolve it in the above solution. Stir thoroughly until the solution turns emerald green.

[0048] (3) Weigh 0.157 g of CuCl2·H2O2 and dissolve it in the above solution, shake thoroughly, and then immediately transfer it to a round-bottom flask;

[0049] (4) The round-bottom flask was placed in an oil bath with continuous stirring and heating at 90 °C for 2 h, and then centrifuged and washed three times with deionized water to obtain the copper metal organic framework material.

[0050] The above-mentioned specific preparation process is a "one-pot method". The luminol-functionalized copper metal organic framework material prepared by the "one-pot method" not only significantly improves its luminous efficiency, but also saves the amount of luminol used, which has certain environmental significance.

[0051] Preferably, in subsequent use, the copper metal organic framework material needs to be dispersed in water.

[0052] The label-free, self-enhanced ECL sensor of the present invention is prepared based on the aforementioned copper metal-organic framework material. The working electrode layer of this label-free, self-enhanced ECL sensor is coated with a luminescent material layer, which in turn is coated with a film layer. The luminescent material layer comprises the aforementioned copper metal-organic framework material. This ECL sensor not only enables high-throughput detection of human erythropoietin, but also effectively improves detection efficiency and accuracy. It also offers the advantages of simplicity, portability, and ease of preparation. Furthermore, this ECL sensor is also suitable for detecting dried blood spot samples, broadening its application and making the detection process more convenient.

[0053] Preferably, the working electrode layer is an ITO electrode, and the membrane layer is a perfluorosulfonic acid polymer (Nafion).

[0054] Compared to other common working electrode layers (GCE, GE), ITO maintains a relatively stable response across a wide range of rHu EPO concentrations, facilitating EPO detection. Furthermore, ITO electrodes offer a higher signal response than GCE. Using ITO as the working electrode allows for simpler and faster sample pretreatment, while also providing improved ECL stability (lower RSD).

[0055] Preferably, the label-free self-enhanced ECL sensor of the present invention is specifically a detection cell, which is prepared by the following method:

[0056] Cut the ITO conductive glass into uniform squares of 3*3cm in size, clean them with three times water, 0.1M NaOH, and three times water in sequence for 1 minute ultrasonic cleaning, and then blow dry with nitrogen for later use. Punch a hole (Φ=3mm) on the surface of a single-sided polyimide tape, then stick it to the conductive side of the ITO, aligning the hole of the tape with the center of the conductive glass. Stick a polytetrafluoroethylene tube (Φ=8mm) on the insulating tape, concentric with the center hole. Apply 2.5μL Luminol@CuGAMOF to the surface of the ITO detection cell, let it dry naturally, then add 5μL of 5wt% Nafion solution and let it dry naturally overnight. At this point, the preparation of the label-free self-enhanced ECL detection cell is completed.

[0057] The present invention's rapid erythropoietin detection method utilizes the aforementioned label-free, self-enhanced ECL sensor. Due to its rapid and athlete-friendly nature, this method can dynamically monitor human erythropoietin levels in athletes undergoing high-altitude training. This allows coaches to assess athlete performance and adjust training plans accordingly.

[0058] Preferably, the method specifically comprises the following steps:

[0059] S1. An Ag / AgCl electrode was used as a reference electrode, a spiral platinum wire electrode was used as a counter electrode, and a label-free self-enhanced ECL sensor was used as a working electrode, and they were connected to an electrochemiluminescence detection device.

[0060] S2. Using a phosphate buffer solution containing hydrogen peroxide, the electrochemiluminescence signal intensity generated by erythropoietin standards of different concentrations is detected by electrochemiluminescence.

[0061] Preferably, the detection parameters of the chemiluminescence detection device are: the high voltage of the photomultiplier tube is 500V, the scanning rate is 0.2V / s; and the scanning range is -1.0 to 1.2V.

[0062] Preferably, in the phosphate buffer solution, the content of hydrogen peroxide is 200 mM, the content of phosphate is 0.1 M, and the pH value of the phosphate buffer solution is 7.4.

[0063] S3. Draw a working curve based on the linear relationship between the obtained electrochemiluminescence signal intensity value and the corresponding erythropoietin standard concentration.

[0064] S4. Detect the erythropoietin sample to be tested using the method of steps S1 and S2 to obtain the electrochemiluminescence intensity value of the sample, and compare the electrochemiluminescence intensity value of the sample with the working curve to obtain the concentration of erythropoietin in the erythropoietin sample to be tested.

[0065] The above steps are used to detect erythropoietin, offering advantages such as speed and accuracy. Furthermore, the sample volume requirement is relatively low, allowing accurate detection with only a small amount of sample, with a detection limit of 0.56 mU / mL.

[0066] The present invention is illustrated below by means of specific examples.

[0067] Example 1 Preparation and verification of luminol functionalized self-enhanced MOF solid-phase luminescent material

[0068] In this example, the method of the present invention was used to prepare a Luminol@CuGAMOF solid-phase luminophore encapsulating luminol small molecules. The preparation process is as follows:

[0069] (1) Prepare a 30 mM Luminol solution and dissolve it in a 0.4 M NaOH solution.

[0070] (2) Weigh 0.155 g of GA and dissolve it in the above solution. Stir thoroughly until the solution turns emerald green.

[0071] (3) Weigh 0.157 g of CuCl2·H2O2 and dissolve it in the above solution. Shake thoroughly and then immediately transfer it to a round-bottom flask.

[0072] (4) Place the round-bottom flask in an oil bath at 90°C and heat with continuous stirring for 2 h. Then, wash it with deionized water by centrifugation three times and disperse it in water for later use.

[0073] In addition, CuGAMOF without Luminol was prepared according to the same method as a control group.

[0074] Relevant characterization experiments were carried out on the obtained luminol-functionalized self-enhanced MOF solid-phase luminescent material and the control group to verify the successful preparation.

[0075] The specific characterization was carried out by SEM electron microscopy, Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The characterization results are as follows:

[0076] By observation Figures 1 to 3 From the SEM image, it can be found that the structures of Luminol@CuGAMOF and CuGAMOF are basically the same, both of which are uniform rice-grain particles. According to the EDAX results, it can be seen that Luminol@CuGAMOF is rich in nitrogen, which is unique to luminol, while the proportion of nitrogen in CuGAMOF is extremely low. Combining scanning electron microscopy and elemental energy spectrum, it can be inferred that the luminol small molecules are encapsulated in Luminol@CuGAMOF and the crystal structure of MOF itself is not destroyed. Luminol@CuGAMOF was successfully synthesized.

[0077] The structural and composition changes of the materials during the synthesis process were characterized by FT-IR. Figure 4 As shown, in CuGAMOF and Luminol@CuGAMOF, the corresponding hydroxyl (-OH) groups are at 3494, 3350, and 3276 cm -1 The absorption peak at 1664 cm -1 The absorption peak at 1422 cm-1 of GA disappears, which proves that -OH and C=O groups participate in the synthesis of CuGAMOF. In addition, the stretching vibration band of CO can be observed in CuGAMOF from 1422 cm-1 of GA to -1 to 1408 cm of CuGAMOF -1In the CuGAMOF spectrum, the peaks at 1310, 1263, and 1023 cm-1 of the GA spectrum shift to 1314, 1200, and 1047 cm-1, respectively, all of which are related to CO vibrations. The shifts in the spectral bands indicate changes in the bond lengths of these groups, which can be attributed to the coordination between GA and Cu.

[0078] In addition, C=C (1491 cm -1 ), -NH2(701cm -1 ) and C=O(1655cm -1 ) group, and no new absorption peak was generated, which also proved that luminol did not participate in the synthesis reaction of MOF. The above also showed that the existence of luminol in Luminol@CuGAMOF was encapsulated in it.

[0079] The chemical composition and crystal phase purity of the material were characterized by XRD patterns. Figure 5 As shown in the XRD patterns of Luminol@CuGAMOF and CuGAMOF, distinct diffraction peaks can be observed near 10.2, 20.1, 31.4, and 33.4°, indicating that the synthesized self-enhanced luminescent molecules Luminol@CuGAMOF and CuGAMOF both have good crystallinity. In addition, a diffraction peak near 27.1°, which is unique to Luminol, was observed in the XRD pattern of Luminol@CuGAMOF, suggesting that Luminol may be encapsulated in the CuGAMOF material.

[0080] Example 2 Effect of recombinant human erythropoietin on the "luminol-hydrogen peroxide" electrochemiluminescence system

[0081] In this example, the detection results of different working electrodes in the "luminol-hydrogen peroxide" electrochemiluminescence system were compared to screen the best working electrode.

[0082] Specifically, this experiment is based on the classic "luminol-hydrogen peroxide" electrochemiluminescence system to conduct an ECL experiment on recombinant human erythropoietin (rHu EPO) standards with different concentration gradients to determine the effect of rHu EPO on the system.

[0083] The experiment tested the effects of free rHu EPO in a luminol-hydrogen peroxide electrochemiluminescence system using different working electrode layers, including glassy carbon electrodes (GCE), gold electrodes (GE), and indium tin oxide conductive glass (ITO). The experiment used a classic three-electrode system, with a spiral platinum wire electrode (Pt) as the counter electrode and silver-silver chloride (Ag|AgCl) as the reference electrode. Luminol at a concentration of 0.1 mM and hydrogen peroxide at a concentration of 100 mM were used as the luminescence background solution.

[0084] Different concentrations of rHu EPO (1, 10, 100 mU / mL) were added to the "luminol-hydrogen peroxide" electrochemiluminescence system under different working electrodes for testing. The experimental results are as follows:

[0085] like Figure 6 The experimental results show that different concentrations of rHu EPO have an impact on the ECL of "luminol-hydrogen peroxide", and the degree of the impact varies with different electrodes. In the range of rHu EPO concentration of 1-10mU / mL, the ECL under the three electrodes of GCE, GE and ITO was affected to varying degrees by rHu EPO (the slope of ΔI), among which the ΔI effect under GE was the strongest. However, in the range of rHu EPO concentration of 10-100mU / mL, the effect of rHu EPO on ΔI under GE became weaker. In contrast, the effect of rHu EPO on ΔI under the two electrodes of GCE and ITO was relatively stable. Maintaining a relatively stable degree of influence in a wider concentration range is conducive to the detection of the substance, and the ITO electrode has a higher signal response than the GCE.

[0086] In addition, compared with GCE and GE, ITO electrodes have a simpler and faster pretreatment method and better ECL stability (lower RSD). Therefore, ITO is more suitable for preparing sensors.

[0087] Example 3 Preparation of electrochemiluminescence sensor and ECL performance test

[0088] In this embodiment, an ITO electrode is used as the working electrode layer to prepare a portable and simple label-free self-enhanced ECL detection cell, which is an electrochemiluminescence sensor.

[0089] The preparation method is as follows: First, cut the ITO conductive glass into uniform squares of 3*3cm in size, clean it with water, anhydrous ethanol, and 0.1M NaOH in sequence for 10 minutes each, and then blow it dry with nitrogen for use. Punch a hole (Φ=3mm) on the surface of a single-sided polyimide tape, then stick it to the conductive side of the ITO, and align the hole of the tape with the center of the conductive glass. Stick a polytetrafluoroethylene tube (Φ=8mm) on the insulating tape, concentric with the center hole. Apply 2.5μL of Luminol@CuGAMOF prepared in Example 1 to the surface of the ITO detection cell, let it dry naturally, then add 5μL of 5wt% Nafion solution and let it dry naturally overnight. At this point, the preparation of the label-free self-enhanced ECL detection cell of this embodiment is completed.

[0090] At the same time, the same conditions were used to prepare 2+ electrochemical sensor.

[0091] The electrochemical sensors prepared in this example were tested for ECL performance, and the following results were obtained: Figure 6 The ECL intensity-potential diagram shown in the figure shows that the curves are: a: Luminol@CuGAMOF, b: Cu 2+ c: 0.0125 mM Luminol, c: 0.0125 mM Luminol, d: CuGAMOF, e: bare ITO. Testing was performed in 0.1 M PBS (pH 7.4) containing 200 mM H₂O₂. Scan rate: 0.2 V / s. Scan range: -1.0 to 1.2 V. PMT: 500 V.

[0092] according to Figure 7 It can be seen that Luminol@CuGAMOF can show higher ECL emission intensity than free Luminol solution due to the molecular confinement effect of the metal organic framework in the presence of equal amount of luminol. 2+ The presence of promotes the release of more active oxygen intermediates from hydrogen peroxide, which can then promote the "luminol-hydrogen peroxide" ECL system to achieve higher luminescence intensity. Although the CuGAMOF material itself does not emit light in the system, the Cu 2+ The presence of may also be one of the reasons that promote the excellent luminescence effect of Luminol@CuGAMOF.

[0093] Example 4 Verification of the stability and accuracy of electrochemiluminescence sensor in rapid detection of erythropoietin

[0094] The same method as in Example 3 was used to prepare a label-free self-enhanced ECL detection cell.

[0095] Before testing actual samples, the label-free self-enhanced ECL detection cell needs to be characterized. Cyclic voltammetry (CV) was used to characterize each modification step in an aqueous solution containing 5 mM K3Fe(CN)6 and 0.5 M KCl at a scan rate of 0.05 V / s.

[0096] The results are as follows Figure 8 As shown in the figure, the curves are: a) ITO, b) Luminol@CuGAMOF / ITO, and c) Nafion / Luminol@CuGAMOF / ITO. Because the unmodified bare ITO electrode has good conductivity, while the MOF material and Nafion membrane have relatively poor conductivity, the peak current gradually decreases with the gradual modification of the electrode surface, which is consistent with the actual modification situation.

[0097] The characterized label-free self-enhanced ECL detection cell was used to directly detect rHu EPO at multiple concentration gradients. The standard curve is shown in Figure 2. Figure 8 As shown, the ECL signal stability diagram under continuous cyclic potential scanning is as follows Figure 9 The test conditions were as follows: 0.1 M PBS (pH = 7.4) containing 200 mM H2O2, scan rate: 0.2 V / s, scan range: -1.0 to 1.2 V, PMT: 500 V.

[0098] according to Figure 9 and Figure 10 It can be seen that the test results show a good linear relationship in the range of 1 to 90 mU / mL, and the regression equation is I = 16.4C rHu EPO +1305.1, R 2 =0.9945, and the signal-to-noise ratio is 3. The label-free self-enhanced ECL detection cell also exhibits good stability. The ECL signal of the detection cell remains essentially unchanged after 20 consecutive cyclic voltammetry scans, with an RSD of 2.06%.

[0099] The label-free, self-enhanced ECL detection cell was used to detect EPO in samples of known concentration. Testing and calculations showed that the detection method of the present invention has an accuracy rate of 85% to 95%. The limit of detection (LOD) of this method was 0.56 mU / mL, calculated using a regression equation and the standard deviation of the blank value.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a copper metal organic framework material, characterized in that: Dissolve luminol and gallic acid in sodium hydroxide solution to obtain solution A. 2+ The solution is mixed with the A solution, heated and reacted to obtain the copper metal organic framework material; luminol, sodium hydroxide, gallic acid and Cu 2+ The molar ratio is 1:3:3:6.

67.

2. The method for preparing a copper metal organic framework material according to claim 1, characterized in that: The heating temperature is 85° C. to 95° C., the heating time is 1.5 to 3 hours, and stirring is performed simultaneously during the heating process.

3. A copper metal organic framework material, characterized in that The method according to claim 1 or 2 is used for preparation.

4. A label-free self-enhanced ECL sensor, characterized in that: It comprises a working electrode layer, a luminescent material layer is coated on the working electrode layer, and a film layer is coated on the luminescent material layer; wherein the composition of the luminescent material layer is the copper metal organic framework material as claimed in claim 3.

5. The label-free self-enhanced ECL sensor according to claim 4, characterized in that: The working electrode layer is an ITO electrode, and the material of the film layer is a perfluorosulfonic acid polymer.

6. A rapid detection method for recombinant human erythropoietin, characterized in that: The label-free self-enhanced ECL sensor according to claim 4 or 5 is used for detection, and the rapid detection method is not directly intended for disease diagnosis or health status assessment.

7. The rapid detection method for recombinant human erythropoietin according to claim 6, characterized in that: The following steps are involved: S1, connecting an Ag / AgCl electrode as a reference electrode, a spiral platinum wire electrode as a counter electrode, and the label-free self-enhanced ECL sensor as a working electrode in a chemiluminescence detection device; S2. Using a phosphate buffer solution containing hydrogen peroxide, the electrochemiluminescence signal intensity generated by erythropoietin standards of different concentrations is detected by electrochemiluminescence; S3. drawing a working curve based on the obtained linear relationship between the electrochemiluminescence signal intensity value and the corresponding erythropoietin standard concentration; S4. Detect the erythropoietin sample to be tested using the method of steps S1 and S2 to obtain an electrochemiluminescence intensity value of the sample, and compare the electrochemiluminescence intensity value of the sample with the working curve to obtain the concentration of erythropoietin in the erythropoietin sample to be tested.

8. The rapid detection method for recombinant human erythropoietin according to claim 7, characterized in that: The detection parameters of the chemiluminescence detection device are: the high voltage of the photomultiplier tube is 500 V, the scanning rate is 0.2 V / s; and the scanning range is -1.0~1.2V.

9. The rapid detection method for recombinant human erythropoietin according to claim 7, characterized in that: In step S2, the content of hydrogen peroxide in the phosphate buffer solution is 200 mM, the content of phosphate is 0.1 M, and the pH value of the phosphate buffer solution is 7.4.

Images