阅读说明：本技术 一种肿瘤标志物8-羟基脱氧鸟苷的检测方法 (Detection method of tumor marker 8-hydroxydeoxyguanosine ) 是由 邱彬 赵华楠 罗子伊 傅志宏 林敏� 于 2020-06-12 设计创作，主要内容包括：本发明公开了一种肿瘤标志物8-羟基脱氧鸟苷的检测方法,包括以下步骤：准备试剂和设备：所用的主要试剂包括β-环糊精(β-CD)、五水硫酸铜(CuSO<Sub>4</Sub>·5H<Sub>2</Sub>O)、L(+)-抗环血酸(AA)、尿酸(UA)、羧基化多壁碳纳米管(MWCNTSs)、Nafion?全氟化树脂溶液(Nafion)、8-羟基脱氧鸟苷(8-OHdG)、氯化钾(KCl)；本发明通过设置准备试剂和设备、CuNCs材料合成、CuNCs-MWCNTSs材料的合成、CuNCs-MWCNTSs-nafion材料的合成、电极的修饰、8-OHdG的检测和电化学测试的工艺流程,解决了传统的8-OHdG测定方法预处理步骤复杂,使用的仪器昂贵,操作程序十分繁琐,这些限制了传统的8-OHdG测定方适用性的问题,该肿瘤标志物8-羟基脱氧鸟苷的检测方法,具备预处理步骤十分简单、操作程序比较容易仪器较为普通的优点。(The invention discloses a detection method of tumor marker 8-hydroxydeoxyguanosine, which comprises the following steps of preparing reagents and equipment, wherein the main reagents comprise β -cyclodextrin (β -CD) and copper sulfate pentahydrate (CuSO) 4 ·5H 2 O), L (+) -Ascorbic Acid (AA), Uric Acid (UA), carboxylated multi-walled carbon nanotubes (MWCNTSs), Nafion perfluorinated resin solution (Nafion), 8-hydroxydeoxyguanosine (8-OHdG) and potassium chloride (KCl); the invention solves the problems of complex pretreatment steps, expensive used instruments and complicated operation procedures of the traditional 8-OHdG measuring method by setting the preparation reagent and equipment, the synthesis of CuNCs materials, the synthesis of CuNCs-MWCNTSs materials, the synthesis of CuNCs-MWCNTs-nafion materials, the modification of electrodes, the detection of 8-OHdG and the process flow of electrochemical testsThe method for detecting the tumor marker 8-hydroxydeoxyguanosine has the advantages of very simple pretreatment steps and relatively easy operation procedures and relatively common instruments.)

1. A detection method of a tumor marker 8-hydroxydeoxyguanosine is characterized by comprising the following steps: the method comprises the following steps:

step 1 preparation of reagents and apparatus the main reagents used included β -cyclodextrin (β -CD), copper sulfate pentahydrate (CuSO)₄·5H₂O), L (+) -Ascorbic Acid (AA), Uric Acid (UA), and carboxylated multi-walled carbon nanotube (A)MWCNTSs), Nafion perfluorinated resin solution (Nafion), 8-hydroxydeoxyguanosine (8-OHdG), potassium chloride (KCl) and potassium ferricyanide (K)₃Fe(CN)₆) Potassium ferrocyanide (K)₄Fe(CN)₆·3H₂O), sodium dihydrogen phosphate (NaH)₂PO₄·2H₂O), disodium hydrogen phosphate dodecahydrate (Na)₂HPO₄·12H₂O), the main instruments used comprise a Milli-Q ultrapure water system, an analytical balance, a vortex mixer, a precision pipetting gun, a transmission electron microscope, an electrochemical workstation, a digital display constant temperature multi-head magnetic stirrer, a numerical control ultrasonic cleaner and a desk-top acidimeter;

step 2, CuNCs material synthesis, firstly weighing 0.011 g β -CD to be dissolved in 3.0 mL water, secondly adding 400.0 muL 10.0mmol/L CuSO₄And 100.0. mu.L of 1mol/L AA; reacting for 10 hours in water bath stirring at 40 ℃ to obtain light yellow CuNCs;

and step 3: synthesis of CuNCs-MWCNTSs material: dissolving 0.65 mg of MWCNTSs in 1400.0 mu L of water, ultrasonically treating and stirring for 15 min respectively, quickly adding CuNCs, and stirring at normal temperature to obtain a uniform CuNCs-MWCNTSs suspension;

and 4, step 4: synthesis of CuNCs-MWCNTSs-nafion material: adding 20.0 mu L of 0.5% nafion solution into 80.0 mu L of CuNCs-MWCNTSs, and uniformly mixing by vortex for preparing a modified electrode;

and 5: modification of the electrode: first, a Glassy Carbon Electrode (GCE) was prepared using 0.3 μm and 0.05 μm Al in this order₂O₃Polishing the powder to be a mirror surface, washing the mirror surface by using distilled water, then respectively performing ultrasonic treatment on absolute ethyl alcohol and ultrapure water for 3 min, finally drying the washed electrode by using nitrogen for later use, taking CuNCs-MWCNTs-nafion dispersed liquid to be dripped on the surface of a clean glassy carbon electrode, naturally drying the dispersed liquid to obtain CuNCs-MWCNTs-nafion/GCE, and simultaneously preparing MWCNTs-nafion/GCE, CuNCs-nafion/GCE, &lttttTtranslation = beta' &gtTgtTpbeta &ltlTttg/T &ttgtTgt-CD-MWCNTs-nafion/GCE, wherein the prepared electrode is stored at 4 ℃ before use;

step 6: detection of 8-OHdG: selecting a three-electrode working system for measurement: CuNCs-MWCNTSs-nafion/GCE is used as a working electrode; an Ag/AgCl electrode is used as a reference electrode; the platinum wire electrode is a counter electrode, 7.0 mu L of CuNCs-MWCNTSs-nafion is dripped on the surface of the GCE, the GCE is placed into PBS containing 8-OHdG with different concentrations and stands for 9 min, and DPV detection is carried out on the 8-OHdG;

and 7: electrochemical testing: the modified working electrode is placed in a test cell for electrochemical testing, and Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV) are selected as electrochemical detection methods.

2. The method for detecting 8-hydroxydeoxyguanosine as a tumor marker according to claim 1, wherein the method comprises the following steps: in step 1, the reagents are all analytically pure and can be used without further purification.

3. The method for detecting 8-hydroxydeoxyguanosine as a tumor marker according to claim 1, wherein the method comprises the following steps: in step 1, the water was ultrapure water (resistivity 18.2 M.OMEGA.cm) purified by Millipore Milli-Q system, Phosphate Buffer Solution (PBS): 0.2 mol/L NaH₂PO₄·2H₂O，0.2 mol/LNa₂HPO₄·12H₂O，0.1 mol/L KCl。

4. The method for detecting 8-hydroxydeoxyguanosine as a tumor marker according to claim 1, wherein the method comprises the following steps: in step 3, the amount of CuNCs added was 300.0. mu.L, and stirred at room temperature for 6 hours.

5. The method for detecting 8-hydroxydeoxyguanosine as a tumor marker according to claim 1, wherein the method comprises the following steps: in step 5, the 8-OHdG oxidation peak current value is gradually increased along with the increase of the dropping volume, and when the dropping volume is 7.0 μ L, the peak current value is not increased any more, so that 7.0 μ L of CuNCs-MWCNTs-nafion is selected as the optimal dropping volume.

6. The method for detecting 8-hydroxydeoxyguanosine as a tumor marker according to claim 1, wherein the method comprises the following steps: in step 6, the oxidation peak current of 8-OHdG is increased and then slightly decreased with the increase of the PBS concentration, and the 8-OHdG peak current value is the largest when the PBS concentration is 0.1mol/L, so that 0.1mol/L PBS is selected as the optimal concentration of the supporting electrolyte.

7. The method for detecting 8-hydroxydeoxyguanosine as a tumor marker according to claim 1, wherein the method comprises the following steps: in step 7, the cyclic voltammetry test is performed at a concentration of 1 mmol/L [ Fe (CN)₆]^3−/4−And KCl of 0.1mol/L, wherein the scanning range is-0.1V-0.6V, and the scanning speed is 50 mV/s.

8. The method for detecting 8-hydroxydeoxyguanosine as a tumor marker according to claim 1, wherein the method comprises the following steps: in step 7, the electrochemical impedance test is carried out in a reactor containing 5 mmol/L [ Fe (CN)₆]^3−/4−And 0.1mol/L KCl, initial potential: 0.243V, amplitude width: 5.0 mV, frequency range: 0.1 Hz-10 Hz⁴Hz。

9. The method for detecting 8-hydroxydeoxyguanosine as a tumor marker according to claim 1, wherein the method comprises the following steps: in step 7, the DPV potential test is performed in 0.1mol/L PBS (pH = 7.0) containing different target concentrations, the scanning range is 0.1V to-0.8V, and the amplitude width: 5.0 mV, pulse width 50, rest time 2 min.

Technical Field

The invention relates to the technical field of medical detection, in particular to a detection method of a tumor marker 8-hydroxydeoxyguanosine.

Background

Research has shown that 8-OHdG is an oxidative adduct formed by the C-atom at position 8 of the guanine base in human DNA molecules binding ∙ OH under excessive ROS attack. The generation and modification of 8-OHdG are not influenced by factors such as diet and the like, so that the method is a high-efficiency index for measuring oxidative stress and DNA oxidative damage which is accepted by academia at present. More and more researches show that the content of 8-OHdG is also closely related to the occurrence and development of tumors. The 8-OHdG has higher expression in body fluid and cancer tissues of patients with liver cancer, breast cancer, gastric cancer, ovarian cancer, lung cancer, esophageal cancer and the like. Measurement of 8-OHdG is therefore a key marker to assess the extent of endogenous oxidative damage to DNA and as an initiating and contributing factor to carcinogenesis.

Conventional 8-OHdG assay: such as high performance liquid chromatography electrochemical detection (HPLC-ECD), liquid chromatography-tandem mass spectrometry (LC-MS/MS), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis-electrochemical detection (CE-ECD), 32P labeling and the like. However, the pretreatment steps of the traditional 8-OHdG measuring method are complex, the used instrument is expensive, the operation procedure is very complicated, and the method limits the applicability of the traditional 8-OHdG measuring method, so that a method for detecting the tumor marker 8-hydroxydeoxyguanosine is provided for solving the problem.

Disclosure of Invention

The invention aims to provide a detection method of a tumor marker 8-hydroxydeoxyguanosine, which solves the problems that the traditional 8-OHdG detection method has complicated pretreatment steps, expensive used instruments and very complicated operation procedures, and the applicability of the traditional 8-OHdG detection method is limited.

In order to achieve the purpose, the invention provides the following technical scheme: a detection method of a tumor marker 8-hydroxydeoxyguanosine comprises the following steps:

step 1 preparation of reagents and apparatus the main reagents used included β -cyclodextrin (β -CD), copper sulfate pentahydrate (CuSO)₄·5H₂O), L (+) -Ascorbic Acid (AA), Uric Acid (UA), carboxylated multi-walled carbon nanotubes (MWCNTSs) and Nafion perfluorinated resin solubleLiquid (Nafion), 8-hydroxydeoxyguanosine (8-OHdG), potassium chloride (KCl) and potassium ferricyanide (K)₃Fe(CN)₆) Potassium ferrocyanide (K)₄Fe(CN)₆·3H₂O), sodium dihydrogen phosphate (NaH)₂PO₄·2H₂O), disodium hydrogen phosphate dodecahydrate (Na)₂HPO₄·12H₂O), the main instruments used comprise a Milli-Q ultrapure water system, an analytical balance, a vortex mixer, a precision pipetting gun, a transmission electron microscope, an electrochemical workstation, a digital display constant temperature multi-head magnetic stirrer, a numerical control ultrasonic cleaner and a desk-top acidimeter;

step 2, CuNCs material synthesis, firstly weighing 0.011 g β -CD to be dissolved in 3.0 mL water, secondly adding 400.0 muL 10.0mmol/L CuSO₄And 100.0. mu.L of 1mol/L AA; reacting for 10 hours in water bath stirring at 40 ℃ to obtain light yellow CuNCs;

and step 3: synthesis of CuNCs-MWCNTSs material: dissolving 0.65 mg of MWCNTSs in 1400.0 mu L of water, ultrasonically treating and stirring for 15 min respectively, quickly adding CuNCs, and stirring at normal temperature to obtain a uniform CuNCs-MWCNTSs suspension;

and 4, step 4: synthesis of CuNCs-MWCNTSs-nafion material: adding 20.0 mu L of 0.5% nafion solution into 80.0 mu L of CuNCs-MWCNTSs, and uniformly mixing by vortex for preparing a modified electrode;

and 5: modification of the electrode: first, a Glassy Carbon Electrode (GCE) was prepared using 0.3 μm and 0.05 μm Al in this order₂O₃Polishing the powder to be a mirror surface, washing the mirror surface by using distilled water, then respectively performing ultrasonic treatment on absolute ethyl alcohol and ultrapure water for 3 min, finally drying the washed electrode by using nitrogen for later use, taking CuNCs-MWCNTs-nafion dispersed liquid to be dripped on the surface of a clean glassy carbon electrode, naturally drying the dispersed liquid to obtain CuNCs-MWCNTs-nafion/GCE, and simultaneously preparing MWCNTs-nafion/GCE, CuNCs-nafion/GCE, &lttttTtranslation = beta' &gtTgtTpbeta &ltlTttg/T &ttgtTgt-CD-MWCNTs-nafion/GCE, wherein the prepared electrode is stored at 4 ℃ before use;

step 6: detection of 8-OHdG: selecting a three-electrode working system for measurement: CuNCs-MWCNTSs-nafion/GCE is used as a working electrode; an Ag/AgCl electrode is used as a reference electrode; the platinum wire electrode is a counter electrode, 7.0 mu L of CuNCs-MWCNTSs-nafion is dripped on the surface of the GCE, the GCE is placed into PBS containing 8-OHdG with different concentrations and stands for 9 min, and DPV detection is carried out on the 8-OHdG;

and 7: electrochemical testing: the modified working electrode is placed in a test cell for electrochemical testing, and Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV) are selected as electrochemical detection methods.

Preferably, in step 1, the reagents are all analytically pure and can be used without further purification.

Preferably, in step 1, the water is ultrapure water (resistivity 18.2 M.OMEGA.cm) purified by a Millipore Milli-Q system, Phosphate Buffer Solution (PBS): 0.2 mol/LNaH₂PO₄·2H₂O，0.2 mol/L Na₂HPO₄·12H₂O，0.1 mol/L KCl。

Preferably, in step 3, the amount of CuNCs added is 300.0. mu.L, and stirring is performed at normal temperature for 6 hours.

Preferably, in step 5, the 8-OHdG oxidation peak current value is gradually increased along with the increase of the dropping volume, and when the dropping volume is 7.0 μ L, the peak current value is not increased any more, so that 7.0 μ L of CuNCs-MWCNTs-nafion is selected as the optimal dropping volume.

Preferably, in step 6, the oxidation peak current of 8-OHdG is increased and then slightly decreased with the increase of the PBS concentration, and the 8-OHdG peak current value is the largest when the PBS concentration is 0.1mol/L, so that 0.1mol/L PBS is selected as the optimal concentration of the supporting electrolyte.

Preferably, in step 7, the cyclic voltammetry test is performed in the presence of 1 mmol/L [ Fe (CN)₆]^3−/4−And KCl of 0.1mol/L, wherein the scanning range is-0.1V-0.6V, and the scanning speed is 50 mV/s.

Preferably, in step 7, the electrochemical impedance test is performed in a sample containing 5 mmol/L [ Fe (CN)₆]^3−/4−And 0.1mol/L KCl, initial potential: 0.243V, amplitude width: 5.0 mV, frequency range: 0.1 Hz-10 Hz⁴Hz。

Preferably, in step 7, the DPV potential test is performed in PBS (pH = 7.0) containing 0.1mol/L of different target concentrations, the scanning range is 0.1V to-0.8V, and the amplitude width: 5.0 mV, pulse width 50, rest time 2 min.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, by setting the preparation reagent and equipment, the synthesis of CuNCs materials, the synthesis of CuNCs-MWCNTSs-nafion materials, the modification of electrodes, the detection of 8-OHdG and the process flow of electrochemical test, the problems of complex pretreatment steps, expensive used instruments and complicated operation procedures of the traditional 8-OHdG determination method and the limitation of the applicability of the traditional 8-OHdG determination method are solved, and the detection method of the tumor marker 8-hydroxydeoxyguanosine has the advantages of simple pretreatment steps and easier operation procedures and is more common in instruments.

Detailed Description

The present invention will now be described in more detail by way of examples, which are given by way of illustration only and are not intended to limit the scope of the present invention in any way.

The invention provides a technical scheme that: a detection method of a tumor marker 8-hydroxydeoxyguanosine comprises the following steps:

step 1 preparation of reagents and apparatus the main reagents used included β -cyclodextrin (β -CD), copper sulfate pentahydrate (CuSO)₄·5H₂O), L (+) -Ascorbic Acid (AA), Uric Acid (UA), carboxylated multi-walled carbon nanotubes (MWCNTSs), Nafion perfluorinated resin solution (Nafion), 8-hydroxydeoxyguanosine (8-OHdG), potassium chloride (KCl) and potassium ferricyanide (K) in sequence₃Fe(CN)₆) Potassium ferrocyanide (K)₄Fe(CN)₆·3H₂O), sodium dihydrogen phosphate (NaH)₂PO₄·2H₂O), disodium hydrogen phosphate dodecahydrate (Na)₂HPO₄·12H₂O), the main instruments used comprise a Milli-Q ultrapure water system, an analytical balance, a vortex mixer, a precision pipetting gun, a transmission electron microscope, an electrochemical workstation and a digital displayA constant-temperature multi-head magnetic stirrer, a numerical control ultrasonic cleaner and a desk-top acidimeter;

step 2, CuNCs material synthesis, firstly weighing 0.011 g β -CD to be dissolved in 3.0 mL water, secondly adding 400.0 muL 10.0mmol/L CuSO₄And 100.0. mu.L of 1mol/L AA; reacting for 10 hours in water bath stirring at 40 ℃ to obtain light yellow CuNCs;

and step 3: synthesis of CuNCs-MWCNTSs material: dissolving 0.65 mg of MWCNTSs in 1400.0 mu L of water, ultrasonically treating and stirring for 15 min respectively, quickly adding CuNCs, and stirring at normal temperature to obtain a uniform CuNCs-MWCNTSs suspension;

and 4, step 4: synthesis of CuNCs-MWCNTSs-nafion material: adding 20.0 mu L of 0.5% nafion solution into 80.0 mu L of CuNCs-MWCNTSs, and uniformly mixing by vortex for preparing a modified electrode;

and 5: modification of the electrode: first, a Glassy Carbon Electrode (GCE) was prepared using 0.3 μm and 0.05 μm Al in this order₂O₃Polishing the powder to be a mirror surface, washing the mirror surface by using distilled water, then respectively performing ultrasonic treatment on absolute ethyl alcohol and ultrapure water for 3 min, finally drying the washed electrode by using nitrogen for later use, taking CuNCs-MWCNTs-nafion dispersed liquid to be dripped on the surface of a clean glassy carbon electrode, naturally drying the dispersed liquid to obtain CuNCs-MWCNTs-nafion/GCE, and simultaneously preparing MWCNTs-nafion/GCE, CuNCs-nafion/GCE, &lttttTtranslation = beta' &gtTgtTpbeta &ltlTttg/T &ttgtTgt-CD-MWCNTs-nafion/GCE, wherein the prepared electrode is stored at 4 ℃ before use;

step 6: detection of 8-OHdG: selecting a three-electrode working system for measurement: CuNCs-MWCNTSs-nafion/GCE is used as a working electrode; an Ag/AgCl electrode is used as a reference electrode; the platinum wire electrode is a counter electrode, 7.0 mu L of CuNCs-MWCNTSs-nafion is dripped on the surface of the GCE, the GCE is placed into PBS containing 8-OHdG with different concentrations and stands for 9 min, and DPV detection is carried out on the 8-OHdG;

and 7: electrochemical testing: the modified working electrode is placed in a test cell for electrochemical testing, and Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV) are selected as electrochemical detection methods.