Electrode based on PLL / MWCNTs-NH2 electrode modification material and preparation method and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to an electrode based on a PLL / MWCNTs-NH2 electrode modification material and a preparation method and application of the electrode. A glassy carbon electrode (GCE) is used as a substrate, and polylysine (PLL) and aminated multi-walled carbon nanotubes (MWCNTs-NH2) are sequentially modified. By optimizing detection conditions, such as pH, scanning speed, PBS concentration and the like, the sensor has good linear response to 8-OHdG in a range of 0.0044 mu M-14.12 mu M, and the detection limit is as low as 0.2 nM. The sensor has strong anti-interference capability, can effectively eliminate the influence of interferents such as uric acid, and has good stability and repeatability. In actual urine sample detection, the detection result is similar to that of an ELISA method, and the kit has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and specifically to an electrode based on PLL / MWCNTs-NH 2 electrode modification material, its preparation method and application. Background Art

[0002] 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is an important biomarker of DNA oxidative damage. The change of its content in vivo is closely related to the occurrence and development of various diseases, such as cancer, neurodegenerative diseases, etc. Accurately detecting the content of 8-OHdG in biological samples is of great significance for the early diagnosis of diseases, the monitoring of disease conditions and the evaluation of treatment effects.

[0003] Currently, the methods for detecting 8-OHdG mainly include high performance liquid chromatography (HPLC), mass spectrometry technology, etc. However, these methods have disadvantages such as expensive equipment, complex operation, long detection cycle, etc., which limit their application in clinical and on-site detection. Electrochemical sensors have advantages such as low cost, high sensitivity, fast response speed, simple operation, etc., and show great application potential in the field of biomolecule detection. However, the existing electrochemical sensors still have problems such as insufficient sensitivity and poor anti-interference ability when detecting 8-OHdG, and need to be further improved. Summary of the Invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides an electrode based on PLL / MWCNTs-NH 2 electrode modification material, its preparation method and application, making up for the deficiencies of existing detection methods and realizing high-sensitivity, high-selectivity and rapid detection of 8-OHdG.

[0005] To achieve the above purpose, the present invention provides the following technical solutions: An electrode based on PLL / MWCNTs-NH 2 electrode modification material, with a glassy carbon electrode as the substrate, and polylysine and amino-functionalized multi-walled carbon nanotubes are sequentially modified on the surface of the glassy carbon electrode.

[0006] A preparation method of an electrode,

[0007] (1) Preparation of PLL (polylysine) deposition solution:

[0008] Dissolve lysine in phosphate buffer solution (PBS), and after fully dissolving and mixing, obtain the deposition solution of PLL;

[0009] (2) Preparation of MWCNTs-NH 2 suspension:

[0010] Disperse MWCNTs-NH 2Dissolve it in deionized water, stir for 12 h, and after the stirring is completed, place it in an ultrasonic instrument and ultrasonicate for 30 min;

[0011] (3) Electrode modification:

[0012] Pretreat the bare glassy carbon electrode;

[0013] Take out the PLL deposition solution in (1), scan 8 cycles under the condition of -1.5 V - 2.5 V to obtain a PLL deposition layer on the surface of the glassy carbon electrode (GCE); take the MWCNTs-NH 2 suspension and drop-coat it on the PLL deposition layer modified electrode, and wait for natural drying to obtain PLL / MWCNTs-NH 2 electrode.

[0014] Furthermore, the PBS in (1) is 0.1 M and pH 9.0.

[0015] Furthermore, the concentration of lysine is 10 mM.

[0016] Furthermore,

[0017] the concentration of MWCNTs-NH 2 in (2) is 1 mg / mL.

[0018] Furthermore,

[0019] The pretreatment of the bare glassy carbon electrode in (3) is as follows: The bare glassy carbon electrode is polished with 0.3 μm and 0.05 μm alumina slurries respectively, and then the electrode is ultrasonically washed in nitric acid (1:1), ethanol and deionized water for 3 min in sequence.

[0020] Furthermore,

[0021] The volume ratio of the PLL deposition solution to the MWCNTs-NH 2 suspension is 500:1.

[0022] The application of an electrode as a sensor for detecting 8-OHdG, wherein the electrode is prepared by the above method.

[0023] The application of a sensor in the detection of 8-hydroxy-2'-deoxyguanosine in environmental monitoring.

[0024] Further, the detection conditions of the sensor are as follows: a three-electrode system is adopted, where the working electrode, reference electrode, and counter electrode are a glassy carbon electrode modified with the experimental material, a silver chloride electrode soaked in saturated potassium chloride, and a platinum electrode, respectively; the electrolyte solution is 0.1 M PBS with pH 7.0 containing 8-OHdG; the test conditions for cyclic voltammetry (CV) are a scanning voltage of 0.1 V - 0.65 V and a scanning speed of 100 mV / s; the test conditions for differential pulse voltammetry (DPV) are a scanning voltage of 0.1 V - 0.65 V, an amplitude of 50 mV, a pulse width of 50 ms, and a scanning speed of 50 mV / s.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The polylysine PLL of the present invention has good biocompatibility and film-forming properties, and can provide stable support for the loading of amino-functionalized multi-walled carbon nanotubes MWCNTs-NH 2 while MWCNTs-NH 2 has a large specific surface area and excellent electrical conductivity. The PLL / MWCNTs-NH 2 composite material formed by the combination of the two can significantly improve the electron transfer efficiency and sensitivity of the sensor. In addition, the structure of PLL matches the spatial conformation of 8-OHdG, improving the specificity of recognition; MWCNTs-NH 2 enhances the electrical conductivity of the electrode. The two are combined through electrostatic interaction, and the formed complex has the strongest electroactivity towards 8-OHdG. The large specific surface area, multiple active sites of the composite material, and the synergistic effect between the components make the sensor have excellent performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 CV curves of the bare GCE electrode, MWCNTs-NH 2 , PLL, and PLL / MWCNTs-NH 2 modified electrodes of the present invention in 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG.

[0028] Figure 2 A is the CV diagram of the PLL / MWCNTs-NH 2 modified electrode of the present invention at different pH values;

[0029] Figure 2 B is the peak current calibration curve of the oxidation peak potential of the PLL / MWCNTs-NH 2 modified electrode of the present invention at different pH values;

[0030] Figure 3 A is the CV diagram of the PLL / MWCNTs-NH 2 modified electrode of the present invention at different scanning speeds;

[0031] Figure 3 B is the PLL / MWCNTs-NH of the present invention 2 Calibration curve of the oxidation peak current of the modified electrode at different scanning rates;

[0032] Figure 4 is the PLL / MWCNTs-NH of the present invention 2 Change of oxidation peak current of the modified electrode in PBS with different concentrations;

[0033] Figure 5 is the change of oxidation peak current of PLL of the present invention under different deposition conditions;

[0034] Figure 6 is different MWCNTs-NH of the present invention 2 Change of oxidation peak current of PLL / MWCNTs-NH modified electrode under different drop-coating amounts of 2 ;

[0035] Figure 7 is the PLL / MWCNTs-NH of the present invention 2 Change of oxidation peak current of the modified electrode at different incubation times;

[0036] Figure 8 is the PLL / MWCNTs-NH of the present invention 2 DPV diagram and calibration curve of the modified electrode in PBS solution with different concentrations of 8-OHdG;

[0037] Figure 9 is the change of current signal of 8-OHdG in the presence of interfering substances of the present invention;

[0038] Figure 10 A is the CV diagram of UA and 8-OHdG of the present invention,

[0039] Figure 10 B is the CV diagram of UA, 8-OHdG and uricase of the present invention;

[0040] Figure 11 is the test result of the anti-interference experiment of the present invention;

[0041] Figure 12 is the test result of the stability of the present invention. Detailed implementation manners

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0043] Embodiment 1:

[0044] An electrode based on a PLL / MWCNTs-NH 2 electrode modification material, with a glassy carbon electrode as the substrate, and polylysine and amino-functionalized multi-walled carbon nanotubes are sequentially modified on the surface of the glassy carbon electrode.

[0045] A preparation method of an electrode,

[0046] (1) Synthesis of PLL: Prepare 0.1 M PBS (pH 9.0), and then dissolve 0.01462 g of lysine in the above PBS to make the concentration of lysine 10 mM. After fully dissolving and mixing evenly, a deposition solution of PLL is obtained and stored in a refrigerator at 4 °C for standby.

[0047] (2) Preparation of MWCNTs-NH 2 suspension: Take 8 mg of MWCNTs-NH 2 , dissolve it in 8 mL of deionized water, put magnetic beads into the mixed solution, place it on a magnetic stirrer and stir for 12 h, then put it into an ultrasonic instrument and ultrasonicate for 30 min. After the ultrasonic treatment is completed, store it in a refrigerator at 4 °C for standby.

[0048] (3) Construction of the electrochemical sensor interface

[0049] Before modifying the electrode, the bare GCE is polished with 0.3 μm and 0.05 μm alumina slurries respectively. Then, the electrode is ultrasonically washed in nitric acid (1:1), ethanol and deionized water for 3 min in sequence. Take out the pre-prepared PLL deposition solution, and scan 8 cycles under the condition of -1.5 V - 2.5 V to obtain PLL on the electrode surface. Subsequently, the MWCNTs-NH 2 nanomaterials are fully mixed with a vortex mixer, and 10 μL of the MWCNTs-NH 2 nanomaterial suspension is dropped on the PLL-modified electrode and allowed to dry naturally to obtain the PLL / MWCNTs-NH 2 electrode. The volume ratio of the PLL deposition solution to the MWCNTs-NH 2 suspension is 500:1. Subsequently, the same method is used to modify PLL and MWCNTs-NH 2 on the electrode surface respectively as a control experiment.

[0050] Example 2:

[0051] Application of an electrode in preparing a sensor for detecting 8-OHdG, wherein the electrode is prepared by the method of Example 1.

[0052] The detection principle is as follows: When the PLL / MWCNTs-NH 2 modified electrode is placed in the electrolyte solution containing 8-OHdG, due to the matching of the structure of PLL with the spatial conformation of 8-OHdG, 8-OHdG can specifically adsorb on the electrode surface. At the same time, MWCNTs-NH 2 excellent conductivity provides a good channel for electron transfer. After applying a certain potential, 8-OHdG undergoes an oxidation reaction on the electrode surface, and specific groups (such as hydroxyl groups) in its molecular structure lose electrons, generating an oxidation current. The oxidation current is detected by differential pulse voltammetry (DPV). In a certain concentration range, the oxidation peak current has a linear relationship with the concentration of 8-OHdG, so that the concentration of 8-OHdG can be quantitatively analyzed according to the detected oxidation peak current value by using the pre-established linear equation.

[0053] Application of a sensor in detecting 8-hydroxy-2'-deoxyguanosine in environmental monitoring.

[0054] Detection is carried out by differential pulse voltammetry (DPV). The modified sensor is placed in 0.1 M PBS (pH 7.0) containing 8-OHdG, and scanned according to the optimized detection conditions, and the oxidation peak current is recorded. The oxidation peak current has a good linear relationship with the concentration of 8-OHdG in the ranges of 0.0044 μM - 0.0706 μM, 0.0706 μM - 3.53 μM, and 3.53 μM - 14.12 μM, and the concentration of 8-OHdG can be quantitatively analyzed according to the linear equation.

[0055] 1. Characterization of the electrochemical sensor interface based on PLL / MWCNTs-NH 2 Characterization of the electrochemical sensor interface

[0056] (1) Electrochemical test system

[0057] Characterization is carried out by an electrochemical workstation. A traditional three-electrode system is selected, which consists of a working electrode, a reference electrode, and a counter electrode, corresponding to the glassy carbon electrode modified with the experimental material, the silver chloride electrode soaked in saturated potassium chloride, and the platinum electrode respectively. The electrolyte solution is 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG; CV test conditions, scanning voltage: 0.1 V - 0.65 V; scanning speed: 100 mV / s. DPV test conditions, scanning voltage: 0.1 V - 0.65 V; amplitude: 50 mV; pulse width: 50 ms; scanning speed: 50 mV / s.

[0058] (2) Characterization of the Electrochemical Sensor Interface

[0059] The bare GCE electrode and the electrodes modified with different modified materials PLL, MWCNTs-NH 2 , PLL / MWCNTs-NH 2 were respectively placed in the electrolyte of 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG for CV scanning to compare the electrochemical responses of different materials to 8-OHdG. As Figure 1 shown, the results showed that the bare GCE electrode (curve a) exhibited a weak irreversible oxidation peak in the presence of 8-OHdG, and the electrodes modified with MWCNTs-NH 2 (curve b) and PLL (curve c) both exhibited obvious oxidation peaks. The oxidation peak current of the electrode modified with PLL / MWCNTs-NH 2 (curve d) was the highest.

[0060] 2. Optimization of the Detection Conditions of the Electrochemical Sensor

[0061] (1) Testing the Influence of Different pH on the Detection of 8-OHdG

[0062] PBS solutions with pH values of 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0 were prepared, 8-OHdG was added to the PBS solutions with different pH values, and the final concentration of 8-OHdG was quantified to be 7.06 μM. Then, the PLL / MWCNTs-NH 2 modified electrode was subjected to CV scanning in the above PBS solutions, and different CV curves were obtained and analyzed. From experimental result Figure 2 A, it can be seen that in the range of pH 6.0 - 9.0 for the oxidation peak current of 8-OHdG, the oxidation peak current reached the highest at pH 7.0. Therefore, pH 7.0 was selected as the optimal pH value. Figure 2 Figure B is the calibration curve of the pH value and the oxidation peak potential (Epa) of 8-OHdG, showing that as the pH value continuously increased, the oxidation peak potential of 8-OHdG continuously decreased, and there was a linear correlation between the two (R 2 = 0.999), indicating that protons participated in the oxidation process of 8-OHdG.

[0063] (2) Testing the Influence of Different Scanning Rates on the Detection of 8-OHdG

[0064] The PLL / MWCNTs-NH 2The modified electrode was placed in 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG for electrochemical testing. The scan rates were changed to 5 mV / s, 30 mV / s, 50 mV / s, 80 mV / s, 100 mV / s, 120 mV / s, 150 mV / s, 200 mV / s, 250 mV / s, 300 mV / s, 350 mV / s to obtain different CV curves for analysis. From Figure 3 As can be seen from A, between the scan rates of 5 - 350 mV / s, the oxidation peak current of 8-OHdG gradually increases with the increase of the scan rate. However, considering that the background current should not be too large, the subsequent experiments were finally carried out under the condition of 100 mV / s. Figure 3 B is the calibration curve of the scan rate and the oxidation peak current of 8-OHdG. The results show that with the continuous increase of the scan rate, the oxidation peak current of 8-OHdG continuously increases, and there is a linear correlation between the two (R 2 = 0.992), indicating that the oxidation of 8-OHdG on GCE / PLL / MWCNTs-NH 2 is an adsorption-controlled electrode process.

[0065] (3) Test the influence of different PBS concentrations on the detection of 8-OHdG

[0066] Prepare PBS with concentrations of 0.025 M, 0.05 M, 0.1 M, 0.15 M, and 0.2 M. Add a specific volume of 8-OHdG to PBS with different concentrations so that each PBS contains 7.06 μM of 8-OHdG. Then perform CV scans on the PLL / MWCNTs-NH 2 modified electrode in the above PBS solutions to obtain different CV curves and analyze the optimal PBS concentration. From Figure 4 it can be seen that in the range of 0.025 M - 0.1 M, the oxidation current of 8-OHdG gradually increases with the increase of the PBS concentration. Between 0.1 M - 0.2 M, the oxidation current of 8-OHdG gradually decreases with the increase of the PBS concentration. The oxidation peak current reaches the highest value and the peak shape is the best at 0.1 M PBS. Therefore, 0.1 M PBS is selected as the optimal electrolyte concentration.

[0067] (4) Test the influence of different deposition conditions of PLL on the detection of 8-OHdG

[0068] Prepare a lysine solution with a pH of 9.0, and change the deposition potential to scan 7 cycles from -1.0 V to -2.2 V, scan 8 cycles from -1.5 V to -2.5 V, scan 8 cycles from -1.0 V to -2.2 V, and scan 8 cycles from -1.5 V to -2.5 V. Perform CV tests on the obtained electrodes in 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG to obtain different CV curves and screen the optimal PLL deposition conditions. From Figure 5 It can be seen that when scanning 8 cycles from -1.5 V to -2.5 V, the oxidation peak current is the highest, which is the optimal deposition condition.

[0069] (5) Test the effect of the drop-coating amount on the detection of 8-OHdG

[0070] Drop 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, 12 μL, 14 μL, 16 μL of MWCNTs-NH 2 nanomaterials on the electrode modified with PLL, perform CV scans in 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG, and analyze the optimal drop-coating amount. From Figure 6 It can be seen that as the drop-coating amount of the nanomaterials continuously increases, the oxidation peak current also gradually increases. When the drop-coating amount is from 8 μL to 10 μL, the increase amplitude of the oxidation peak current reaches the maximum. Continuing to increase the drop-coating amount, the increase amplitude of the oxidation peak current decreases. Since the peak current increase amplitude is the largest and the peak shape is the best when the drop-coating amount is 10 μL, the drop-coating amount of 10 μL is finally selected.

[0071] (6) Test the effect of different preconcentration conditions on the detection of 8-OHdG

[0072] Perform electrochemical tests on the PLL / MWCNTs-NH 2 electrode in 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG, change the incubation time of the modified electrode in the electrolyte solution to 4 min, 6 min, 8 min, 10 min, 12 min, 14 min, 16 min, obtain different CV curves and analyze them, and select the optimal incubation time. From Figure 7 It can be seen that within the range of 4 min - 16 min, as the incubation time increases, the oxidation current of 8-OHdG also continuously increases. When it is from 8 min to 10 min, the increase amplitude of the oxidation peak current is the largest. Within the range of 10 min - 16 min, the increase amplitude of the oxidation current of 8-OHdG decreases. Therefore, 10 min is selected as the optimal incubation time.

[0073] 3. Performance analysis of the electrochemical sensor

[0074] (1) Linear range and LOD of the electrochemical sensor for 8-OHdG

[0075] Prepare PBS solutions containing 0.0044 μM, 0.0176 μM, 0.0235 μM, 0.0353 μM, 0.0706 μM, 0.176 μM, 0.353 μM, 0.706 μM, 1.02 μM, 1.76 μM, 2.65 μM, 3.53 μM, 5.3 μM, 7.06 μM, 10.6 μM, 14.12 μM of 8-OHdG respectively. Immerse the PLL / MWCNTs-NH 2 electrode in the above electrolyte for DPV scanning to obtain different DPV curves and analyze them. From Figure 8 A, it can be seen that the oxidation peak current increases with the increase of the 8-OHdG concentration, and the oxidation peak current value of 8-OHdG shows a good linear relationship with the concentration of 8-OHdG in the ranges of 0.0044 μM - 0.0706 μM, 0.0706 μM - 3.53 μM, and 3.53 μM - 14.12 μM (as shown in Figure 8 B, 8C and 8D). The linear equations are respectively: Ipa(μA) = 37.35 + 74.03C(μM), R 2 = 0.999; Ipa(μA) = 42.13 + 18.31C(μM), R 2 = 0.993; Ipa(μA) = 92.18 + 3.61C(μM), R 2 = 0.994, and the detection limit (LOD) is 0.2 nM.

[0076] (2) Anti-interference experiment of the electrochemical sensor

[0077] Test the influence of some possible interfering substances in urine samples on the detection of 8-OHdG under the same conditions, including UA, AA, A, Na + , Zn 2+ , K + . Among them, set the concentration of 8-OHdG to 7.06 μM, the concentration of UA to 20 μM, the concentrations of AA and A to 100 μM each, and the concentrations of Na + , Zn 2+ , K + to 400 μM each. Immerse the PLL / MWCNTs-NH 2 modified electrode in the above electrolyte for CV scanning detection to obtain different CV curves and conduct comparative analysis. From Figure 9 it can be seen that in the presence of interference, the current signal of 8-OHdG does not change significantly.

[0078] In Figure 10In A, curve (a) represents the CV curve of 7.06 μM 8-OHdG, which shows the electrochemical characteristics of single 8-OHdG under the current test conditions. Curve (b) represents the CV curve of 7.06 μM 8-OHdG + 20 μM UA. It can be seen from this curve that in the presence of 7.06 μM 8-OHdG and 20 μM UA, an obvious oxidation peak current appears for UA.

[0079] In view of the obvious oxidation peak current shown by UA when coexisting with 8-OHdG, in-depth research was carried out subsequently. A PBS solution containing 400 μM UA and 7.06 μM 8-OHdG was prepared for electrochemical testing. Subsequently, different concentrations of uricase were added to this solution and reacted for 30 min before testing again. Figure 10 In B, curve (a) represents the CV curve of 7.06 μM 8-OHdG + 400 μM UA, presenting the electrochemical state of the system without adding uricase. Curve (b) represents the CV curve of 7.06 μM 8-OHdG + 400 μM UA + 10 μg / mL uricase. After adding 10 μg / mL uricase, the electrochemical signal of the system begins to change. Curve (c) represents the CV curve of 7.06 μM 8-OHdG + 400 μM UA + 15 μg / mL uricase. With the increase in the concentration of uricase, the change is further manifested. Curve (d) represents the CV curve of 7.06 μM 8-OHdG + 400 μM UA + 20 μg / mL uricase. Figure 10 It can be known from the comparison of a series of curves in B that as the concentration of uricase increases from 10 - 20 μg / mL, the interference of UA gradually decreases. When the concentration of uricase reaches 20 μg / mL, the oxidation peak of UA almost disappears, which fully proves that adding uricase can effectively eliminate the interference of UA in urine.

[0080] (3) Reproducibility experiment of the electrochemical sensor

[0081] Prepare 5 groups of electrodes modified with PLL / MWCNTs-NH 2 and place the above electrodes in 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG for electrochemical testing. Each electrode is tested in parallel three times to obtain CV curves and calculate the RSD ( Figure 11 ). The experimental results show that the calculated RSD is 2.81%, indicating that the sensor has good reproducibility.

[0082] (4) Stability experiment of the electrochemical sensor

[0083] Place the PLL / MWCNTs-NH 2The electrode was stored at 4 °C for one week. The modified electrode was placed in 0.1 M PBS (pH 7.0) containing 7.06 μM 8-OHdG for electrochemical testing to obtain a CV curve and calculate the RSD( Figure 12 ). The experimental results showed that the oxidation peak current remained above 90% of the original response, indicating that the sensor had good stability.

[0084] 4. Analysis of actual samples

[0085] The collected urine samples were placed in a 4 °C refrigerator for 12 h to precipitate impurities in the urine. Subsequently, the supernatant urine was taken and centrifuged at a speed of 12000 rpm for 15 min to further remove impurities, and then the supernatant was collected. Then, the urine was diluted five times with 0.1 M PBS (pH 7.0), and uricase was added with a quantitative uricase concentration of 20 μg / mL and reacted for 30 min to eliminate the interference of uric acid. The PLL / MWCNTs-NH 2 electrode was subjected to DPV scanning in the processed urine. The content of 8-OHdG in the urine was measured by the standard addition method and compared with the ELISA detection results, and the recovery rate was calculated. The experimental results are shown in Table 1. The standard recovery rate of 8-OHdG in urine samples was between 91.72% - 102.97%. 8-OHdG could be detected in the urine samples of breast cancer patients, but not in the samples of healthy people. Each sample was detected in parallel 3 times, and the RSD was less than 10%. At the same time, an ELISA kit was used to detect 8-OHdG in the same urine specimens, and the data obtained by the two methods were compared. As shown in Table 2, the results showed that the detection results of the two methods were close. The t-test was used to analyze the difference between the two detection methods, and P = 0.907, P > 0.05, indicating no significant statistical difference, indicating that there was no obvious difference between the two detection methods.

[0086] Table 1 Results of detecting 8-OHdG in urine based on PLL / MWCNTs-NH 2 sensor

[0087]

[0088] Table 2 Results of detecting 8-OHdG in urine by two methods of PLL / MWCNTs-NH 2 sensor and ELISA kit

[0089]

[0090]

[0091] In summary, through optimizing the preparation process and detection conditions, the present invention has successfully constructed an 8-OHdG electrochemical sensor based on a PLL / MWCNTs-NH 2 modified electrode. The sensor has the advantages of high sensitivity, wide linear range, strong anti-interference ability, good stability and repeatability, etc. In the detection of actual urine samples, it shows detection results similar to those of traditional ELISA methods, has good application prospects, and is expected to play an important role in the fields of biomedical detection and environmental monitoring.

[0092] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An electrode based on PLL / MWCNTs-NH2 electrode modification material, characterized in that: Taking glassy carbon electrode as substrate, the surface of the glassy carbon electrode is modified with poly-lysine and amino-modified multi-walled carbon nanotubes in sequence.

2. A method for preparing the electrode according to claim 1, characterized in that: (1) Preparation of polylysine (PLL) deposition solution: Dissolve lysine in PBS, and mix thoroughly to obtain a PLL deposition solution; (2) Preparation of MWCNTs-NH2 suspension: Dissolve MWCNTs-NH2 in deionized water and stir for 12 h. After stirring, place it in an ultrasonicator for 30 min. (3) Electrode modification: The bare glassy carbon electrode is pretreated; Take out the PLL deposition liquid in (1), scan 8 circles under the condition of -1.5V-2.5V, and obtain a PLL deposition layer on the surface of the glassy carbon electrode; take the MWCNTs-NH2 suspension in (2) and drop it on the PLL deposition layer modified electrode, wait for natural drying, and obtain a PLL / MWCNTs-NH2 electrode.

3. The method for preparing an electrode according to claim 2, characterized in that: The PBS in (1) is 0.1 M, pH 9.

0.

4. The method for preparing an electrode according to claim 2, characterized in that: The concentration of lysine was 10 mM.

5. The method for preparing an electrode according to claim 2, characterized in that: The concentration of MWCNTs-NH2 in (2) is 1 mg / mL.

6. The method for preparing an electrode according to claim 2, characterized in that: The bare glassy carbon electrode in (3) is pretreated as follows: the bare glassy carbon electrode is polished with 0.3 μm and 0.05 μm alumina slurries, respectively, and then the electrode is ultrasonically washed in nitric acid (1:1), ethanol and deionized water for 3 minutes respectively.

7. The method for preparing an electrode according to claim 2, characterized in that: The volume ratio of PLL deposition solution and MWCNTs-NH2 suspension was 500:

1.

8. Use of the electrode according to claim 1 as a sensor for detecting 8-OHdG, wherein the electrode is prepared by the method according to any one of claims 2 to 7.

9. Use of the sensor according to claim 8 for detecting 8-hydroxy-2'-deoxyguanosine in environmental monitoring.

10. The use according to claim 9, characterized in that: The detection conditions of the sensor are as follows: a three-electrode system is adopted, in which the working electrode, reference electrode and counter electrode are a glassy carbon electrode modified with experimental materials, a silver chloride electrode soaked in saturated potassium chloride and a platinum electrode, respectively; the electrolyte solution is 0.1 M PBS, pH 7.0, containing 8-OHdG; the cyclic voltammetry (CV) test conditions are a scanning voltage of 0.1 V-0.65 V and a scanning speed of 100 mV / s; the DPV test conditions are a scanning voltage of 0.1 V-0.65 V, an amplitude of 50 mV, a pulse width of 50 ms, and a scanning speed of 50 mV / s.

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