MWCNTs-COOH / AuNPs / CS / Apt electrode modification material and electrode as well as preparation method and application of MWCNTs-COOH / AuNPs / CS / Apt electrode modification material

Abstract

The invention relates to the technical field of biological detection, in particular to an MWCNTs-COOH / AuNPs / CS / Apt electrode modification material, an electrode and a preparation method and application of the MWCNTs-COOH / AuNPs / CS / Apt electrode modification material. A glassy carbon electrode is used as a substrate, and a carboxylated multi-walled carbon nanotube / gold nanoparticle / chitosan (MWCNTs-COOH / AuNPs / CS) nano composite material layer, an aptamer (Apt) recognition layer and a mercaptohexanol (MCH) closing layer are sequentially modified. The preparation method comprises the following steps: hydrothermally synthesizing AuNPs, preparing an MWCNTs-COOH / AuNPs / CS compound through ultrasonic dispersion, modifying the surface of an electrode layer by layer and the like. The aptamer immobilization technology and detection conditions are optimized, the sensor has high-sensitivity linear response to 8-OHdG within the range of 0.0706-7.06 mu M, the detection limit reaches 0.33 mu M, and the anti-interference performance is high (interference signal lt; 5%). The method solves the problems that a traditional detection method is high in cost and long in consumed time and an existing sensor is insufficient in sensitivity, and is suitable for rapid and accurate analysis of 8-OHdG in biomedical detection and environmental monitoring.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a MWCNTs-COOH / AuNPs / CS / Apt electrode modification material and electrode, and a preparation method and application thereof. Background Art

[0002] 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a key biomarker of DNA oxidative damage. Its content changes in vivo are closely related to a variety of major diseases. During the occurrence and development of cancer, the level of oxidative stress in cells increases, leading to DNA damage, and the content of 8-OHdG increases accordingly. By detecting its content changes, it can provide important clues for the early diagnosis of cancer. In neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, oxidative stress is also one of the important pathogenesis. The detection of 8-OHdG is of great significance for evaluating the progression of the disease and the treatment effect. However, traditional 8-OHdG detection methods, such as high-performance liquid chromatography (HPLC) and mass spectrometry, although with high accuracy, have many limitations. These devices are usually expensive, with a purchase cost of hundreds of thousands or even millions of yuan, and require professional maintenance and operation teams. In addition, traditional detection methods are complicated to operate and have a long detection cycle. It often takes hours or even days from sample processing to obtaining results, which is difficult to meet the needs of rapid clinical diagnosis and large-scale screening.

[0003] Electrochemical sensors have attracted much attention in the field of biomolecule detection in recent years due to their advantages such as low cost, high sensitivity and fast response speed. However, there are still many problems with existing electrochemical sensors. On the one hand, the sensitivity of the sensor is insufficient, and it is difficult to detect trace amounts of 8-OHdG in biological samples, which affects the accuracy and reliability of the test results. On the other hand, the sensor has poor anti-interference ability. In actual detection, there are often a variety of interfering substances in biological samples, such as uric acid (UA), ascorbic acid (AA), adenine (A), metal ions, etc. These interfering substances will compete with the target for binding sites, or affect the electron transfer process on the electrode surface, resulting in deviations in the test results. In the prior art, the combination of nanomaterials and aptamers has not been fully optimized, and the synergistic effect of the two cannot be fully exerted, which further limits the performance improvement of the sensor. Therefore, the development of a new type of composite nanomaterial modified electrode to achieve efficient detection of 8-OHdG has become a key issue to be solved in this field. Summary of the invention

[0004] In view of the above situation, to overcome the defects of the prior art, the present invention provides a MWCNTs-COOH / AuNPs / CS / Apt electrode modification material and an electrode, as well as a preparation method and an application thereof, to solve the problems of low sensitivity and poor anti-interference ability in the prior art, and to achieve rapid and high-precision detection of 8-OHdG.

[0005] To achieve the above object, the present invention provides the following technical solutions: A MWCNTs-COOH / AuNPs / CS / Apt electrode modification material, comprising a MWCNTs-COOH / AuNPs / CS nanocomposite and Apt attached to the surface of the MWCNTs-COOH / AuNPs / CS nanocomposite.

[0006] Preparation method of the electrode modification material

[0007] (1) Preparation of Au NPs:

[0008] HAuCl4 solution is added to water, heated and stirred until the temperature reaches 100 °C, sodium citrate solution is quickly added, stirring is continued and the boiling state of the solution is maintained. After the solution color turns dark red, boiling is continued for 5 min, and then it is naturally cooled to room temperature under continuous stirring to obtain an AuNPs colloidal solution;

[0009] (2) Preparation of MWCNTs-COOH / AuNPs / CS nanocomposite:

[0010] Weigh MWCNTs-COOH and slowly add it to the AuNPs colloidal solution in (1), stir and mix; then ultrasonically disperse; centrifuge the mixed solution and discard the upper liquid; add chitosan (CS) solution for redissolution and mix well to form a stable MWCNTs-COOH / AuNPs / CS composite;

[0011] (3) Apt pretreatment:

[0012] The thiol-modified 100 μM aptamer (Apt) is dissolved in Tris-HCl PBS, then placed in a water bath for heating, quickly placed in ice water at 4 °C for cooling for 15 min, and finally incubated at room temperature for 20 min to obtain an Apt solution. Tris(2-carboxyethyl)phosphine (TCEP) is added to the Apt solution to break the disulfide bond;

[0013] (4) Preparation of MWCNTs-COOH / AuNPs / CS / Apt:

[0014] Deposit the MWCNTs-COOH / AuNPs / CS nanocomposite deposition solution obtained in (2) on the surface of the substrate and let it dry naturally at room temperature to obtain the MWCNTs-COOH / AuNPs / CS deposition layer. Dropwise coat the Apt solution obtained in (3) on the surface of the MWCNTs-COOH / AuNPs / CS deposition layer and incubate. After the incubation is completed, dropwise coat 6-mercaptohexanol (MCH) solution to block the non-specific sites on the surface, and obtain the MWCNTs-COOH / AuNPs / CS / Apt nanocomposite material.

[0015] Furthermore, in the (1), the mass concentration of the HAuCl4 solution is 2%, the mass concentration of the trisodium citrate solution is 5%, and the volume ratio of water to the HAuCl4 solution and the trisodium citrate solution is 48:1:1; the prepared AuNPs have a particle size of 13 ± 2 nm;

[0016] During the preparation process of (1), a glass container is used, and the treatment steps of the glass container are to soak it in aqua regia overnight, then thoroughly rinse it with a large amount of deionized water, and finally dry it in an oven at 60 °C.

[0017] Furthermore, in the (2), 1 mL of the AuNPs colloidal solution and 0.1 mL of the CS solution are added to each milligram of MWCNTs-COOH;

[0018] The stirring condition in (2) is at a speed of 300 rpm for 12 h; the ultrasonic condition is ultrasonic treatment at a power of 100 W for 30 min; the centrifugation condition is centrifugation at 12000 rpm for 15 min.

[0019] Furthermore, in the (3), the heating condition in the water bath is 90 °C for 10 min; the volume ratio of the thiol-modified Apt to Tris-HCl PBS is 1:199; the concentration of TCEP is 2 mM, and the volume ratio of TCEP to the Apt solution is 2:1.

[0020] Furthermore, in the (4), the volume ratio of the MWCNTs-COOH / AuNPs / CS nanocomposite to the Apt solution is 1:1;

[0021] The concentration of the Apt solution is 1 μM, the concentration of the MCH solution is 2 mM, and the volume ratio of the Apt solution to the MCH solution is 1:1.

[0022] An electrode containing the above electrode modification material.

[0023] A method for preparing an electrode,

[0024] (1) Pretreatment of bare glassy carbon electrode: The GCE was polished with alumina slurries of different particle sizes, and the electrode was successively placed in nitric acid, ethanol, and deionized water, and ultrasonically washed in an ultrasonic cleaner for 3 min each;

[0025] (2) Preparation of MWCNTs-COOH / AuNPs / CS nanocomposite layer: The MWCNTs-COOH / AuNPs / CS nanocomposite was uniformly drop-coated on the surface of the glassy carbon electrode and naturally dried at room temperature;

[0026] (3) Preparation of Apt layer: The Apt solution was drop-coated on the surface of the electrode modified with the nanocomposite layer and incubated in a constant temperature and humidity chamber at 25 °C and 60% humidity for 16 h to firmly fix the aptamer on the surface of the nanocomposite layer through covalent bonding with amino or mercapto groups; after incubation, 10 μL of 2 mM MCH solution was drop-coated and incubated for 30 min to block the non-specific sites on the electrode surface.

[0027] Furthermore,

[0028] In (1), the particle sizes of the alumina slurries are 0.3 μm and 0.05 μm respectively, and the volume ratio of nitric acid to water in the nitric acid solution is 1:1.

[0029] Application of an electrode as described above in the preparation of a sensor for detecting 8-OHdG.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] In the present invention, MWCNTs-COOH, AuNPs, and CS are compounded together to form a nanocomposite with unique properties. The high conductivity and large specific surface area of MWCNTs-COOH provide an ideal platform for the loading of AuNPs. The introduction of AuNPs enhances the electron transfer rate and improves the sensitivity of the sensor. The addition of CS not only improves the dispersibility of the composite material but also enhances its biocompatibility, providing a stable support structure for the immobilization of aptamers. The synergistic effect of the three significantly improves the performance of the sensor, enabling rapid, accurate, and sensitive detection of trace levels of 8-OHdG in biological samples, and providing a more efficient and reliable technical support for evaluating the level of DNA oxidative damage. Description of the Drawings

[0032] Figure 1 It is the TEM and EDS characterization of the nanocomposite of the present invention (MWCNTs-COOH, AuNPs, MWCNTs-COOH / AuNPs / CS);

[0033] Figure 2 It is the CV response curve of different modified electrodes of the present invention;

[0034] Figure 3 is the optimization curve of the aptamer concentration of the present invention;

[0035] Figure 4 is the optimization curve of the incubation time of the present invention;

[0036] Figure 5 is the optimization curve of the reaction time of the present invention;

[0037] Figure 6 is the DPV response of the sensor of the present invention to 8-OHdG and the calibration curve of the oxidation peak current;

[0038] Figure 7 is the test result of the anti-interference experiment of the present invention.

[0039] Figure 8 is the test result of the stability of the present invention. Detailed implementation manners

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with 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; based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0041] Example 1:

[0042] A kind of MWCNTs-COOH / AuNPs / CS / Apt electrode modification material, including MWCNTs-COOH / AuNPs / CS nanocomposite, and Apt attached to the surface of the MWCNTs-COOH / AuNPs / CS nanocomposite.

[0043] The preparation method of the electrode modification material,

[0044] (1) Preparation of AuNPs by hydrothermal reduction method:

[0045] Au NPs are prepared according to the sodium citrate reduction method. Before preparation, all glass containers are soaked in aqua regia overnight, then thoroughly rinsed with a large amount of deionized water, and finally dried in an oven at 60 °C. Take 96 mL of deionized water, add 2 mL of HAuCl4 with a mass concentration of 2%, heat and stir until 100 °C, then add 2 mL of sodium citrate with a mass concentration of 5%, continue to stir and keep boiling. After the solution color turns dark red, continue to boil for 5 min, and then naturally cool it to room temperature (25 °C) under stirring, and store it in a brown reagent bottle in a 4 °C refrigerator.

[0046] (2) Preparation of MWCNTs-COOH / AuNPs / CS nanocomposites: Accurately weigh 8 mg of MWCNTs-COOH with an electronic balance with a precision of 0.01 mg, and slowly add it to 8 mL of the prepared AuNPs solution. Place magnetic beads in the mixed solution, put it on a magnetic stirrer, set the stirring speed to 300 rpm, and stir for 12 h to preliminarily mix MWCNTs-COOH and AuNPs evenly. After stirring, put it into an ultrasonic instrument, set the ultrasonic power to 100 W, and the ultrasonic time to 30 min, and use the cavitation effect of ultrasound to further promote the dispersion of the material. After the ultrasonic treatment, divide the mixed solution into centrifuge tubes, put it into a high-speed centrifuge, centrifuge at a speed of 12000 rpm for 15 min, take out the upper layer of liquid and discard it, and then add deionized water to redissolve. According to the ratio of adding 100 μL of CS per 1 mL of MWCNTs-COOH, add CS and mix well to prepare a MWCNTs-COOH / AuNPs / CS suspension, and finally store it in a refrigerator at 4 °C for later use.

[0047] (3) The 8-OHdG aptamer was synthesized and purified by Shanghai Sangon Biotech Co., Ltd., and the sequence is as follows: 5’-SH-(CH2)6-GCGGGC GAT CGG CGG GGG GTG CGT GCG CTC TGT GCCAGG GGG TGG GACAGATCA TAT GGG GGTGCT-3’

[0048] Carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) are tightly bound to gold nanoparticles (AuNPs) through physical adsorption. Chitosan (CS) is used as a dispersant and a fixed matrix to enhance the stability and dispersibility of the composite material;

[0049] Experimental results:

[0050] Figure 1 The TEM image of... clearly shows the microstructure of MWCNTs-COOH, presenting a small bundle or single-tube structure with irregular and intertwined lengths; the successfully prepared AuNPs have a particle size of about 13 nm and are evenly loaded on the surface of MWCNTs-COOH; after adding CS, the dispersibility of MWCNTs-COOH / AuNPs is significantly improved, and there is no obvious aggregation phenomenon. The EDS diagram further confirms that the MWCNTs-COOH / AuNPs / CS nanocomposite is mainly composed of elements such as C, O, N, and Au, providing an important basis for the structural and compositional analysis of the nanocomposite material.

[0051] Example 2:

[0052] An electrode contains electrode modification materials, including MWCNTs-COOH / AuNPs / CS nanocomposite materials and Apt attached to the surface of the MWCNTs-COOH / AuNPs / CS nanocomposite materials.

[0053] Method for preparing the electrode,

[0054] (1) Fix the glassy carbon electrode (GCE) on a special fixture, and gently polish the electrode surface in the same direction for 5 minutes with 0.3μm alumina slurry on a polishing cloth, then replace with 0.05μm alumina slurry and continue polishing for 3 minutes to make the electrode surface present a mirror gloss. The polished electrode is placed in nitric acid (1:1), ethanol and deionized water in turn, and ultrasonically washed in an ultrasonic oscillator for 3 minutes respectively, with the ultrasonic power set to 80W, to completely remove impurities on the electrode surface and have good adhesion.

[0055] (2) The MWCNTs-COOH / AuNPs / CS nanocomposite was evenly drop-coated on the surface of the glassy carbon electrode and allowed to dry naturally at room temperature;

[0056] (3) Apt pretreatment:

[0057] 100 μM thiol-modified Apt was dissolved in Tris-HCl PBS, and the volume ratio of thiol-modified Apt to Tris-HCl PBS was 1:199; then it was placed in a 90°C water bath, heated for 10 minutes, quickly placed in 4°C ice water to cool for 15 minutes, and finally incubated at room temperature for 20 minutes to obtain an Apt solution, and 2 mM TCEP was added to the Apt solution to destroy the disulfide bond; the volume ratio of TCEP to Apt solution was 2:1.

[0058] The treated 1μM Apt solution was drop-coated on the electrode surface modified with MWCNTs-COOH / AuNPs / CS nanocomposite material, with a drop-coating amount of 10μL, and then the electrode was placed in a constant temperature and humidity chamber, set at 25°C, humidity at 60%, and incubated for 16h, so that Apt can be fully combined with MWCNTs-COOH / AuNPs / CS through amino or thiol covalent bonding. After the incubation, 2mM MCH solution was drop-coated again, with a drop-coating amount of 10μL, and incubated for 30min to block the excess active sites on the electrode surface, reduce nonspecific adsorption, and reduce background interference. The volume ratio of MWCNTs-COOH / AuNPs / CS nanocomposite to Apt solution was 1:1, and the volume ratio of Apt solution to MCH solution was 1:1.

[0059] An electrochemical sensor for highly sensitive and selective detection of 8-OHdG based on MWCNTs-COOH / AuNPs / CS / Apt nanocomposites. The sensor is applied in the early diagnosis of cancer, the evaluation of neurodegenerative diseases or the monitoring of environmental oxidative damage. By using this sensor to rapidly and accurately detect 8-OHdG in biological samples or environmental samples, and through detecting the concentration change of 8-OHdG, it provides key data support for the early detection of cancer, the evaluation of the condition of neurodegenerative diseases and the monitoring of the degree of environmental oxidative damage, assisting doctors in formulating disease diagnosis and treatment plans, and at the same time providing a scientific basis for environmental quality assessment and pollution prevention and control.

[0060] The detection steps include:

[0061] Differential pulse voltammetry (DPV) is used for detection. The modified sensor is placed in an electrolyte containing 0.1M KCl and 5.0mM [Fe(CN)6] 3- / 4- and scanned within a specific potential range. The DPV test conditions are: scanning voltage 0V - 0.6V, amplitude 50mV, pulse width 50ms, scanning speed 50mV / s. Under these test conditions, the sensor has a good response to 8-OHdG, and the oxidation peak current has a good linear relationship with the concentration of 8-OHdG in the range of 0.0706 μM - 7.06 μM. The detection limit (LOD) is 0.33 μM (S / N = 3), and it can accurately detect trace amounts of 8-OHdG in the sample.

[0062] Detection principle:

[0063] The detection of this sensor is based on the specific binding of the aptamer to the target and the change of electron transfer on the electrode surface. The aptamer (Apt) is a single-stranded oligonucleotide that can specifically recognize 8-OHdG after screening. When not bound to 8-OHdG, the aptamer is in a relatively extended state, and at this time, the electron transfer on the electrode surface is relatively smooth. In the electrolyte containing [Fe(CN)6] 3- / 4- [Fe(CN)6] 3- / 4- can relatively easily approach the electrode surface for redox reactions, generating a certain oxidation peak current.

[0064] When the sensor contacts a sample containing 8-OHdG, the aptamer specifically binds to 8-OHdG, and the conformation of the aptamer changes from an extended state to a more compact state. This conformational change hinders the diffusion and electron transfer process of [Fe(CN)6] 3- / 4- to the electrode surface, reducing the rate of the redox reaction, and thus resulting in a decrease in the oxidation peak current.

[0065] The change value (ΔI%) of this oxidation peak current is detected by differential pulse voltammetry (DPV). Within a certain concentration range, the change value of the oxidation peak current has a linear relationship with the concentration of 8-OHdG. Therefore, by measuring the change value of the oxidation peak current and using the pre-established linear equation, the quantitative analysis of the concentration of 8-OHdG in the sample can be achieved.

[0066] Experimental results:

[0067] To explore the electrochemical behavior of the aptamer sensor during the preparation process, Figure 2 shows the cyclic voltammetry (CV) responses of different modified electrodes in an electrolyte containing 0.1 M KCl and 5.0 mM [Fe(CN)6], 3- / 4- including the MWCNTs-COOH / AuNPs / CS electrode (curve a) constructed with multi-walled carbon nanotubes-carboxyl (MWCNTs-COOH), gold nanoparticles (AuNPs), and chitosan (CS) as modification materials; the MWCNTs-COOH / AuNPs / CS / Apt electrode (curve b) further modified with aptamer (Apt) based on the MWCNTs-COOH / AuNPs / CS electrode; the MWCNTs-COOH / AuNPs / CS / Apt / MCH electrode (curve c) further modified with 6-mercaptohexanol (MCH) based on the MWCNTs-COOH / AuNPs / CS / Apt electrode; and the MWCNTs-COOH / AuNPs / CS / Apt / MCH / 8-OHdG modified electrode (curve d) further introducing 8-OHdG based on the MWCNTs-COOH / AuNPs / CS / Apt / MCH electrode. By comparing the oxidation peak currents and peak potentials of different curves, the influence of the modification process on the electron transfer rate and electrochemical activity of the electrode can be intuitively understood, providing key data for sensor interface characterization and performance optimization. The results show that the MWCNTs-COOH / AuNPs / CS electrode exhibits a reversible oxidation peak in the electrolyte, and the oxidation current signal is the highest. The oxidation current of the MWCNTs-COOH / AuNPs / CS / Apt electrode gradually decreases because Apt itself has a negative resistance that hinders electron transfer. The current of the MWCNTs-COOH / CS / AuNPs / Apt / MCH electrode further decreases, indicating that MCH successfully blocks the electrode surface and reduces the active sites on the electrode surface. In addition, due to the presence of 8-OHdG, 8-OHdG will bind to Apt, causing Apt to change from a straight chain to a compact structure, making it more difficult for the negatively charged [Fe(CN)6] 3- / 4- to be transferred to the electrode surface, so the oxidation peak current decreases.

[0068] Optimization process:

[0069] By changing the aptamer concentration ( Figure 3 ), incubation time ( Figure 4 ), and the reaction time between the aptamer and 8-OHdG ( Figure 5 ), the effects of these parameters on the sensor performance were systematically studied. In the aptamer concentration optimization experiment, Apt solutions with concentrations of 0.01 μM, 0.1 μM, 0.5 μM, 1.0 μM, 2.0 μM, and 5.0 μM were prepared and respectively drop-coated on the surface of the modified electrode. When the Apt concentration varied in the range of 0.01 μM - 5 μM, with the increase in concentration, the change value of the oxidation peak current intensity (△I% = (I0 - I) / I0%) caused by the modification of 8-OHdG gradually increased. This was because more aptamer molecules could bind to 8-OHdG, resulting in a more obvious change in the electrochemical signal. However, when the Apt concentration exceeded 1 μM, △I% began to decrease. This might be due to the enhanced intermolecular interaction caused by the excessive concentration of aptamer, which affected its effective binding to 8-OHdG, or the formation of an overly thick film layer on the electrode surface, hindering electron transfer. Therefore, 1 μM was determined as the optimal Apt concentration.

[0070] In the Apt incubation time optimization experiment, the incubation time of Apt on the electrode surface was respectively set to 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h. The results showed that within the range of 4 h - 24 h, with the increase in the incubation time, △I% continuously increased. This was because the longer the incubation time, the more time the Apt molecules had to bind to the active sites on the surface of the MWCNTs-COOH / AuNPs / CS nanocomposite, thereby increasing the immobilization amount of Apt on the electrode surface. At 16 h, the growth rate of △I% decreased, indicating that the Apt on the electrode surface was basically saturated, and further extending the incubation time had little effect on improving the sensor performance. Therefore, 16 h was selected as the optimal incubation time.

[0071] For the optimization of the reaction time between the aptamer and 8-OHdG, the reaction time was set to 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, and 50 min. As the reaction time increased from 5 min to 35 min, △I% continuously increased. This was because the binding reaction between 8-OHdG and Apt required a certain time to reach equilibrium, and the longer the reaction time, the more sufficient the binding, and the more obvious the change in the electrochemical signal. After 35 min, the growth rate of △I% gradually stabilized, indicating that the reaction between 8-OHdG and Apt tended to be stable, and further extending the reaction time had little effect on the detection result. Therefore, 35 min was determined as the optimal reaction time.

[0072] Sensor performance test:

[0073] 1. Experimental procedure:

[0074] To verify the detection performance of the aptasensor, under the optimized optimal conditions, differential pulse voltammetry (DPV) was used to test the oxidation peak current (Ipa) of 8-OHdG at different concentrations, and the performance of the sensor was comprehensively tested ( Figure 6 ). PBS solutions containing 0.0706 μM, 0.176 μM, 1.76 μM, 3.53 μM, and 7.06 μM 8-OHdG were prepared respectively. 10 μL of each solution was taken and drop-coated on the prepared MWCNTs-COOH / CS / AuNPs / Apt / MCH modified electrode. The modified electrode was placed in an electrolyte containing 0.1 M KCl and 5.0 mM [Fe(CN)6] 3- / 4- and scanned according to the DPV test conditions (scanning voltage 0 V - 0.6 V, amplitude 50 mV, pulse width 50 ms, scanning speed 50 mV / s), and the DPV curves corresponding to different concentrations of 8-OHdG were recorded.

[0075] To accurately determine the response time of the sensor, the sensor needs to be placed in a solution containing a specific and known concentration of 8-OHdG. After the sensor is in full contact with the solution, the current change is monitored in real time, and the duration experienced when the current rises from the initial value to 90% of the current value when it reaches a steady state after interacting with 8-OHdG at this concentration is recorded. This duration is the response time of the sensor for 8-OHdG at this concentration.

[0076] In the anti-interference experiment ( Figure 7 ), an interference solution containing 7.06 μM 8-OHdG and 400 μM UA, 400 μM AA, 400 μM A, 800 μM Zn 2+ , 800 μM K + , and 800 μM Na + was prepared. The MWCNTs-COOH / AuNPs / CS / Apt / MCH modified electrode was incubated in the above interference solution for 35 min and then placed in the electrolyte for cyclic voltammetry (CV) scanning. The CV curve was recorded and compared with the test results in a solution containing only 7.06 μM 8-OHdG.

[0077] To test the repeatability of the sensor, 5 parallel MWCNTs-COOH / CS / AuNPs / Apt nanocomposite modified electrodes were prepared according to the above method, and the response of the modified electrodes to 7.06 μM 8-OHdG in an electrolyte containing 0.1 M KCl and 5.0 mM [Fe(CN)6] 3- / 4- was tested respectively. Each electrode was tested in parallel three times ( Figure 8 A). The relative standard deviation (RSD) of ΔI% of the 5 electrodes was calculated to evaluate the repeatability.

[0078] The electrode modified with MWCNTs-COOH / AuNPs / CS / Apt nanocomposite was stored at 4 °C for one week, and then placed in an electrolyte containing 0.1 M KCl and 5.0 mM 3- / 4- [Fe(CN)6], and the current change value of the above electrode for 7.06 μM 8-OHdG was tested ( Figure 8 B), and the CV curve was obtained and the RSD was calculated to evaluate the stability of the sensor.

[0079] 2. Experimental results:

[0080] The DPV test results showed that the oxidation peak current decreased with the increase of the 8-OHdG concentration, and there was a good linear relationship between the 8-OHdG oxidation peak current value and the 8-OHdG concentration in the range of 0.0706 μM - 7.06 μM. The linear equation was Ipa(μA) = 4.98 + 1.085C(μM), R 2 = 0.995, the LOD was 0.33 μM (S / N = 3), and the sensitivity was 1.085 μA / μM, indicating that the sensor could achieve accurate quantitative detection of 8-OHdG.

[0081] The experimental results showed that the response time of the sensor was about 2.332 s, indicating that the sensor had a fast response ability and could meet the requirements of rapid detection.

[0082] The anti-interference experimental results showed that in the solution containing interfering substances, the detection signal interference of the sensor for 8-OHdG < 5%, indicating that the sensor had a strong anti-interference ability to common interferents and could accurately detect 8-OHdG in complex biological samples.

[0083] The RSD calculated from the repeatability experiment for 5 electrodes was 1.83%, indicating that the sensor had good repeatability and high consistency of multiple detection results. In the stability experiment, after the sensor was stored at 4 °C for one week, its signal retention rate > 80% and the RSD was 3.2%, proving that the sensor had stable performance during storage and could meet the storage requirements in actual detection.

[0084] In summary, through the detection of the sensor's own properties such as sensitivity, linear range, selectivity, response time, and stability, the performance of the sensor modified with MWCNTs-COOH / AuNPs / CS / Apt electrode prepared by the present invention was comprehensively evaluated, further proving that the sensor had the advantages of high sensitivity, high selectivity, fast response, and good stability in the detection of 8-OHdG, and had broad application prospects.

[0085] Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will appreciate 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. A MWCNTs-COOH / AuNPs / CS / Apt electrode modification material, characterized in that: It includes MWCNTs-COOH / AuNPs / CS nanocomposite materials and Apt attached to the surface of the MWCNTs-COOH / AuNPs / CS nanocomposite materials.

2. The method for preparing the electrode modifying material according to claim 1, characterized in that: (1) Preparation of AuNPs: Add HAuCl4 solution to water, heat and stir until the temperature reaches 100°C, quickly add trisodium citrate solution, continue stirring and keep the solution boiling. After the solution color turns to dark red, continue boiling for 5 minutes, then cool naturally to room temperature while continuing stirring to obtain AuNPs colloidal solution; (2) Preparation of MWCNTs-COOH / AuNPs / CS nanocomposites: Weigh MWCNTs-COOH and slowly add it to the AuNPs colloidal solution in (1), stirring to mix; Then ultrasonic dispersion; The mixed solution was centrifuged and the supernatant liquid was discarded; CS solution was added for redissolution and mixed thoroughly to form a stable MWCNTs-COOH / AuNPs / CS composite; (3) Aptamer (Apt) pretreatment: The thiol-modified Apt was dissolved in Tris-HCl PBS, then heated in a water bath, quickly placed in 4°C ice water to cool for 15 min, and finally incubated at room temperature for 20 min to obtain an Apt solution. TCEP was added to the Apt solution to destroy the disulfide bonds. (4) Preparation of MWCNTs-COOH / AuNPs / CS / Apt: The MWCNTs-COOH / AuNPs / CS nanocomposite deposition liquid obtained in (2) is deposited on the surface of the substrate and dried naturally at room temperature to obtain a MWCNTs-COOH / AuNPs / CS deposition layer. The Apt solution obtained in (3) is drop-coated on the surface of the MWCNTs-COOH / AuNPs / CS deposition layer and incubated. After the incubation, 6-mercaptohexanol (MCH) solution is drop-coated to block nonspecific sites on the surface to obtain a MWCNTs-COOH / AuNPs / CS / Apt nanocomposite material.

3. The method for preparing the electrode modifying material according to claim 2, characterized in that: The mass concentration of the HAuCl4 solution in (1) is 2%, the mass concentration of the trisodium citrate solution is 5%, and the volume ratio of water to the HAuCl4 solution and the trisodium citrate solution is 48:1:1; the particle size of the prepared AuNPs is 13±2nm; In the preparation process of (1), a glass container is used. The treatment steps of the glass container are as follows: soaking in aqua regia overnight, then thoroughly rinsing with a large amount of deionized water, and finally drying in an oven at 60°C.

4. The method for preparing an electrode modifying material according to claim 2, characterized in that: For every mg of MWCNTs-COOH in (2), 1 mL of AuNPs colloidal solution and 0.1 mL of CS solution were added; In the step (2), the stirring condition is 300 rpm for 12 h; the ultrasonic condition is 100 W ultrasonic power for 30 min; and the centrifugal condition is 12000 rpm for 15 min.

5. The method for preparing the electrode modifying material according to claim 2, characterized in that: The water bath heating condition in (3) is 90° C. for 10 min; the volume ratio of thiol-modified Apt to Tris-HCl PBS is 1:199; the concentration of tris(2-carboxyethyl)phosphine (TCEP) is 2 mM, and the volume ratio of TCEP to Apt solution is 2:

1.

6. The method for preparing an electrode modifying material according to claim 2, characterized in that: In (4), in (4), the volume ratio of MWCNTs-COOH / AuNPs / CS nanocomposite to Apt solution is 1:1; The concentration of Apt solution was 1 μM, the concentration of MCH solution was 2 mM, and the volume ratio of Apt solution to MCH solution was 1:

1.

7. An electrode, characterized in that: Contains the electrode modifying material according to any one of claims 2 to 6.

8. A method for preparing the electrode according to claim 7, characterized in that: (1) Bare glassy carbon electrode pretreatment: The GCE was polished using alumina slurries of different particle sizes, and the electrode was placed in nitric acid, ethanol, and deionized water in turn, and ultrasonically cleaned in an ultrasonic cleaner for 3 min each; (2) Preparation of MWCNTs-COOH / AuNPs / CS nanocomposite layer: The MWCNTs-COOH / AuNPs / CS nanocomposite was evenly drop-coated on the surface of the glassy carbon electrode and dried naturally at room temperature; (3) Preparation of Apt layer: Apt solution was drop-coated on the surface of the electrode modified with the nanocomposite layer, and incubated in a constant temperature and humidity chamber at 25°C and 60% for 16 h to allow the aptamer to be firmly fixed on the surface of the nanocomposite layer through covalent bonding of amino or thiol groups. After the incubation, 10 μL of 2 mM MCH solution was drop-coated and incubated for 30 min to block the nonspecific sites on the electrode surface.

9. The method for preparing an electrode according to claim 8, characterized in that: The particle sizes of the alumina slurry in (1) are 0.3 μm and 0.05 μm respectively, and the volume ratio of nitric acid to water in the nitric acid solution is 1:

1.

10. Use of the electrode according to claim 7 as a sensor for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG).

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