Preparation method and application of a biosensor based on a red blood cell-carbon nanotube hybrid

Abstract

The present invention discloses a preparation method and application of a biosensor based on a red blood cell-carbon nanotube hybrid. The biosensor is composed of a reference electrode, a counter electrode, and a working electrode modified with a red blood cell-carbon nanotube hybrid. The modified working electrode is composed of a carbon cloth electrode and a red blood cell-carbon nanotube hybrid coated on the surface of the carbon cloth electrode. The red blood cell-carbon nanotube hybrid uses red blood cells as a matrix, and by mixing carbon nanotubes after ultrasonic dispersion with red blood cells evenly, a red blood cell-carbon nanotube hybrid with a carbon nanotube artificial shell layer coated on the surface of the red blood cell membrane is obtained. The biosensor prepared with the synthesized red blood cell-carbon nanotube hybrid as a catalyst can simultaneously detect the concentration levels of dopamine and uric acid, and has a wide detection range and a low detection limit for dopamine and uric acid. At the same time, it exhibits good selectivity, reproducibility, stability, and strong anti-interference ability.

Description

Technical Field

[0001] The present invention belongs to the technical fields of bioinorganic chemistry and biosensors, and particularly relates to a preparation method and application of a red blood cell-carbon nanotube hybrid-based biosensor capable of simultaneously and quantitatively detecting dopamine and uric acid. Background Art

[0002] Dopamine (DA) and uric acid (UA) are electrochemically active molecules existing in the human body and having important biological research value. Among them, DA is an important catecholamine neurotransmitter and plays important roles in the central nervous system, peripheral nervous system, cardiovascular system, and endocrine system. Diseases such as Parkinson's disease, depression, and drug addiction are all related to abnormal dopamine secretion in the human body. UA is a natural antioxidant, and abnormal levels of its concentration in human body fluids can cause diseases such as hyperuricemia, gout, leukemia, and pneumonia. As the main products of purine metabolism, DA and UA generally coexist in the extracellular fluid of the central nervous system and serum. Therefore, developing a detection method with good biocompatibility, high selectivity, high sensitivity, and capable of simultaneously detecting the concentration levels of dopamine and uric acid is of great significance for the analytical application and diagnostic research of some diseases.

[0003] DA and UA have similar structures and relatively close redox potentials, and it is a huge challenge to detect them simultaneously. Traditional detection techniques for dopamine and uric acid include high performance liquid chromatography, spectrophotometry, fluorescence method, capillary electrophoresis, chemiluminescence method, liquid chromatography-mass spectrometry analysis, etc. However, these methods have complex pre-treatment operation steps, are time-consuming, and require high requirements for the required equipment. Compared with the above methods, biosensors based on electrochemical technology have the advantages of fast response, simple operation, low cost, and high sensitivity, and are an extremely attractive and competitive detection method. A variety of nanomaterials have been reported for the quantitative detection of DA and UA, such as carbon nanomaterials, metal nanomaterials, polymer nanomaterials, etc. Although the electrochemical sensors based on these materials have successfully achieved the separation of the oxidation potentials of DA and UA, they face technical challenges such as cumbersome material preparation steps, high cost, biological toxicity, narrow detection range, and low sensitivity, which form obstacles to the further realization of the real-time detection of DA and UA in vivo. In view of the above technical deficiencies, developing a determination method with good biocompatibility, high selectivity, low detection limit, and capable of simultaneously detecting DA and UA has important research significance.

[0004] Red blood cells (RBCs) are the most abundant cell type in the human body and have attracted much attention due to their inherent electrocatalytic properties and biocompatibility. However, their low catalytic activity and hemolytic properties limit RBC-based applications. Nanomaterials have advantages such as large specific surface area, high electrical conductivity, and catalytic activity. Synthesizing the exoskeleton of RBCs with nanomaterials and combining the natural biological functions of RBCs with the physical and chemical properties of nanomaterials is an effective strategy to improve the stability and catalytic activity of RBCs. Carbon nanotubes are electrochemically active substances with a large specific surface area, high stability, excellent electronic conductivity, and mechanical properties, and may be the best candidate nanomaterials to achieve this goal. Therefore, using carbon nanotubes as a protective shell can not only protect RBCs and improve their stability, but also design and synthesize RBC-based biosensors by combining the biological functions of RBCs with the physical and chemical properties of carbon nanotubes. There is currently no relevant report in this regard. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a preparation method of a biosensor based on a red blood cell-carbon nanotube hybrid (RBC@CNTs) with good biocompatibility, high selectivity, and high sensitivity. This method first synthesizes the RBC@CNTs hybrid and constructs a biosensor using the electrode modified with the RBC@CNTs hybrid for simultaneous quantitative detection of dopamine and uric acid concentrations.

[0006] The present invention adopts the following technical solutions to solve the above technical problems. A preparation method of a biosensor based on a red blood cell-carbon nanotube hybrid, characterized in that: the biosensor is composed of a reference electrode, a counter electrode, and a working electrode modified with a red blood cell-carbon nanotube hybrid. The modified working electrode is composed of a carbon cloth electrode and a red blood cell-carbon nanotube hybrid coated on the surface of the carbon cloth electrode. The red blood cell-carbon nanotube hybrid uses red blood cells as a matrix, and by mixing carbon nanotubes dispersed evenly by ultrasound with red blood cells, a red blood cell-carbon nanotube hybrid with a carbon nanotube artificial shell coated on the surface of the red blood cell membrane is obtained.

[0007] Further defined, the counter electrode is a platinum sheet electrode, and the reference electrode is an Ag / AgCl electrode.

[0008] Further defined, the specific preparation process of the red blood cell-carbon nanotube hybrid is as follows:

[0009] Step S1, perform surface pretreatment on the carbon cloth electrode;

[0010] Step S2, centrifuge and wash the upper plasma from animal blood or human blood to obtain natural red blood cells, then fix the natural red blood cells with a 4wt% glutaraldehyde solution, and then wash them repeatedly with physiological saline and deionized water to obtain fixed red blood cells A;

[0011] Step S3: adding carbon nanotubes into ultrapure water and ultrasonically dispersing the mixture to obtain solution B;

[0012] Step S4: at room temperature, the fixed red blood cells A obtained in step S2 are suspended and dispersed in the solution B obtained in step S3, and then placed in a constant temperature oscillator for reaction, and then the precipitate is collected by centrifugation and washed by ultrapure water to remove the unreacted precursor, and finally the red blood cell-carbon nanotube hybrid is obtained.

[0013] It is further defined that the specific process of the surface pretreatment of the carbon cloth electrode in step S1 is: ultrasonically treating the carbon cloth electrode in acetone and ethanol for 30 minutes in sequence to remove surface impurities, and then immersing the carbon cloth electrode in a mixed acid solution of concentrated sulfuric acid and concentrated nitric acid for treatment after drying, and then repeatedly rinsing the carbon cloth electrode with ultrapure water until the rinsing liquid is neutral, and obtaining a surface-pretreated carbon cloth electrode after drying.

[0014] It is further defined that the animal blood in step S2 is one or more of pig blood, sheep blood, rabbit blood or cow blood.

[0015] It is further defined that the carbon nanotubes in step S3 are one or more of multi-walled carbon nanotubes, carboxylated multi-walled carbon nanotubes, hydroxylated carbon nanotubes or amino carbon nanotubes.

[0016] It is further defined that the specific process of steps S2-S5 is as follows: 500 μL of 3% specific volume natural red blood cells are fixed and then washed with water to obtain fixed red blood cells A; 2 mg of carbon nanotubes are added to 1 mL of ultrapure water, and ultrasonically dispersed in an ice water bath to obtain solution B; the fixed red blood cells A are suspended and dispersed in solution B, and then placed in a constant temperature oscillator at a speed of 110 rpm for 24 hours, and then the precipitate is collected by centrifugation, and washed three times with ultrapure water to remove excess carbon nanotube precursors, and finally restored to a 3% specific volume RBC@CNTs hybrid suspension with ultrapure water, and stored at 4°C for use; 30 μL of 3% specific volume RBC@CNTs hybrid suspension is added dropwise to the surface of the pretreated carbon cloth electrode, and naturally dried at room temperature to obtain a working electrode modified based on red blood cells-carbon nanotube hybrids.

[0017] The invention discloses an application of the red blood cell-carbon nanotube hybrid biosensor in the quantitative detection of dopamine and uric acid.

[0018] It is further defined that the red blood cell-carbon nanotube hybrid biosensor uses a standard three-electrode system to detect the concentration of dopamine and uric acid, that is, the red blood cell-carbon nanotube hybrid modified carbon cloth electrode is used as the working electrode, Ag / AgCl is used as the reference electrode, and a platinum sheet (1 cm 2 ) was the counter electrode, the electrolyte was 0.1 M PBS buffer with pH = 7.4, and all electrochemical tests were carried out in nitrogen-saturated PBS buffer.

[0019] Further defined, the erythrocyte-carbon nanotube hybrid biosensor uses differential pulse voltammetry (DPV) to detect the electrochemical response current, and calculates the detection limits of dopamine and uric acid for the erythrocyte-carbon nanotube hybrid biosensor according to the linear relationship between the electrochemical response current and the dopamine concentration and the linear relationship between the electrochemical response current and the uric acid concentration. The linear detection range of dopamine is 4-400 μM, and the detection limit is 0.10 μM; the linear detection range of uric acid is 2-500 μM, and the detection limit is 0.69 μM, and the erythrocyte-carbon nanotube hybrid biosensor for quantitative detection of dopamine and uric acid has good selectivity, anti-interference ability, reproducibility and stability.

[0020] The present invention has the following advantages and beneficial effects compared with the prior art:

[0021] 1. The present invention uses erythrocytes as the supporting material to synthesize an erythrocyte-carbon nanotube hybrid, and the synthesis process is green and pollution-free.

[0022] 2. The biosensor prepared by using the synthesized erythrocyte-carbon nanotube hybrid as a catalyst can simultaneously detect the concentration levels of dopamine and uric acid, has a wide detection range and a low detection limit for dopamine and uric acid, and at the same time exhibits good selectivity, reproducibility, stability and strong anti-interference ability.

[0023] 3. The erythrocyte-carbon nanotube hybrid biosensor prepared by the present invention has good electrochemical sensing performance. The linear detection range of dopamine is 4-400 μM, and the detection limit is 0.10 μM; the linear detection range of uric acid is 2-500 μM, and the detection limit is 0.69 μM; compared with traditional sensors, it has obvious advantages and is expected to further realize the monitoring of dopamine and uric acid concentrations in the body. Description of the Drawings

[0024] Figure 1 is the FESEM image of the RBC@MWCNTs hybrid prepared in Example 1.

[0025] Figure 2 is the TEM image of the RBC@MWCNTs hybrid prepared in Example 1.

[0026] Figure 3 is the DPV graph (a) of the working electrode modified with the RBC@MWCNTs hybrid prepared in Example 1 in 0.1 M PBS (pH = 7.4) buffer solution containing 50 μM DA with different concentrations of UA and the corresponding linear graph (b).

[0027] Figure 4The response diagram (a) of the working electrode modified with the RBC@MWCNTs hybrid prepared in Example 1 to different concentrations of DA in a 0.1 M PBS (pH = 7.4) buffer solution containing 100 μM UA and the corresponding linear curve diagram (b).

[0028] Figure 5 It is the selectivity detection diagram of the working electrode modified with the RBC@MWCNTs hybrid prepared in Example 1 for UA.

[0029] Figure 6 It is the selectivity detection diagram of the working electrode modified with the RBC@MWCNTs hybrid prepared in Example 1 for DA.

[0030] Figure 7 In (a), it is the cyclic voltammetry (DPV) test curve diagram of eight times at regular intervals of a working electrode modified with the RBC@MWCNTs hybrid prepared in Example 1 in a 0.1 M PBS buffer solution containing 100 μM DA and 200 μM UA; (b) is the DPV diagram of five working electrodes modified with the RBC@MWCNTs hybrid prepared in Example 1 in a 0.1 M PBS buffer solution containing 100 μM DA and 200 μM UA.

[0031] Figure 8 It is the bar chart of the long-term stability test of the working electrode modified with the RBC@MWCNTs hybrid prepared in Example 1. Detailed implementation manners

[0032] The above content of the present invention will be further described in detail through the following examples, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. Any technology implemented based on the above content of the present invention belongs to the scope of the present invention.

[0033] Example 1

[0034] Pretreatment of the carbon cloth electrode

[0035] The commercial carbon cloth (CC) was successively ultrasonicated in acetone and ethanol for 30 min, and after natural drying, the carbon cloth was immersed in a mixed acid solution of concentrated sulfuric acid and concentrated nitric acid (concentrated sulfuric acid: concentrated nitric acid = 3:1, v / v) for 24 h. Then, the mixed acid on the carbon cloth was repeatedly rinsed with ultrapure water until the rinsing solution was neutral, and after drying, the surface-pretreated carbon cloth electrode was obtained.

[0036] Preparation of the RBC@MWCNTs hybrid

[0037] Centrifuge and wash whole pig blood to remove the upper plasma layer, and wash it three times with physiological saline to obtain packed natural red blood cells. Then fix the natural red blood cells with a 4 wt% glutaraldehyde solution, and subsequently centrifuge and wash them multiple times with physiological saline and ultrapure water to obtain fixed red blood cells A. Accurately weigh 2 mg of multiwalled carbon nanotubes (MWCNTs) and add 1 mL of ultrapure water, and place it in an ice-water bath for ultrasonic dispersion for 1 h to obtain solution B. Suspend and disperse fixed red blood cells A in solution B, and then place it in a thermostatic oscillator and react at a speed of 110 revolutions per minute for 24 h. After the reaction, centrifuge to collect the precipitate, and centrifuge and wash it three times with ultrapure water to remove the excess MWCNTs precursor. Finally, reconstitute it with ultrapure water into a 3% specific volume RBC@MWCNTs hybrid suspension and store it at 4 °C for later use.

[0038] Figure 1 Figure 4 is the FESEM image of the RBC@MWCNTs hybrid prepared in this example. It can be seen from the figure that the RBC@MWCNTs hybrid presents a biconcave disc shape, its surface is relatively rough, and the cell surface is evenly covered with a layer of carbon nanotubes, indicating that the CNTs artificial shell layer is successfully formed on the surface of red blood cells. Figure 2 Figure 5 is the TEM image of the RBC@MWCNTs hybrid prepared in this example. It can be seen from the figure that an artificial shell layer formed by a layer of carbon nanotubes is coated on the surface of the red blood cell membrane, and the thickness of this shell layer is about 300 nm.

[0039] Preparation of a working electrode modified with RBC@MWCNTs hybrid

[0040] Drop 30 μL of the 3% specific volume RBC@MWCNTs hybrid suspension onto the surface of the pretreated carbon cloth electrode, and let it dry naturally at room temperature to obtain a working electrode modified with the RBC@MWCNTs hybrid.

[0041] Electrochemical testing

[0042] The working electrode modified with the RBC@MWCNTs hybrid obtained above, a counter electrode, and a reference electrode are combined to form a three-electrode system (a platinum sheet is used as the counter electrode, and Ag / AgCl is used as the reference electrode). Then, electrochemical testing is carried out at room temperature, and the electrolyte is 0.1 M phosphate buffer solution. Before testing, N2 is bubbled for 30 min, and then differential pulse voltammetry (DPV) is used for testing. During the testing process, different concentrations of dopamine or uric acid solutions are added dropwise after stabilization.

[0043] Figure 3DPV graphs of the RBC@MWCNTs hybrid modified working electrode prepared in this example in 0.1 M PBS (pH = 7.4) buffer containing 50 μM DA at different concentrations of UA and the corresponding linear graphs. It can be seen from the graphs that in the range of 2 - 500 μM, the current response of uric acid (UA) increases linearly with the increase of UA concentration. When the UA concentration is 2 - 150 μM, the regression equation is I(mA)=0.0092C UA +0.2850(R 2 =0.9940). When the UA concentration is 150 - 500 μM, the regression equation is I(mA)=0.0045C UA +0.9930(R 2 =0.9890). Using the detection limit calculation formula LOD = 3S / b (where S represents the standard deviation of the blank and b is the slope of the linear equation of concentration vs. peak current), the lower detection limit (LOD) of UA is calculated to be approximately 0.69 μM.

[0044] Figure 4 DPV graphs of the RBC@MWCNTs hybrid modified working electrode prepared in this example in 0.1 M PBS (pH = 7.4) buffer containing 100 μM UA at different concentrations of DA and the corresponding linear graphs. It can be seen from the graphs that in the range of 4 - 400 μM, the current response of dopamine (DA) increases linearly with the increase of DA concentration. When the DA concentration is 4 - 150 μM, the regression equation is I(mA)=0.022C DA +0.384(R 2 =0.9899). When the DA concentration is 150 - 400 μM, the regression equation is I(mA)=0.0098C DA +2.051(R 2 =0.9845). Using the detection limit calculation formula LOD = 3S / b (where S represents the standard deviation of the blank and b is the slope of the linear equation of concentration vs. peak current), the lower detection limit (LOD) of DA is calculated to be approximately 0.10 μM.

[0045] Figure 5 Selective detection graph of uric acid by the RBC@MWCNTs hybrid modified working electrode prepared in this example. This was carried out by adding 0.9 mM KCl, NaCl, K2CO3 and 0.5 mM AA, L-Cys, Glu, GSH, Sucrose to the PBS buffer containing 200 μM UA. The electrochemical test results show that these substances do not cause obvious interference to the determination of UA, indicating that the RBC@MWCNTs hybrid-based biosensor has good selectivity and anti-interference ability for UA.

[0046] Figure 6 This is a graph showing the selective detection of dopamine by the working electrode modified with the RBC@MWCNTs hybrid prepared in this example. This was carried out by adding 0.9 mM KCl, NaCl, K2CO3 and 0.5 mM AA, L-Cys, Glu, GSH, Sucrose to a PBS buffer solution containing 100 μM DA. The results of the electrochemical tests showed that these substances did not cause significant interference in the determination of DA, indicating that the biosensor based on the RBC@MWCNTs hybrid has good selectivity and anti-interference ability for DA.

[0047] Figure 7 This is the reproducibility determination of the working electrode modified with the RBC@MWCNTs hybrid prepared in this example. Figure 7 (a) This is a cyclic voltammetry (DPV) test curve graph of the same working electrode modified with the RBC@MWCNTs hybrid in a 0.1 M PBS buffer solution containing 100 μM DA and 200 μM UA at certain time intervals for eight times. It can be seen from the graph that there is no obvious change in the peak current intensity of DA and UA. The relative standard deviation (RSD) of DA and UA calculated from the results of 8 measurements is 1.29% and 0.89% respectively. Figure 7 (b) These are DPV test graphs of 5 parallelly prepared working electrodes modified with the RBC@MWCNTs hybrid in a 0.1 M phosphate buffer solution containing 100 μM DA and 200 μM UA. It can be seen from the graph that the response current values of DA and UA measured 5 times are basically the same, and no obvious current decay is found. The relative standard deviation (RSD) calculated from the 5 current values of DA and UA is 0.23% and 0.82% respectively. In summary, the two tests above show that the working electrode modified with the RBC@MWCNTs hybrid has good reproducibility in the detection of DA and UA.

[0048] Figure 8 This is a bar graph showing the long-term stability test of the working electrode modified with the RBC@MWCNTs hybrid prepared in this example. It can be seen from the graph that after the working electrode modified with the RBC@MWCNTs hybrid is stored in the refrigerator for 10 days, the peak signals of DA and UA can still maintain 93.6% and 91.4% of their initial values respectively, indicating that the working electrode modified with the RBC@MWCNTs hybrid has good stability in the detection of DA and UA.

[0049] Example 2

[0050] Pretreatment of the carbon cloth electrode, and the specific steps are the same as those in Example 1.

[0051] Preparation of the RBC@MWCNTs-COOH hybrid

[0052] Centrifuge and wash the whole sheep blood to remove the upper plasma and wash it three times with physiological saline to obtain packed natural red blood cells. Then fix the natural red blood cells with 4 wt% glutaraldehyde solution, and then centrifuge and wash them with physiological saline and ultrapure water multiple times to obtain fixed red blood cells A; accurately weigh 2 mg of carboxylated multi-walled carbon nanotubes and add 1 mL of ultrapure water, and place them in an ice-water bath for ultrasonic dispersion for 1 h to obtain solution B; suspend and disperse the fixed red blood cells A in solution B, and then place them in a thermostatic oscillator and react at a rotation speed of 110 revolutions per minute for 24 h. After the reaction, centrifuge to collect the precipitate, and wash it three times with ultrapure water by centrifugation to remove the excess MWCNTs-COOH precursor. Finally, restore it to a 3% specific volume RBC@MWCNTs-COOH hybrid suspension with ultrapure water and store it at 4 °C for later use.

[0053] Prepare a working electrode modified with RBC@MWCNTs-COOH hybrid, and the specific steps are the same as in Example 1.

[0054] Biosensing test, the specific steps are the same as in Example 1. The detection limit of dopamine by the prepared working electrode modified with RBC@MWCNTs-COOH hybrid is 0.10 μM, and the detection limit of UA is about 0.69 μM, and it has good selectivity, reproducibility and stability.

[0055] Example 3

[0056] Pretreatment of the carbon cloth electrode, and the specific steps are the same as in Example 1.

[0057] Prepare RBC@MWCNTs-NH2 hybrid

[0058] Centrifuge and wash the whole rabbit blood to remove the upper plasma and wash it three times with physiological saline to obtain packed natural red blood cells. Then fix the natural red blood cells with 4 wt% glutaraldehyde solution, and then centrifuge and wash them with physiological saline and ultrapure water multiple times to obtain fixed red blood cells A; accurately weigh 2 mg of amino-functionalized multi-walled carbon nanotubes and add 1 mL of ultrapure water, and place them in an ice-water bath for ultrasonic dispersion for 1 h to obtain solution B; suspend and disperse the fixed red blood cells A in solution B, and then place them in a thermostatic oscillator and react at a rotation speed of 110 revolutions per minute for 24 h. After the reaction, centrifuge to collect the precipitate, and wash it three times with ultrapure water by centrifugation to remove the excess MWCNTs-NH2 precursor. Finally, restore it to a 3% specific volume RBC@MWCNTs-NH2 hybrid suspension with ultrapure water and store it at 4 °C for later use.

[0059] Prepare a working electrode modified with RBC@MWCNTs-NH2 hybrid, and the specific steps are the same as in Example 1.

[0060] Biosensing test, the specific steps are the same as those in Example 1. The detection limit of dopamine by the prepared working electrode modified with RBC@MWCNTs-NH2 hybrid is 0.10 μM, and the detection limit of UA is about 0.69 μM. Moreover, it has good selectivity, reproducibility and stability.

[0061] Example 4

[0062] Pretreatment of the carbon cloth electrode, the specific steps are the same as those in Example 1.

[0063] Preparation of RBC@MWCNTs-OH hybrid

[0064] Centrifuge and wash the upper plasma from whole human blood and wash it three times with normal saline to obtain packed natural red blood cells. Then fix the natural red blood cells with 4 wt% glutaraldehyde solution. Subsequently, centrifuge and wash them with normal saline and ultrapure water multiple times to obtain fixed red blood cells A. Accurately weigh 2 mg of hydroxylated multi-walled carbon nanotubes and add 1 mL of ultrapure water, and place them in an ice-water bath for ultrasonic dispersion for 1 h to obtain solution B. Suspend and disperse fixed red blood cells A in solution B, and then place them in a constant temperature oscillator and react at a speed of 110 revolutions per minute for 24 h. After the reaction, centrifuge to collect red blood cells, and centrifuge and wash them three times with ultrapure water to remove the excess MWCNTs-OH precursor. Finally, restore them to a 3% specific volume suspension of RBC@MWCNTs-OH hybrid with ultrapure water and store it at 4 °C for standby.

[0065] Preparation of the working electrode modified with RBC@MWCNTs-OH hybrid, the specific steps are the same as those in Example 1.

[0066] Biosensing test, the specific steps are the same as those in Example 1. The detection limit of dopamine by the prepared working electrode modified with RBC@MWCNTs-OH hybrid is 0.10 μM, and the detection limit of UA is about 0.69 μM. Moreover, it has good selectivity, reproducibility and stability.

[0067] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A preparation method of a biosensor based on a red blood cell-carbon nanotube hybrid, characterized in that: The biosensor is composed of a reference electrode, a counter electrode, and a working electrode modified with a red blood cell-carbon nanotube hybrid. The modified working electrode is composed of a carbon cloth electrode and a red blood cell-carbon nanotube hybrid coated on the surface of the carbon cloth electrode. The red blood cell-carbon nanotube hybrid uses red blood cells as the matrix. By mixing the carbon nanotubes after ultrasonic dispersion with red blood cells evenly, a red blood cell-carbon nanotube hybrid with a carbon nanotube artificial shell coated on the surface of the red blood cell membrane is obtained. The red blood cell-carbon nanotube hybrid biosensor can be used for the quantitative detection of dopamine and uric acid. The specific preparation process of the red blood cell-carbon nanotube hybrid is as follows: Step S1, perform surface pretreatment on the carbon cloth electrode. Step S2, centrifuge and wash the upper plasma from animal blood or human blood to obtain natural red blood cells. Then fix the natural red blood cells with a 4wt% glutaraldehyde solution, and then wash them repeatedly with physiological saline and deionized water for multiple times to obtain fixed red blood cells A. Step S3: Add carbon nanotubes to ultrapure water and disperse them by ultrasonic treatment to obtain solution B. Step S4: Under room temperature conditions, suspend and disperse the fixed red blood cells A obtained in step S2 in the solution B obtained in step S3, then place them in a thermostatic oscillator for reaction, then centrifuge to collect the precipitate, and wash it with ultrapure water by centrifugation to remove the unreacted precursors. Finally, a red blood cell-carbon nanotube hybrid is obtained.

2. The preparation method of the biosensor based on the erythrocyte-carbon nanotube hybrid according to claim 1, wherein: The counter electrode is a platinum sheet electrode, and the reference electrode is an Ag / AgCl electrode.

3. The preparation method of the biosensor based on the erythrocyte-carbon nanotube hybrid according to claim 1, characterized in that The specific process of the surface pretreatment of the carbon cloth electrode in step S1 is as follows: Ultrasonically treat the carbon cloth electrode in acetone and ethanol for 30 min in sequence to remove surface impurities. After drying, immerse the carbon cloth electrode in a mixed acid solution of concentrated sulfuric acid and concentrated nitric acid for treatment, and then repeatedly rinse the carbon cloth electrode with ultrapure water until the rinsing solution is neutral. After drying, a carbon cloth electrode with surface pretreatment is obtained.

4. The preparation method of the biosensor based on the erythrocyte-carbon nanotube hybrid according to claim 1, characterized in that: The animal blood in step S2 is one or more of pig blood, sheep blood, rabbit blood, or bovine blood.

5. The preparation method of the biosensor based on the erythrocyte-carbon nanotube hybrid according to claim 1, characterized in that: The carbon nanotubes in step S3 are one or more of carboxylated multi-walled carbon nanotubes, hydroxylated carbon nanotubes, or amino-functionalized carbon nanotubes.

6. The preparation method of the biosensor based on the erythrocyte-carbon nanotube hybrid according to claim 1, characterized in that: Fix 500 μL of 3% specific volume of natural red blood cells and wash them with water to obtain fixed red blood cells A; add 2 mg of carbon nanotubes to 1 mL of ultrapure water, place them in an ice-water bath and disperse them by ultrasonic treatment to obtain solution B; suspend and disperse the fixed red blood cells A in solution B, then place them in a thermostatic oscillator and react at a rotation speed of 110 revolutions per minute for 24 h, then centrifuge to collect the precipitate, and wash it with ultrapure water by centrifugation three times to remove the excess carbon nanotube precursors. Finally, restore it to a 3% specific volume of RBC@CNTs hybrid suspension with ultrapure water, and store it at 4 °C for standby; drop 30 μL of 3% specific volume of RBC@CNTs hybrid suspension onto the surface of the pretreated carbon cloth electrode, and let it dry naturally at room temperature to obtain a working electrode modified with a red blood cell-carbon nanotube hybrid.

7. Application of the red blood cell-carbon nanotube hybrid biosensor prepared by the method according to any one of claims 1-6 in the quantitative detection of dopamine and uric acid.

8. The application according to claim 7, characterized in that: The biosensor based on the erythrocyte-carbon nanotube hybrid uses a standard three-electrode system to detect the concentrations of dopamine and uric acid. The carbon cloth electrode modified with the erythrocyte-carbon nanotube hybrid is used as the working electrode, Ag / AgCl is used as the reference electrode, and a platinum sheet is used as the counter electrode. The electrolyte is a 0.1 M PBS buffer solution with a pH of 7.

4. All electrochemical tests are carried out in a nitrogen-saturated PBS buffer solution.

9. The application according to claim 7, characterized in that: The biosensor based on the erythrocyte-carbon nanotube hybrid uses differential pulse voltammetry to detect the electrochemical response current. The detection limits of the biosensor based on the erythrocyte-carbon nanotube hybrid for dopamine and uric acid are calculated according to the linear relationship between the electrochemical response current and the dopamine concentration and the linear relationship between the electrochemical response current and the uric acid concentration. The linear detection range of dopamine is 4 - 400 μM, and the detection limit is 0.10 μM; the linear detection range of uric acid is 2 - 500 μM, and the detection limit is 0.69 μM. Moreover, the biosensor based on the erythrocyte-carbon nanotube hybrid has good selectivity, anti-interference ability, reproducibility, and stability for the quantitative detection of dopamine and uric acid.

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