Method for preparing electrochemiluminescence sensor and application thereof

Abstract

The application relates to a preparation method of an electrochemiluminescence sensor and application thereof, and belongs to the technical field of electrochemiluminescence sensors, and specifically comprises the following steps: step 1, firstly, a bare glassy carbon electrode is repeatedly polished with aluminum oxide powder, and then the electrode is thoroughly cleaned before use; Au@CN solution is coated on the electrode; step 2, after the electrode is dried, a conconavalin A solution is added dropwise, and after 1 hour, the electrode is washed with a phosphate solution to block the non-specific binding between the conconavalin A and the electrode surface; step 3, NMS / CNT@Glu solution is coated on the electrode surface, and after 1 hour, the obtained electrode is washed to remove the unbound NMS / CNT@Glu on the outer surface, so that an electrochemiluminescence sensor for detecting conconavalin A is obtained. When the sensor detects conconavalin A, a wide linear range and a low detection limit are exhibited, so that the sensor can be applied to actual detection.

Description

Technical Field

[0001] This invention relates to a method for preparing an electrochemiluminescence sensor and its application, and more particularly to a method for preparing an electrochemiluminescence sensor for detecting concanavalin A and its application, which belongs to the field of electrochemiluminescence sensor technology. Background Technology

[0002] Electrochemiluminescence (ECL) is a chemiluminescence phenomenon triggered by electrochemical methods, possessing the advantages of both chemiluminescence and electrochemistry. Due to its unique advantages such as simple operation, fast response, high sensitivity, strong controllability, and low background signal, ECL has become a research hotspot and has been widely applied in clinical diagnosis, environmental and food analysis, and immunoassay. Commonly used luminescent materials in ECL systems include ruthenium complexes, luminol, metal nanoclusters, and semiconductor quantum dots (CdS, CdSe, CdTe, and PbSe). Furthermore, graphitic carbon nitride compounds have also attracted widespread interest due to their large surface area, good biocompatibility, strong ECL intensity, low cost, and good chemical stability. Researchers have found that the ECL stability of graphitic carbon nitride compounds decreases significantly with increasing scan potential because electrode passivation occurs and subsequent ECL emission is blocked. These factors limit the reusability of graphitic carbon nitride compounds and their application in highly sensitive ECL sensors, while metal nanoparticle-doped carbon nitride may be an effective way to overcome this problem. Some studies on gold-doped carbon nitride-based electrochemiluminescence sensors have been reported, and some electrochemiluminescence quenchers have been investigated with the aim of expanding the application of graphitic carbon nitride compounds in electrochemiluminescence, including ferrocene and polyaniline. However, the quenching mechanism of molybdenum disulfide nanoflowers has not been studied.

[0003] Recently, layered transition metal sulfides (TMDs) have attracted widespread interest as a novel electrode material due to their inherent properties. Molybdenum disulfide (MoS2), a typical member of the TMD family, has drawn considerable attention and is widely used in energy storage, hydrogen production, and biosensors. However, MoS2 tends to form nanotube structures or fullerene-like nanoparticles during its fabrication. Combining MoS2 with high-surface-area carbon materials can overcome these drawbacks and result in better conductivity and a more stable structure. Furthermore, Ni- or Co-doped MoS2 can generate strong electron transfer.

[0004] Concanavalin A (Con A) is a globulin isolated from beet meal and is one of the most frequently studied lectins with four sugar-binding sites, exhibiting a unique affinity between the lectin and carbohydrate ligands. The interaction between lectins and carbohydrates plays a crucial role in biological processes such as cell surface recognition, intercellular communication, cancer, and host-pathogen infection. Based on carbohydrate-lectin interactions, numerous detection methods exist for Concanavalin A, including electrochemical analysis, photoelectrochemistry, surface plasmon resonance, and fluorescence spectroscopy. However, reports on the detection of Concanavalin A using electrochemiluminescence sensors are relatively few. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for preparing an electrochemiluminescence sensor for detecting concanavalin A and its application. The method uses gold-doped carbon nitride prepared by in-situ growth as the electrochemiluminescent agent and glucose-functionalized carbon nanotube-supported nickel sulfide and molybdenum disulfide nanosheet composite material prepared by hydrothermal method as the quencher, thereby constructing a highly sensitive and selective electrochemiluminescence sensor for detecting the content of concanavalin A.

[0006] A method for preparing a functionalized carbon nanotube composite material includes the following steps:

[0007] Step 1: Preparation of glucose-functionalized carbon nanotube-supported nickel sulfide and molybdenum disulfide nanosheet composite material: Acidified multi-walled carbon nanotubes were ultrasonically treated for 40 minutes and then dissolved in 0.05 mol / L glucose aqueous solution; then, nickel sulfate, ammonium molybdate and thiourea were added to the above mixed solution and stirred for 1 hour. The solution was then transferred to a polytetrafluoroethylene-lined autoclave and kept in a forced-air drying oven at 200°C for 24 hours. The resulting mixture was thoroughly washed three times by centrifugation with distilled water and ethanol.

[0008] Step 2: Preparation of glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material modified with bovine serum albumin: 2 mg of glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material was dispersed in 1 mL of pH 7.4 phosphate buffer solution. Then, 100 μL of 1% bovine serum albumin solution was added and the mixture was shaken for 2 hours to block non-specific adsorption sites. Finally, the solid was collected by centrifugation to obtain the glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material modified with bovine serum albumin, i.e., NMS / CNT@Glu. The NMS / CNT@Glu solution was redispersed in 1 mL of pH 7.4 phosphate buffer solution for later use. When not in use, it was stored at 4°C.

[0009] Furthermore, in step (1), the amount of multi-walled carbon nanotubes added is 25-35 mg, the amount of glucose aqueous solution added is 36-45 mL, the amount of nickel sulfate added is 0.18-0.21 g, the amount of ammonium molybdate added is 0.14-0.18 g, and the amount of thiourea added is 0.3-0.4 g.

[0010] A method for fabricating an electrochemiluminescence sensor based on a prepared functionalized carbon nanotube composite material includes the following steps:

[0011] Step 1: First, polish the bare glassy carbon electrode repeatedly with alumina powder, then clean it thoroughly before use, and coat the electrode with 5μL of 1mg / mL Au@CN solution.

[0012] Step 2: After the electrode is dry, add 5 μL of concanavalin A solution. After 1 hour, wash the electrode with phosphate solution to block the non-specific binding between concanavalin A and the electrode surface.

[0013] Step 3: Coat the electrode surface with 5 μL of 1 mg / mL NMS / CNT@Glu solution. After 1 hour, rinse the obtained electrode to remove unbound NMS / CNT@Glu from the outer surface, and obtain an electrochemiluminescent sensor for detecting concanavalin A.

[0014] In step 1, the preparation method of the Au@CN solution includes the following steps:

[0015] Step (1), Preparation of carbon nitride nanosheets: First, place 3-5g of melamine in a crucible and heat it at a rate of 2-5℃ / min, and heat it at 550℃ for 4 hours to prepare loose carbon nitride lumps. After grinding the resulting yellow lumps into powder, put a certain amount of carbon nitride lumps into a porcelain crucible, heat it at a rate of 5℃ / min and keep it at 550℃ for 4 hours to obtain carbon nitride nanosheets;

[0016] Step (2), Preparation of gold-doped carbon nitride nanosheets: First, the carbon nitride nanosheets from step 1 were ultrasonically dispersed into a carbon nitride aqueous solution. 1 mL of 0.5 mmol / L HAuCl4 aqueous solution was added to 10 mL of 2.0 mg / mL carbon nitride aqueous solution and stirred vigorously for 2 hours. Then, 1 mL of freshly prepared 0.01 mol / L NaBH4 solution was added dropwise to the mixture to reduce trivalent gold ions. The resulting suspension was centrifuged at 10000 r / min to remove unreacted / unbound substances including individual gold nanoparticles. Finally, the obtained gold-doped carbon nitride nanosheets, i.e., Au@CN, were washed with distilled water and redispersed in 10 mL of distilled water to obtain an Au@CN solution for later use.

[0017] Applications based on the fabricated electrochemiluminescence sensor include the following steps:

[0018] (1) Using the Ag / AgCl electrode as the reference electrode, the platinum wire electrode as the counter electrode, and the prepared electrochemiluminescence sensor as the working electrode, the electrochemical workstation and the chemiluminescence detector are connected in the dark box of the chemiluminescence detector.

[0019] (2) Using 10 mL of phosphate buffer solution containing 0.1 mol / L K2S2O8 as a co-reactant, the intensity of the electrochemiluminescence signal generated by different concentrations of concanavalin A was detected by electrochemiluminescence method.

[0020] (3) Optimization of measurement conditions:

[0021] By controlling a single variable, the pH value of the phosphate buffer solution containing 0.1 mol / L K2S2O8, the concentration of Au@CN solution, and the concentration of NMS / CNT@Glu solution were optimized through experiments. The optimal pH value of the phosphate buffer solution containing 0.1 mol / L K2S2O8, the concentration of Au@CN solution, and the concentration of NMS / CNT@Glu solution were obtained, which are the best experimental conditions.

[0022] (4) Plotting working curves:

[0023] A series of standard concentrations of concanavalin A solutions were prepared. Under optimal experimental conditions, the electrochemiluminescence intensity of the series of standard concentrations of concanavalin A was measured. The working curve was plotted with the logarithm of the concanavalin A concentration as the x-axis and the electrochemiluminescence intensity after loading NMS / CNT@Glu as the y-axis.

[0024] (5) Anti-interference performance study:

[0025] The interference of ascorbic acid, uric acid, glucose, and bovine serum albumin on the detection of concanavalin A was investigated. Under optimal experimental conditions, the electrochemiluminescence intensity of the concanavalin A standard solution in the presence of different interfering substances was measured to study the anti-interference ability of the sensor.

[0026] (6) Actual sample testing:

[0027] Under optimal experimental conditions, the content of concanavalin A in human serum samples was detected, and a spiked recovery experiment was performed. First, different concentrations of concanavalin A were added to human serum samples. Then, before analysis, 100 μL of human serum was diluted 10 times with 0.01 mol / L phosphate buffer and then modified onto the electrode for testing.

[0028] Furthermore, the optimal experimental conditions are as follows: a phosphate buffer solution containing 0.1 mol / L K2S2O8 with a pH of 7.0, a NMS / CNT@Glu solution concentration of 0.5 mg / mL, and an Au@CN solution concentration of 1 mg / mL.

[0029] Furthermore, the phosphate buffer solution is prepared from 0.1 mol / L disodium hydrogen phosphate and 0.1 mol / L potassium dihydrogen phosphate, and its pH is 7.4.

[0030] Furthermore, the parameters of the chemiluminescence detector are set as follows: the high voltage of the photomultiplier tube is set to 700-800V, and the scanning rate is set to 0.15V / s; the parameters of the electrochemical workstation are set as follows: the cyclic voltammetric scan potential range is -1.3V to 0V, and the scanning rate is set to 0.1V / s.

[0031] The beneficial effects of this invention are as follows:

[0032] 1. The gold-doped carbon nitride prepared in this invention can emit strong electrochemiluminescence intensity and bind concanavalin A through the interaction between Au and N bonds;

[0033] 2. In this invention, gold-doped carbon nitride is used as an electrochemiluminescent agent, and layered nickel sulfide and molybdenum disulfide nanosheets supported by bovine serum albumin-modified glucose-functionalized carbon nanotubes are used as quenchers. By combining these with electrochemiluminescence technology, a highly sensitive and selective electrochemiluminescence sensor for detecting concanavalin A is constructed.

[0034] 3. The sensor exhibits a wide linear range and low detection limit when detecting concanavalin A. It demonstrates high accuracy and precision in the detection of concanavalin A in human serum samples, making it possible to apply to practical detection and having important biological significance in food safety and human health. Attached Figure Description

[0035] Figure 1 Here is a scanning electron microscope image of the NMS / CNT@Glu;

[0036] Figure 2 The image shows a scanning electron microscope (SEM) image of the carbon nitride nanosheets.

[0037] Figure 3 Electrochemiluminescence curves for (a) bare electrode, (b) Au@CN / GCE, (c) NiS / Au@CN / GCE, (d) MoS2 / Au@CN / GCE and (e) NMS / CNT@Glu / Au@CN / GCE;

[0038] Figure 4This is a flowchart illustrating the fabrication process of the electrochemiluminescence sensor.

[0039] Figure 5 Electrochemiluminescence curves of (a) GCE, (b) Au@CN / GCE, (c) Con A / Au@CN / GCE, (d) BSA / Con A / Au@CN / GCE and (e) NMS / CNT / BSA / Con A / Au@CN / GCE in 0.1 mol / L phosphate buffer solution (pH 7.4) containing 0.1 mol / L KCl and 10 mmol / L K2S2O8;

[0040] Figure 6 The electrochemical impedance spectroscopy spectra of (a) GCE, (b) Au@CN / GCE, (c) Con A / Au@CN / GCE, (d) BSA / Con A / Au@CN / GCE and (e) NMS / CNT / BSA / Con A / Au@CN / GCE in 2 mmol / L K3[Fe(CN)6] and 0.1 mol / L KCl solutions are shown.

[0041] Figure 7 A schematic diagram showing the effect of pH of a phosphate buffer solution containing 0.1 mol / L K2S2O8 on the detection of 1 ng / mL concanavalin A by an electrochemiluminescent sensor.

[0042] Figure 8 This is a schematic diagram showing the effect of the concentration of Au@CN solution on the detection of 1 ng / mL concanavalin A by an electrochemiluminescence sensor.

[0043] Figure 9 This is a schematic diagram showing the effect of the concentration of NMS / CNT@Glu solution on the detection of 1 ng / mL concanavalin A by an electrochemiluminescent sensor.

[0044] Figure 10 Electrochemiluminescence (ECL) curves of the electrochemiluminescence sensor for different concentrations of concanavalin A in 0.1 mol / L PBS solution (pH 7.0) containing 0.1 mol / L KCl and 0.1 mol / L K₂S₂O₈ (from a to k: 5 × 10⁻⁶). -5 1×10 -4 1×10 -3 ,0.005,0.01,0.05,0.1,1,10,50,100ng / mL);

[0045] Figure 11 The calibration curves of the electrochemiluminescence sensor for different concanavalin A concentrations are shown.

[0046] Figure 12The image shows the electrochemiluminescence curve of the electrochemiluminescence sensor for 1 ng / mL concanavalin A. Detailed Implementation

[0047] The following will be combined with the appendix Figure 1 To be continued Figure 12 The technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0048] A method for preparing a functionalized carbon nanotube composite material includes the following steps:

[0049] Step 1: Preparation of glucose-functionalized carbon nanotube-supported nickel sulfide and molybdenum disulfide nanosheet composite material: 30 mg of acidified multi-walled carbon nanotubes were ultrasonically treated for 40 minutes and then dissolved in 36 mL of 0.05 mol / L glucose aqueous solution; then, 0.18 g of nickel sulfate (NiSO4·6H2O), 0.14 g of ammonium molybdate ((NH4)2MoO4·4H2O) and 0.3 g of thiourea were added to the above mixed solution and stirred for 1 hour. The solution was then transferred to a polytetrafluoroethylene-lined autoclave and kept in a forced-air drying oven at 200 °C for 24 hours. The resulting mixture was thoroughly washed three times by centrifugation with distilled water and ethanol.

[0050] Step 2: Preparation of glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material modified with bovine serum albumin: 2 mg of glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material was dispersed in 1 mL of pH 7.4 phosphate buffer solution. Then, 100 μL of 1% bovine serum albumin solution was added and the mixture was shaken for 2 hours to block non-specific adsorption sites. Finally, the solid was collected by centrifugation to obtain the glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material modified with bovine serum albumin, i.e., NMS / CNT@Glu. The NMS / CNT@Glu solution was redispersed in 1 mL of pH 7.4 phosphate buffer solution for later use. When not in use, it was stored at 4°C.

[0051] A method for preparing gold-doped carbon nitride nanosheets includes the following steps:

[0052] Step 1, Preparation of carbon nitride nanosheets: First, 3g of melamine was placed in a crucible and heated at a rate of 2℃ / min, and heated at 550℃ for 4 hours to prepare loose carbon nitride lumps. After grinding the resulting yellow lumps into powder, a certain amount of carbon nitride lumps were placed in a porcelain crucible and heated at a rate of 5℃ / min and held at 550℃ for 4 hours to obtain carbon nitride nanosheets.

[0053] Step 2, Preparation of gold-doped carbon nitride nanosheets: First, the carbon nitride nanosheets from Step 1 were ultrasonically dispersed into a carbon nitride aqueous solution. 1 mL of 0.5 mmol / L HAuCl4 aqueous solution was added to 10 mL of the 2.0 mg / mL carbon nitride aqueous solution, and the mixture was stirred vigorously for 2 hours. Then, 1 mL of freshly prepared 0.01 mol / L NaBH4 solution was added dropwise to the mixture to reduce trivalent gold ions, i.e., Au(III). The resulting suspension was centrifuged at 10000 r / min to remove unreacted / unbound substances, including individual gold nanoparticles. Finally, the obtained gold-doped carbon nitride nanosheets, i.e., Au@CN, were washed with distilled water and redispersed in 10 mL of distilled water to obtain an Au@CN solution for later use.

[0054] To verify that NMS / CNT@Glu can quench Au@CN, the electrochemiluminescence performance of the prepared materials was validated using electrochemiluminescence technology. Figure 3 As shown:

[0055] (1) The bare glassy carbon electrode exhibits weak electrochemiluminescence intensity. Figure 3 Curve a), and then Au@CN is modified on the electrode. Figure 3 Curve b) shows that the electrochemiluminescence intensity is greatly increased compared to the electrode.

[0056] (2) Electrochemiluminescence signals of different quenchers, including NiS, MoS2, CNT, and NMS / CNT@Glu, were also investigated: Figure 3 The results show that CNT, NiS, and MoS2 can effectively quench Au@CN ( Figure 3 (Curves c and d), however, NMS / CNT@Glu showed the best quenching effect on Au@CN ( Figure 3 The curve e) may be related to the synergistic effect between NiS and MoS2.

[0057] A method for detecting concanavalin A using a bovine serum albumin-modified glucose-functionalized carbon nanotube-supported nickel sulfide and molybdenum disulfide nanosheets and gold-doped carbon nitride nanosheets in an electrochemiluminescence sensor: This composite material is used in conjunction with electrochemiluminescence technology. The changes in electrochemiluminescence intensity caused by different concentrations of concanavalin A are used to quantitatively detect concanavalin A. The preparation method of the electrochemiluminescence sensor includes the following steps:

[0058] Step 1: First, polish the bare glassy carbon electrode repeatedly with alumina powder, then clean it thoroughly before use, and coat the electrode with 5μL of 1mg / mL Au@CN solution.

[0059] Step 2: After the electrode is dry, add 5 μL of concanavalin A solution. After 1 hour, wash the electrode with phosphate solution to block the non-specific binding between concanavalin A and the electrode surface.

[0060] Step 3: Coat the electrode surface with 5 μL of 1 mg / mL NMS / CNT@Glu solution. After 1 hour, rinse the obtained electrode to remove unbound NMS / CNT@Glu from the outer surface, and obtain an electrochemiluminescent sensor for detecting concanavalin A.

[0061] To characterize the fabrication process of the electrochemiluminescence sensor, the changes in electrochemiluminescence intensity and impedance after each step were recorded. The electrochemiluminescence intensity was as follows: Figure 5 As shown:

[0062] (1) In S2O8 2- In the / O2 system, the bare glassy carbon electrode produces weak electrochemiluminescence ( Figure 5 Curve a) shows that when the electrode is modified with Au@CN, the electrochemiluminescence intensity is significantly increased compared to the bare electrode. Figure 5 Curve b);

[0063] (2) After concanavalin A and bovine serum albumin were sequentially immobilized on the modified electrode, the electrochemiluminescence intensity decreased continuously because they both inhibited S2O8. 2- / O2 and Au@CN electron transfer ( Figure 5 (Curves c and d);

[0064] (3) When the sensor is incubated with NMS / CNT@Glu ( Figure 5 As shown in curve e), the electrochemiluminescence intensity decreased significantly because NMS / CNT@Glu effectively quenched the electrochemiluminescence of Au@CN. These results demonstrate that the electrochemiluminescence sensor has been successfully fabricated.

[0065] Electrochemical impedance spectroscopy as shown Figure 6 As shown:

[0066] (1) The bare glassy carbon electrode exhibits a small semi-circular region ( Figure 6 (Curve a) This is due to the transfer of free electrons. After modifying the electrode with Au@CN, a smaller semi-circular region was obtained compared to the electrode itself. Figure 6 Curve b) shows that gold doping can effectively promote electron transfer;

[0067] (2) Concanavalin A, bovine serum albumin, and NMS / CNT@Glu were sequentially assembled onto the modified electrode, with the semicircular region gradually increasing in size. Figure 6(Curves c, d, and e are shown, respectively). This may be due to the formation of a protein hysteresis layer, which significantly hinders the [Fe(CN)6] process. 3- / 4- The diffusion to the electrode surface forms an inert electron and mass transfer barrier layer, indicating that the electrochemiluminescence sensor has been successfully fabricated.

[0068] The application of the electrochemiluminescence sensor prepared by the method for preparing the electrochemiluminescence sensor for detecting concanavalin A includes the following steps:

[0069] (1) Using the Ag / AgCl electrode as the reference electrode, the platinum wire electrode as the counter electrode, and the prepared electrochemiluminescence sensor as the working electrode, the electrochemical workstation and the chemiluminescence detector are connected in the dark box of the chemiluminescence detector.

[0070] (2) Using 10 mL of phosphate buffer solution containing 0.1 mol / L K2S2O8 as a co-reactant, the intensity of the electrochemiluminescence signal generated by different concentrations of concanavalin A was detected by electrochemiluminescence method.

[0071] (3) Optimization of measurement conditions:

[0072] By controlling a single variable, the pH value, Au@CN concentration, and NMS / CNT@Glu concentration of the phosphate buffer solution containing 0.1 mol / L K2S2O8 were optimized through experiments. The optimal pH value, Au@CN concentration, and NMS / CNT@Glu concentration of the phosphate buffer solution containing 0.1 mol / L K2S2O8 were obtained, which are the best experimental conditions.

[0073] like Figure 7 As shown:

[0074] (1) The electrochemiluminescence intensity reaches its maximum value when the pH is 7.0. This is because a highly acidic or alkaline environment can damage immobilized proteins. Therefore, pH 7.0 was chosen for the following experiment.

[0075] (2) The effect of Au@CN solution concentration was also studied: Figure 8 The results showed that the electrochemiluminescence intensity increased in the range of 0.1–1 mg / mL as the concentration of Au@CN solution increased; when the concentration was further increased, the electrochemiluminescence intensity decreased, which may be attributed to the self-absorption effect of the luminescent material. Therefore, 1 mg / mL Au@CN was selected for the following experiments.

[0076] (3) Figure 9As shown, the electrochemiluminescence intensity increased with increasing NMS / CNT@Glu solution concentration from 0.1 mg / mL to 0.5 mg / mL, while the electrochemiluminescence intensity decreased with further increasing NMS / CNT@Glu solution concentration. Therefore, 0.5 mg / mL NMS / CNT@Glu solution was used for the following experiments.

[0077] In summary, the optimal experimental conditions are: the optimal pH value of the phosphate buffer solution containing 0.1 mol / L K2S2O8 is 7.0, the concentration of NMS / CNT@Glu solution is 0.5 mg / mL, and the concentration of Au@CN solution is 1 mg / mL.

[0078] (4) Plotting working curves:

[0079] A series of standard concentrations of concanavalin A solutions were prepared. Under optimal experimental conditions, the electrochemiluminescence intensity of the series of standard concentrations of concanavalin A was measured. The working curve was plotted with the logarithm of the concanavalin A concentration as the x-axis and the electrochemiluminescence intensity after loading NMS / CNT@Glu as the y-axis.

[0080] Under optimal experimental conditions, the electrochemiluminescent response of this sensor to different concentrations of concanavalin A was shown in... Figure 10 In the study, the electrochemiluminescence intensity showed a linear relationship with the concentration of concanavalin A in the range of 0.005 ng / mL to 100 ng / mL. Figure 11 The linear equation is y = 1095.72 - 348.35lgc, R = 0.981, and the detection limit is 0.005 ng / mL, proving that the stability of the sensor is acceptable. Figure 12 The sensor exhibits high sensitivity and a wide linear range.

[0081] (5) Anti-interference performance study:

[0082] The interference of ascorbic acid, uric acid, glucose, and bovine serum albumin on the detection of concanavalin A was investigated. Under optimal experimental conditions, the electrochemiluminescence intensity of the concanavalin A standard solution in the presence of different interfering substances was measured to study the anti-interference ability of the sensor.

[0083] The results showed that the current change was almost unchanged under the same concentrations of ascorbic acid, uric acid, glucose, bovine serum albumin and concanavalin A, which means that the sensor can be used to directly measure concanavalin A in human serum samples.

[0084] (6) Actual sample testing:

[0085] Under optimal experimental conditions, the content of concanavalin A in human serum samples was detected, and a spiked recovery experiment was performed. First, different concentrations of concanavalin A were added to human serum samples, with concentrations of 1.0, 5.0, and 10.0 ng / mL, respectively. Then, before analysis, 100 μL of human serum was diluted 10 times with 0.01 mol / L phosphate buffer and then modified onto the electrode for testing.

[0086] The biosensor was tested for practical analytical applications using the standard addition method: Different concentrations of concanavalin A (1.0, 5.0, and 10.0 ng / mL) were added to human serum samples. The recoveries ranged from 98.6% to 101.0%, and the relative standard deviations ranged from 0.99% to 5.26%, as shown in Table 1.

[0087] The recovery rates of the prepared biosensors are shown in Table 1.

[0088]

[0089]

[0090] The sensor has proven effective in identifying concanavalin A in human serum samples, demonstrating its potential for widespread application.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for fabricating an electrochemiluminescence sensor based on functionalized carbon nanotube composite materials, characterized in that: The preparation method of the functionalized carbon nanotube composite material includes the following steps: Step 1) Preparation of glucose-functionalized carbon nanotube-supported nickel sulfide and molybdenum disulfide nanosheet composite material: Acidified multi-walled carbon nanotubes were ultrasonically treated for 40 minutes and then dispersed in a 0.05 mol / L glucose aqueous solution to obtain a mixed solution; then, nickel sulfate, ammonium molybdate and thiourea were added to the above mixed solution and stirred for 1 hour. The solution was then transferred to a polytetrafluoroethylene-lined autoclave and kept in a 200ºC forced-air drying oven for 24 hours. The resulting mixture was thoroughly washed three times by centrifugation with distilled water and ethanol. Step 2) Preparation of glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material modified by bovine serum albumin: 2 mg of glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material was dispersed in 1 mL of pH 7.4 phosphate buffer solution, then 100 μL of 1% bovine serum albumin solution was added and shaken for 2 hours to block non-specific adsorption sites. Finally, the solid was collected by centrifugation to obtain the glucose-functionalized carbon nanotube-loaded nickel sulfide / molybdenum disulfide nanosheet composite material modified by bovine serum albumin, i.e., NMS / CNT@Glu. It was then redispersed in 1 mL of pH 7.4 phosphate buffer solution to obtain NMS / CNT@Glu solution for later use. When not in use, it was stored at 4°C. The method for preparing the electrochemiluminescence sensor includes the following steps: Step 1: First, polish the bare glassy carbon electrode repeatedly with alumina powder, then clean it thoroughly before use, and coat the electrode with 5 μL of 1 mg / mL gold-doped carbon nitride nanosheet Au@CN solution. Step 2: After the electrode is dry, add 5 μL of concanavalin A solution. After 1 hour, wash the electrode with phosphate solution to block the non-specific binding between concanavalin A and the electrode surface. Step 3: Coat the electrode surface with 5 μL of 1 mg / mL NMS / CNT@Glu solution. After 1 hour, rinse the obtained electrode to remove unbound NMS / CNT@Glu from the outer surface, and obtain an electrochemiluminescent sensor for detecting concanavalin A.

2. The method for preparing the electrochemiluminescence sensor according to claim 1, characterized in that: In step (1), the amount of multi-walled carbon nanotubes added is 25-35 mg, the amount of glucose aqueous solution added is 36-45 mL, the amount of nickel sulfate added is 0.18-0.21 g, the amount of ammonium molybdate added is 0.14-0.18 g, and the amount of thiourea added is 0.3-0.4 g.

3. The method for preparing the electrochemiluminescence sensor according to claim 1, characterized in that: In step 1, the preparation method of the gold-doped carbon nitride nanosheet Au@CN solution includes the following steps: Step (1), Preparation of carbon nitride nanosheets: First, place 3-5g of melamine in a crucible and heat it at a rate of 2-5℃ / min, and heat it at 550℃ for 4 hours to prepare loose carbon nitride lumps. After grinding the resulting yellow lumps into powder, put a certain amount of carbon nitride lumps into a porcelain crucible, heat it at a rate of 5℃ / min and keep it at 550℃ for 4 hours to obtain carbon nitride nanosheets; Step (2), Preparation of gold-doped carbon nitride nanosheets Au@CN solution: First, the carbon nitride nanosheets from step 1 were ultrasonically dispersed into a carbon nitride aqueous solution. 1 mL of 0.5 mmol / L HAuCl4 aqueous solution was added to 10 mL of 2.0 mg / mL carbon nitride aqueous solution and stirred vigorously for 2 hours. Then, 1 mL of freshly prepared 0.01 mol / L NaBH4 solution was added dropwise to the mixture to reduce trivalent gold ions. The resulting suspension was centrifuged at 10000 r / min to remove unreacted / unbound substances, including single gold nanoparticles. Finally, the obtained gold-doped carbon nitride nanosheets Au@CN were washed with distilled water and redispersed in 10 mL of distilled water to obtain the Au@CN solution for later use.

4. An application of an electrochemiluminescence sensor obtained based on the preparation method of the electrochemiluminescence sensor according to claim 1, characterized in that, Includes the following steps: (1) Using the Ag / AgCl electrode as the reference electrode, the platinum wire electrode as the counter electrode, and the prepared electrochemiluminescence sensor as the working electrode, the electrochemical workstation and the chemiluminescence detector are connected in the dark box of the chemiluminescence detector. (2) Using 10 mL of phosphate buffer solution containing 0.1 mol / L K2S2O8 as a co-reactant, the intensity of the electrochemiluminescence signal generated by different concentrations of concanavalin A was detected by electrochemiluminescence method. (3) Optimization of measurement conditions: By controlling a single variable, the pH value of the phosphate buffer solution containing 0.1 mol / L K2S2O8, the concentration of Au@CN solution, and the concentration of NMS / CNT@Glu solution were optimized through experiments to obtain the optimal pH value, Au@CN concentration, and NMS / CNT@Glu concentration, which are the best experimental conditions. (4) Plotting working curves: A series of standard concentrations of concanavalin A solutions were prepared. Under optimal experimental conditions, the electrochemiluminescence intensity of the series of standard concentrations of concanavalin A was measured. The working curve was plotted with the logarithm of the concanavalin A concentration as the x-axis and the electrochemiluminescence intensity after loading NMS / CNT@Glu as the y-axis. (5) Anti-interference performance study: The interference of ascorbic acid, uric acid, glucose, and bovine serum albumin on the detection of concanavalin A was investigated. Under optimal experimental conditions, the electrochemiluminescence intensity of the concanavalin A standard solution in the presence of different interfering substances was measured to study the anti-interference ability of the sensor. (6) Actual sample testing: Under optimal experimental conditions, the content of concanavalin A in human serum samples was detected, and a spiked recovery experiment was performed.

5. The application of the electrochemiluminescence sensor according to claim 4, characterized in that: The optimal experimental conditions were: a phosphate buffer solution containing 0.1 mol / L K2S2O8 with a pH of 7.0, an NMS / CNT@Glu solution concentration of 0.5 mg / mL, and an Au@CN solution concentration of 1 mg / mL.

6. The application of the electrochemiluminescence sensor according to claim 4, characterized in that: The phosphate buffer solution was prepared from 0.1 mol / L disodium hydrogen phosphate and 0.1 mol / L potassium dihydrogen phosphate, and its pH was 7.

4.

7. The application of the electrochemiluminescence sensor according to claim 4, characterized in that: The parameters of the chemiluminescence detector are set as follows: the high voltage of the photomultiplier tube is set to 700-800V, and the scanning rate is set to 0.15V / s; the parameters of the electrochemical workstation are set as follows: the cyclic voltammetric scan potential range is -1.3V to 0V, and the scanning rate is set to 0.1V / s.

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