Nitrogen-doped quantum dot graphene composite sensor, its preparation method and application

Abstract

This invention relates to a nitrogen-doped quantum dot graphene composite sensor, comprising a substrate electrode and a nitrogen-doped quantum dot graphene composite material fixed on the surface of the substrate electrode. The nitrogen-doped quantum dot graphene composite material is prepared by the following method: carbon quantum dots and graphene oxide are mixed in a solvent, ultrasonically treated, and the resulting suspension is heated at 175-185°C for 13-14 hours, the solvent is removed, and the mixture is dried to obtain the composite material. The composite material is then mixed with a nitrogen source, heated in an inert gas atmosphere, cooled, washed, and dried to obtain the final product. This sensor exhibits a wide linear detection range for hydrogen peroxide, with an average response time of 5 s, and good anti-interference properties, making it directly applicable to the detection of hydrogen peroxide. A malic acid biosensor constructed based on this sensor using chitosan-loaded NADH oxidase and malic acid dehydrogenase can effectively respond to malic acid, with a linear detection range of 0.4 mM.

Description

Technical Field

[0001] This invention belongs to the technical field of biosensors, specifically relating to a sensor based on nitrogen-doped quantum dot graphene composite material, its preparation method, and its application. Background Technology

[0002] Hydrogen peroxide (H2O2) is a reactive oxygen species and a byproduct of cellular metabolism, playing a crucial role in physiological processes. It is widely used in the food, textile and paper industries, pharmaceutical formulation, clinical analysis, mining, and chemical industries. In biological systems, H2O2 is essential for intracellular signal transduction, cell proliferation, protein synthesis, and the control of biological activity. It is associated with cell damage, oxidative stress, diabetes, and cardiovascular diseases. Simultaneously, H2O2 is also an important electrochemical sensing active material, exhibiting electrochemical redox reaction capabilities at electrodes. Combined with oxidases, it can indirectly detect the concentration of responding substances. Therefore, accurate and reliable detection of H2O2 has become an important research topic.

[0003] Currently, methods for determining H2O2 include colorimetry, chemiluminescence, high-performance liquid chromatography (HPLC), fluorescence, and electrochemical sensors. Among these, electrochemical sensing strategies have received more attention due to their speed, accuracy, portability, cost-effectiveness, high sensitivity, and selectivity. Currently, non-enzymatic H2O2 sensors have gained more attention in practical applications than enzyme sensors because they offer rapid detection, high sensitivity, stability, and ease of operation. However, enzyme-based sensors are prone to denaturation, leading to loss of activity. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a sensor based on a nitrogen-doped quantum dot graphene composite material, an electrochemical sensing material capable of rapid response to hydrogen peroxide, and its preparation method. The sensor described in this invention can be directly used for the detection of hydrogen peroxide, laying a theoretical and applied foundation for the subsequent development of online detection devices for substances such as hydrogen peroxide.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0006] A nitrogen-doped quantum dot graphene composite sensor includes a substrate electrode and a nitrogen-doped quantum dot graphene composite material fixed to the surface of the substrate electrode.

[0007] The nitrogen-doped quantum dot graphene composite material was prepared by the following method:

[0008] (1) Carbon quantum dots (CQD) and graphene oxide (GO) are mixed in a solvent and ultrasonically treated to obtain a suspension;

[0009] (2) The suspension obtained in step (1) is heated at 175-185°C for 13-14 hours to remove the solvent and dry it to obtain the composite material;

[0010] (3) The composite material is mixed with a nitrogen source, heated in an inert gas, cooled, washed and dried to obtain the nitrogen-doped quantum dot graphene composite material.

[0011] Preferably, the fixing is performed using Nafion.

[0012] Preferably, the fixing method is as follows: the suspension of the nitrogen-doped quantum dot graphene composite material is mixed with Nafion solution, then drop-coated onto the surface of the substrate electrode, and dried to obtain the sensor.

[0013] Preferably, the substrate electrode is a glassy carbon electrode.

[0014] Preferably, the concentration of the nitrogen-doped quantum dot graphene composite material suspension is 1 to 1.2 mg / ml.

[0015] Preferably, the volume ratio of the nitrogen-doped quantum dot graphene composite material suspension to the Nafion solution is 1:1.

[0016] Preferably, the solvent for the suspension of the nitrogen-doped quantum dot graphene composite material is ethanol.

[0017] Preferably, the Nafion solution contains 0.5% Nafion by mass.

[0018] Preferably, the mass ratio of carbon quantum dots to graphene oxide in step (1) is 2:1.

[0019] Preferably, the solvent in step (1) is double-distilled water.

[0020] Preferably, the ultrasound time in step (1) is 1.5 to 2 hours.

[0021] Preferably, the heating described in step (2) is carried out in a polytetrafluoroethylene-lined autoclave.

[0022] Preferably, the solvent removal method in step (2) is centrifugation, wherein the centrifugation speed is 8000 rpm and the centrifugation time is 6 min.

[0023] Preferably, the mass ratio of the composite material to the nitrogen source in step (3) is 1:10.

[0024] Preferably, the nitrogen source in step (3) is thiourea.

[0025] Preferably, the heating temperature in step (3) is 800°C and the heating time is 40 min.

[0026] Preferably, the heating rate in step (3) to reach the heating temperature is 5°C / min. -1 .

[0027] Preferably, the flow rate of the inert gas in step (3) is 10-15 mL / min. -1 .

[0028] Preferably, the inert gas in step (3) is nitrogen.

[0029] The present invention also provides an application of the above-mentioned nitrogen-doped quantum dot graphene composite material sensor, including:

[0030] A three-electrode system was constructed using the nitrogen-doped quantum dot graphene composite sensor as the working electrode, and hydrogen peroxide was detected by chronoamperometry in PBS buffer.

[0031] This invention also provides another application of the above-mentioned nitrogen-doped quantum dot graphene composite material sensor, including:

[0032] (1) After mixing the enzyme solution of NADH oxidase, the enzyme solution of malate dehydrogenase and chitosan solution to obtain a mixture, the mixture is dropped onto the nitrogen-doped quantum dot graphene composite material sensor and dried to obtain a malate sensor.

[0033] (2) A three-electrode system was constructed using the malic acid sensor as the working electrode, containing flavin mononucleotide and NAD. + The malic acid sensor was detected using a chronoamperometric method in PBS buffer. The construction method of this malic acid sensor is the same as that in CN115308285 A, the only difference being the material used to modify the electrode.

[0034] Preferably, the chitosan solution contains 0.5-5% chitosan by mass; the chitosan solution and enzyme solution are mixed at a volume ratio of 1:0.8-1.2.

[0035] Preferably, the chitosan solution contains 1.25% chitosan by mass; the chitosan solution and enzyme solution are mixed at a volume ratio of 1:1.

[0036] Compared with existing technologies, the nitrogen-doped quantum dot graphene composite material prepared in this invention can respond to hydrogen peroxide, thus enabling the construction of a hydrogen peroxide biosensor. The sensor prepared using this method has a wide detection limit and a short response time, laying a theoretical and applied foundation for the subsequent development of online detection devices for hydrogen peroxide / malic acid, etc. Immobilization of NADH oxidase and malate dehydrogenase with chitosan also shows good performance, demonstrating superior capabilities for rapid and effective detection of malic acid. Attached Figure Description

[0037] Figure 1 This is a comparison of the CV curves of a blank glassy carbon electrode (empty GCE) and the nitrogen-doped quantum dot graphene composite sensor prepared in this invention, using 5.0 mM H2O2 in PBS (pH = 7.0).

[0038] Figure 2 This is an anti-interference test of the nitrogen-doped quantum dot graphene composite sensor prepared in Example 1.

[0039] Figure 3 The image shows the current response curves of the nitrogen-doped quantum dot graphene composite sensor prepared in Example 1 to different concentrations of hydrogen peroxide.

[0040] Figure 4 This is a standard curve of the nitrogen-doped quantum dot graphene composite sensor prepared in Example 1 responding to different concentrations of hydrogen peroxide-current.

[0041] Figure 5 The images show the current response curves of the corresponding malic acid sensing electrodes prepared in Example 2 to different concentrations of malic acid.

[0042] Figure 6 The standard curves of the current response of the corresponding malic acid sensing electrode prepared in Example 2 to different concentrations of malic acid are shown. Detailed Implementation

[0043] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention. All components not mentioned in this invention are commercially available.

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific examples, which will help to better understand the invention. The descriptions of the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims. The technical features of the technical solutions provided by the present invention are further clearly and completely described, but are not intended to limit the scope of protection thereof.

[0045] Example 1

[0046] (1) 20 mg of carbon quantum dots and 10 mg of graphene oxide were mixed in 15 mL of double-distilled water and sonicated for 2 h. The resulting suspension was then poured into a 20 mL polytetrafluoroethylene-lined autoclave and heated at 180 °C for 14 h. The resulting aqueous solution was centrifuged at 8000 rpm for 6 min to obtain a solid precipitate. Finally, the precipitate was dried in a vacuum oven at 60 °C for 14 h.

[0047] (2) Grind 20 mg of the powder obtained in step (1) with 200 mg of thiourea using a mortar and pestle until thoroughly mixed. Then, transfer the mixture to a quartz boat and heat it in a muffle furnace. The heating rate is 5 °C / min. -1 The temperature was raised to 800℃ and maintained at 800℃ for 40 minutes. N2 was introduced during heating at a flow rate of 10 mL / min. -1 The mixture was naturally cooled in N2. Then it was washed three times by centrifugation with water, and then dried in a vacuum oven at 60°C for 20 hours to obtain nitrogen-doped quantum dot graphene composite material.

[0048] (3) Dissolve 1 mg of the obtained nitrogen-doped quantum dot graphene composite material in 1 ml of ethanol to obtain a suspension of 1 mg / ml. Mix the suspension with 0.5% wt Nafion solution to obtain a mixed solution, then titrate the mixed solution onto the substrate electrode and dry it to obtain a nitrogen-doped quantum dot graphene composite material sensor. The suspension and 0.5% wt Nafion solution are mixed at a volume ratio of 1:1.

[0049] (4) An electrochemical workstation (model CHI650EA15655) from Shanghai Zhenhua was used, employing a three-electrode system for detection. The nitrogen-doped quantum dot graphene composite sensor was used as the working electrode, the Ag / AgCl electrode as the reference electrode, and a platinum wire as the counter electrode. The detection environment consisted of 40 mL of 50 mM PBS. Current changes were detected using the Chi650e software, and the chronoamperometric method was employed for testing.

[0050] (4-1) CV curve test:

[0051] First, the glassy carbon electrode was connected to the electrochemical workstation to scan the CV spectrum. 1 mol / L hydrogen peroxide was prepared using pure water as solvent and added to the reaction cell to prepare a 5 mmol / L hydrogen peroxide solution. Then, the hydrogen peroxide sensing electrode obtained in Example 2 was connected to the electrochemical workstation to scan the CV spectrum and observe the changes in voltage and current.

[0052] The obtained nitrogen-doped quantum dot graphene composite sensor exhibits a more significant redox current response to hydrogen peroxide solution compared to an empty GCE electrode, such as... Figure 1 As shown.

[0053] (4-2) Anti-interference test:

[0054] 1 mol / L solutions of hydrogen peroxide, ascorbic acid, uric acid, and glucose were prepared using pure water as the solvent. The resulting nitrogen-doped quantum dot graphene composite sensor was then connected to an electrochemical workstation to scan the IT graph. After the baseline stabilized, 8 μL of the prepared hydrogen peroxide solution was added to the reaction cell. Once the current response stabilized, the remaining solutions were added in 8 μL increments to the detection cell, reacting for 100 seconds, and the current changes were observed. Finally, another 8 μL of hydrogen peroxide solution was added to verify the repeatability of the response; the current changes after each solution addition were recorded.

[0055] The obtained nitrogen-doped quantum dot graphene composite sensor only shows a significant current response to hydrogen peroxide solution, proving that this hydrogen peroxide sensor only responds to hydrogen peroxide solution and has good anti-interference ability. Figure 2 As shown.

[0056] (4-3) Response time test:

[0057] A 1 mol / L hydrogen peroxide solution was prepared using pure water as a solvent. The resulting nitrogen-doped quantum dot graphene composite sensor was then connected to an electrochemical workstation to scan the IT graph. Once the baseline stabilized, 8 μL of the prepared hydrogen peroxide solution was added to the reaction cell. After the baseline stabilized, the response time was recorded.

[0058] Repeat the hydrogen peroxide addition step and calculate the average response time, such as... Figure 3 As shown, the average response time is 5 seconds. This proves that the sensor has the characteristics of short response time, fast speed, and high efficiency.

[0059] (4-4) Sensor linear range test:

[0060] A 1 mol / L hydrogen peroxide solution was prepared using pure water as the solvent. The resulting nitrogen-doped quantum dot graphene composite sensor was then connected to an electrochemical workstation to scan the IT graph. Once the baseline stabilized, 8 μL of the prepared hydrogen peroxide solution was added to the reaction cell and allowed to stabilize. This process of adding hydrogen peroxide was repeated several times, and the magnitude of the response current was calculated each time. The maximum limit of the same response value was identified, and the final concentration and linear range were calculated. Figure 4 As shown. The linear range is 0.1 mM to 1 mM, and the linear formula is y = 1.139x + 1.441, R. 2 =0.996.

[0061] Example 2

[0062] The nitrogen-doped quantum dot graphene composite sensor obtained in Example 1 was prepared according to the method for preparing the malic acid sensing electrode described in one of the patents CN115308285 A. The NADH oxidase enzyme solution, malic acid dehydrogenase enzyme solution, and chitosan solution were mixed to obtain a mixture. 6 μL of this mixture was then dropped onto the nitrogen-doped quantum dot graphene composite sensor and dried to obtain the malic acid sensing electrode. The NOX and MDH enzyme activity ratio in the mixture was configured as 1:1, the chitosan solution contained 1.25% chitosan by mass, and the chitosan solution and enzyme solution were mixed at a volume ratio of 1:1.

[0063] The electrochemical detection performance of the prepared malic acid sensing electrode was evaluated. A three-electrode system was used in the detection process with a Shanghai Zhenhua CHI650EA15655 electrochemical workstation. The malic acid sensing electrode was the working electrode, the Ag / AgCl electrode was the reference electrode, and the platinum wire was the counter electrode. The detection environment consisted of 40 mL of 50 mM PBS and 0.2 mM FAD and 2 mM NAD. + The test environment was used, and the current change was detected using the Chi650e software. The chronoamperometry method was employed, with a detection voltage of -0.05V.

[0064] (1) Sensor linear range test:

[0065] A 1 mol / L malic acid solution was prepared using pure water as the solvent. The resulting malic acid sensing electrode was then connected to an electrochemical workstation to scan the IT graph. After the baseline stabilized, 20 μL of the prepared malic acid solution was added to the reaction cell, and the process was repeated several times. The magnitude of the response current was calculated for each step. The induced current is as follows: Figure 5 As shown, the standard curve is as follows Figure 6 As shown, the linear formula is y = 0.566x + 0.294, R0 2 =0.99.

[0066] The results show that the malic acid sensor based on nitrogen-doped quantum dot graphene composite material exhibits a good response to malic acid. This sensor has promising practical applications in the field of electrochemical sensing.

[0067] Example 3

[0068] (1) 20 mg of carbon quantum dots and 10 mg of graphene oxide were mixed in 15 mL of double-distilled water and sonicated for 2 h. The resulting suspension was then poured into a 20 mL polytetrafluoroethylene-lined autoclave and heated at 185 °C for 13 h. The resulting aqueous solution was centrifuged at 8000 rpm for 6 min to obtain a solid precipitate. Finally, the precipitate was dried in a vacuum oven at 60 °C for 14 h.

[0069] (2) Grind 20 mg of the powder obtained in step (1) with 200 mg of thiourea using a mortar and pestle until thoroughly mixed. Then, transfer the mixture to a quartz boat and heat it in a muffle furnace. The heating rate is 5 °C / min. -1 The temperature was raised to 800℃ and maintained at 800℃ for 40 minutes. N2 was introduced during heating at a flow rate of 10 mL / min. -1 The mixture was naturally cooled in N2. Then it was washed three times by centrifugation with water, and then dried in a vacuum oven at 60°C for 20 hours to obtain nitrogen-doped quantum dot graphene composite material.

[0070] (3) Dissolve 1.2 mg of the obtained nitrogen-doped quantum dot graphene composite material in 1 ml of ethanol to obtain a suspension of 1 mg / ml. Mix the suspension with 0.5% wt Nafion solution to obtain a mixed solution, then titrate the mixed solution onto the substrate electrode and dry it to obtain a nitrogen-doped quantum dot graphene composite material sensor. The suspension and 0.5% wt Nafion solution are mixed at a volume ratio of 1:1.

[0071] The tested sensor has a linear range of 0.1mM to 1mM.

[0072] Example 4

[0073] (1) 20 mg of carbon quantum dots and 10 mg of graphene oxide were mixed in 15 mL of double-distilled water and sonicated for 1.5 h. The resulting suspension was then poured into a 20 mL polytetrafluoroethylene-lined autoclave and heated at 175 °C for 14 h. The resulting aqueous solution was centrifuged at 8000 rpm for 6 min to obtain a solid precipitate. Finally, the precipitate was dried in a vacuum oven at 60 °C for 14 h.

[0074] (2) Grind 20 mg of the powder obtained in step (1) with 200 mg of thiourea using a mortar and pestle until thoroughly mixed. Then, transfer the mixture to a quartz boat and heat it in a muffle furnace. The heating rate is 5 °C / min. -1The temperature was raised to 800℃ and maintained at 800℃ for 40 minutes. N2 was introduced during heating at a flow rate of 15 mL / min. -1 The mixture was naturally cooled in N2. Then it was washed three times by centrifugation with water, and then dried in a vacuum oven at 60°C for 20 hours to obtain nitrogen-doped quantum dot graphene composite material.

[0075] (3) Dissolve 1 mg of the obtained nitrogen-doped quantum dot graphene composite material in 1 ml of ethanol to obtain a suspension of 1 mg / ml. Mix the suspension with 0.5% wt Nafion solution to obtain a mixed solution, then titrate the mixed solution onto the substrate electrode and dry it to obtain a nitrogen-doped quantum dot graphene composite material sensor. The suspension and 0.5% wt Nafion solution are mixed at a volume ratio of 1:1.

[0076] The tested sensor has a linear range of 0.1mM to 1mM.

[0077] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are merely illustrative of selected implementations based on combinations of all possible embodiments. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. An application of a nitrogen-doped quantum dot graphene composite material sensor, characterized in that, A three-electrode system was constructed using the nitrogen-doped quantum dot graphene composite sensor as the working electrode, and hydrogen peroxide was detected by chronoamperometry in PBS buffer. The nitrogen-doped quantum dot graphene composite material sensor includes a substrate electrode and a nitrogen-doped quantum dot graphene composite material fixed to the surface of the substrate electrode. The nitrogen-doped quantum dot graphene composite material was prepared by the following method: (1) Carbon quantum dots and graphene oxide are mixed in a solvent and ultrasonically treated to obtain a suspension; the mass ratio of carbon quantum dots to graphene oxide is 2:

1. (2) The suspension obtained in step (1) is heated at 175~185℃ for 13~14h to remove the solvent and dry to obtain the composite material; (3) The composite material is mixed with a nitrogen source, heated in an inert gas, cooled, washed and dried to obtain the nitrogen-doped quantum dot graphene composite material.

2. The application of the nitrogen-doped quantum dot graphene composite material sensor according to claim 1, characterized in that, The fixing method is to use Nafion for fixing.

3. The application of the nitrogen-doped quantum dot graphene composite material sensor according to claim 2, characterized in that, The fixation method is as follows: a suspension of the nitrogen-doped quantum dot graphene composite material is mixed with Nafion solution, then drop-coated onto the surface of the substrate electrode, and dried to obtain the sensor.

4. The application of the nitrogen-doped quantum dot graphene composite material sensor according to claim 1, characterized in that, The ultrasound time in step (1) is 1.5~2 hours.

5. The application of the nitrogen-doped quantum dot graphene composite material sensor according to claim 1, characterized in that, The mass ratio of the composite material to the nitrogen source in step (3) is 1:

10.

6. The application of the nitrogen-doped quantum dot graphene composite material sensor according to claim 1, characterized in that, The nitrogen source mentioned in step (3) is thiourea.

7. The application of the nitrogen-doped quantum dot graphene composite material sensor according to claim 1, characterized in that, The heating temperature in step (3) is 800℃ and the heating time is 40min.

8. The application of the nitrogen-doped quantum dot graphene composite material sensor according to claim 1, characterized in that, The heating rate to reach the heating temperature in step (3) is 5°C / min.

9. The application of the nitrogen-doped quantum dot graphene composite material sensor according to claim 1, characterized in that, The applications include: After mixing NADH oxidase solution, malate dehydrogenase solution and chitosan solution to obtain a mixture, the mixture is dropped onto the nitrogen-doped quantum dot graphene composite sensor and dried to obtain a malate sensor. A three-electrode system was constructed using the malic acid sensor as the working electrode, containing flavin mononucleotide and NAD. + The PBS buffer solution was used for detection using a chronoamperometric method.

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