Oxidase-peroxidase cascade system as well as construction method and application thereof

Abstract

The invention discloses a construction method and application of an oxidase-peroxidase cascade system. The system comprises a nitrogen-doped carbon nanocage NC with oxidase activity and CuO with peroxidase activity. According to the invention, a nitrogen-doped carbon nanocage with oxidase activity and copper oxide with peroxidase activity are used for constructing an oxidase-peroxidase cascade system; when a substrate ascorbic acid exists in the system, a color developing agent terephthalic acid can be converted into o-hydroxyterephthalic acid through a series of reactions to achieve output of a fluorescence signal, the fluorescence excitation wavelength is 315 nm, the emission wavelength is 423 nm, the cascade system can successfully achieve detection of ascorbic acid, and the linear range is 0.1-50 [mu] M; furthermore, the system is applied to a micro-fluidic chip, higher-sensitivity detection of ascorbic acid is achieved, and the detection method has the advantages of being high in specificity, high in sensitivity and the like.

Description

Technical Field

[0001] The invention belongs to the field of nanomaterial enzyme simulation, and particularly relates to an oxidase-peroxidase cascade system, a construction method thereof and applications thereof. Background Art

[0002] Ascorbic acid (AA), also known as vitamin C or L-ascorbic acid, is a naturally occurring compound that is widely found in fruits and vegetables. In addition, ascorbic acid can also be used as an antioxidant and anti-browning agent, and is often used as an additive in processed fruits and vegetables. As one of the most common small biological molecules in human blood, ascorbic acid is of great significance to the normal functioning of human physiological functions. Clinical studies have shown that ascorbic acid is not only closely related to human metabolism, but is also associated with mental illness and neurodegenerative diseases related to oxidative stress. Therefore, ascorbic acid concentration is also used as one of the indicators for disease detection. Common ascorbic acid methods mainly include electrophoresis, chemiluminescence, titration, liquid chromatography, etc.

[0003] Enzymes, fundamental biocatalysts of life, are mostly proteins composed of unique amino acid sequences. However, due to the sensitivity of protein structure, enzyme activity is easily affected by pH and temperature. With the development and integration of materials science and biology, a large number of novel nanomaterials with enzymatic activity have emerged. These emerging nanomaterials are commonly referred to as "nanozymes." Compared to natural enzymes, nanozymes offer advantages such as high stability, ease of mass production, and low cost, and hold great potential in areas such as disease treatment, biosensing, and drug interactions. Since the discovery of peroxidase activity in iron oxide (Fe3O4) magnetic nanoparticles, numerous new materials with intrinsic nanozyme activity have been discovered. In the future, nanozymes are expected to become alternatives to traditional enzymes.

[0004] In organisms, natural enzymes work together, and the product of an enzyme-catalyzed reaction is consumed by another enzyme in a cascade manner. This reaction is called a cascade reaction. In a cascade reaction, a single substrate is converted into a single product through one or more intermediates in the process. This cascade process not only helps to save time and reduce waste during the reaction, but also improves the efficiency of the reaction. More importantly, the cascade reaction can achieve selective detection of substrates by gradually screening substrates. At the same time, carrying out cascade reactions in a confined space can prevent intermediates from entering the environmental medium through ineffective diffusion, causing loss and conversion, and can also promote the transfer of substances. In addition, the flow reactor will greatly eliminate possible interference between the substrate and the nanozyme.

[0005] When detecting ascorbic acid in real samples, it may be interfered with by various other substances, such as amino acids, which may cause false positives. Therefore, it is necessary to design a detection system with good selectivity for ascorbic acid detection. Compared with other interfering substances, it only responds to ascorbic acid, thereby improving the selectivity and accuracy of the detection. Summary of the Invention

[0006] Purpose of the invention: To address the problems existing in the prior art, the present invention provides an oxidase-peroxidase cascade system. The cascade reaction system of NC and CuO constructed by the present invention, in which NC catalyzes AA to produce H2O2, forms a cascade system with CuO for fluorescence detection, achieving the goal of highly selective detection of ascorbic acid with significant results.

[0007] The present invention also provides a method for constructing an oxidase-peroxidase cascade system and the application of the cascade reaction construction in an open system and a microfluidic device to realize the detection of AA.

[0008] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides an oxidase-peroxidase cascade system, which includes nitrogen-doped carbon nanocages NC with oxidase activity and CuO with peroxidase activity.

[0009] The nitrogen-doped carbon nanocage with oxidase activity is obtained by calcining pyridine as a precursor at high temperature using a magnesium oxide template method.

[0010] The method for constructing the oxidase-peroxidase cascade system of the present invention comprises the following steps:

[0011] (1) Weighing basic magnesium carbonate and placing it in a reaction vessel, evacuating it to a vacuum and then heating it under an inert gas atmosphere; adding a precursor pyridine and reacting it at a high temperature; after the reaction is completed, placing the product in a concentrated acid solution, stirring it, filtering it, washing it, and drying it to obtain nitrogen-doped carbon nanocages NC with oxidase activity;

[0012] (2) Add acetic acid and deionized water to copper acetate, stir and heat until boiling, then pour concentrated sodium hydroxide solution into the solution. Stop the reaction immediately after a black product is generated. After cooling, centrifuge, wash, and dry to obtain CuO with peroxidase activity:

[0013] (3) The NC in step (1), the CuO in step (2), and the color developing agent terephthalic acid are used to form an oxidase-peroxidase cascade system.

[0014] In step (1), basic magnesium carbonate was weighed and placed in a reaction vessel. After evacuation, a tube furnace was set up under an inert gas atmosphere and the temperature was increased to 800°C at a rate of 10°C / min; then the precursor pyridine was added through a syringe pump at a rate of 1 mL·30 min. -1 After 30 minutes of reaction at high temperature, the injection pump was turned off, the heating was stopped, and the inert gas was no longer introduced after the temperature of the tube furnace dropped. The product was placed in a concentrated hydrochloric acid solution (6M) and stirred for 40-50 hours, then filtered, washed, and dried.

[0015] Application of the oxidase-peroxidase cascade system of the present invention in ascorbic acid detection.

[0016] NC was added to an ascorbic acid solution, incubated for a period of time, and then centrifuged to obtain a supernatant. TA and CuO were dropped into the supernatant after centrifugation and continued to be incubated. Finally, the obtained reaction solution was characterized by a fluorescence spectrophotometer.

[0017] The incubation time for the first step is 3-10 minutes, and the incubation time for the second step is 20-30 minutes; the incubation temperature is 35-65°C.

[0018] Preferably, the first step incubation time is 3 minutes, the second step incubation time is 25 minutes, and the incubation temperature is 45°C.

[0019] Preferably, 10 μL (2.5 mg / mL) of NC is added to 1 mL of ascorbic acid solution (PBS buffer containing 50 μM AA, pH = 6, 0.2 M) and incubated for a period of time. After centrifugation to obtain the supernatant, 25 μL of 120 mM TA and 10 μL of 5 mg / mL CuO are added dropwise to the obtained supernatant. Then, the above solution is incubated for a long time. Finally, the obtained reaction solution is characterized by a fluorescence spectrophotometer; or 10 μL (2.5 mg / mL) of NC is added to ascorbic acid PBS buffer with different concentrations and incubated for a period of time. After centrifugation, a certain amount of TA and 10 μL of 5 mg / mL CuO are added dropwise to the obtained supernatant. Then, the above solution is incubated for a long time. Finally, the obtained reaction solution is characterized by a fluorescence spectrophotometer.

[0020] Application of the oxidase-peroxidase cascade system of the present invention in the preparation of ascorbic acid detection tools or reagents.

[0021] The oxidase-peroxidase cascade system of the present invention is applied to the detection of ascorbic acid in a microfluidic system.

[0022] Among them, all applications include:

[0023] (1) Pour Dow Corning 184 polydimethylsiloxane (containing the basic components and the curing agent, mixed in a weight ratio of 10:1) into the template silicon wafer;

[0024] (2) After curing at 80°C, the chip containing the dual channels (the first channel and the second channel) was peeled off from the template and holes were punched at the inlet and outlet of the channels using a punch. All channels were approximately 70 μm deep, 40 mm long, and 4 mm wide.

[0025] (3) NC and CuO nanoparticles were applied to the channel respectively and then covered with smooth glass;

[0026] (4) Connect each inlet of the microfluidic chip to the syringe pump using polypropylene tubing;

[0027] (5) pumping ascorbic acid solution into the first channel to react;

[0028] (6) Pump the reaction solution into the second reaction channel and simultaneously pump in TA to continue the reaction;

[0029] (7) Detecting the collected reaction solution and detecting its fluorescence signal.

[0030] Among them, in step (3), a glass slide needs to be covered and solidified, and the PDMS and the glass slide need to be bonded.

[0031] Preferably, after reacting at a certain temperature for a period of time in steps (5) and (6), the reaction solution is collected and the fluorescence signal is detected. Compared with the cascade reaction detection of AA in an open system, this method can detect ascorbic acid at a lower concentration and has higher sensitivity.

[0032] The present invention provides a method for constructing and applying an oxidase-peroxidase cascade system, specifically, a method for highly selectively detecting ascorbic acid using a cascaded NC oxidase and CuO peroxidase. Experimental results demonstrate that NC oxidase catalyzes the dehydrogenation of AA to produce H2O2. Under CuO catalysis, hydroxyl radicals (·OH) are generated, which react with TA to form TAOH, exhibiting a fluorescence absorption peak at approximately 423 nm. This invention provides a new solution for highly selective and sensitive ascorbic acid detection.

[0033] Preferably, an oxidase-peroxidase cascade system is used to construct and detect AA, comprising the following steps:

[0034] (1) Magnesium carbonate was weighed and placed in a reaction vessel. After evacuation, a tube furnace was set up under an inert gas atmosphere and the temperature was increased to 800°C at a rate of 10°C / min. Then, the precursor pyridine was added through a syringe pump at a rate of 1 mL / 30 min. -1After 30 minutes of reaction at high temperature, the injection pump was turned off, the heating was stopped, and the inert gas was no longer introduced after the temperature of the tube furnace dropped. The product was placed in a concentrated hydrochloric acid solution (6M) and stirred for 40-50 hours, then filtered, washed, and dried.

[0035] (2) Add 150 mL of 0.02 M Cu(Ac)2 and 0.5 mL of HAc to a flask. Heat and stir until boiling. Quickly add 10 mL of 1 M NaOH to terminate the reaction. Centrifuge, wash three times with ethanol, and dry overnight.

[0036] (3) Use NC and CuO to construct a cascade reaction, add 10uL NC (1mg / mL) into the centrifuge tube -1 ) and a PBS buffer (pH 6) containing a specific concentration of AA (50 μM), followed by incubation at a specific temperature for 3-10 minutes. After centrifugation, the liquid was collected and 25 μL of TA (120 mM) and 10 μL of CuO (5 mg / mL) were added to the system. The mixed reaction solution was incubated at a specific temperature for another 20-30 minutes, after which the fluorescence signal of the reaction solution was measured using a fluorescence spectrometer.

[0037] The present invention is based on the application of the above-mentioned method for constructing an oxidase-peroxidase cascade system, transferring the system to a microfluidic device to further improve the reaction sensitivity. The present invention comprises the following steps:

[0038] Step 1: Coating the corresponding NC and CuO nanozymes on PDMS respectively;

[0039] Step 2: Covering the PDMS with a glass slide by bonding;

[0040] Step 3: Pump PBS buffer containing a certain concentration of ascorbic acid into the first channel of the microfluidic chip and react at a certain temperature;

[0041] Step 4: Pump the reaction solution into the second channel and simultaneously pump the TA into the second reaction channel. Continue the reaction at a certain temperature, then pump the liquid out of the chip and collect it for fluorescence signal detection.

[0042] Application of the oxidase-peroxidase cascade system combined with a microfluidic system in the preparation of a kit or tool for detecting ascorbic acid.

[0043] The catalytic mechanism of the above-mentioned method for constructing an oxidase-peroxidase cascade system was demonstrated using colorimetry and triethanolamine. The specific mechanism is as follows:

[0044] When ascorbic acid is present in the system, NC, due to its oxidase properties, catalyzes the oxidation of ascorbic acid to produce dehydroascorbic acid, while also producing a certain amount of hydrogen peroxide (H2O2). The generated H2O2 can serve as the substrate for the second step of the cascade reaction. Due to the peroxidase activity of CuO, it can catalyze the decomposition of H2O2, generating hydroxyl radicals (·OH). ·OH can then bind to TA, producing o-hydroxyterephthalic acid (TAOH), which then displays a fluorescent signal at around 423 nm, achieving the goal of AA detection.

[0045] The design principle of the present invention is as follows: Nanozymes are widely used due to their advantages of low cost, easy mass synthesis, and convenient storage. However, compared with natural enzymes, nanozymes do not have superior selectivity. In response to this situation, the present invention proposes to achieve highly selective detection of substrates by constructing a cascade reaction and gradually screening the substrates. Specifically: The present invention selects NC with oxidase properties and CuO with peroxidase properties to construct a cascade reaction in series. Figure 1 As shown, the oxidase properties of NC were tested by cascading with HRP and TMB. UV detection results showed that, compared to the NC-HRP-TMB system without AA, TMB did not directly appear blue, and no clear peak was observed around 652 nm. However, when AA was added to the system, TMB was clearly oxidized, producing blue TMBox with a distinct peak at 652 nm. This experiment demonstrated that NC was capable of oxidizing AA, enabling the subsequent cascade reaction. Next, the CuO-TA and CuO-TA-H2O2 systems were compared, and fluorescence signals were measured after each reaction under identical conditions. The results showed that TAOH was only produced when CuO, TA, and H2O2 were present in the system, with a fluorescence signal around 423 nm. No TAOH was observed in the CuO-TA system, confirming the peroxidase properties of CuO.

[0046] This invention proposes a method for constructing an oxidase-peroxidase cascade system and applying it to the highly selective detection of AA. In the present invention, NC can catalyze the reduction of O2 to H2O2 during the ascorbic acid oxidation process. In the subsequent reaction, H2O2 can be further catalyzed to produce ·OH, which combines with TA to produce TAOH with a fluorescent signal, thereby achieving highly selective detection of AA. Subsequently, by further combining this fluorescence detection method with a microfluidic device, the sensing sensitivity is further improved. This invention provides a method for improving the selectivity of nanozymes and enhancing the detection and sensing sensitivity.

[0047] The present invention uses nitrogen-doped carbon nanocages (NCs) with oxidase activity and copper oxide (CuO) with peroxidase activity to construct an oxidase-peroxidase cascade system. When the substrate ascorbic acid (AA) is present in the system, a series of reactions can convert the developer terephthalic acid (TA) into o-hydroxyterephthalic acid (TAOH) to achieve the output of a fluorescent signal. The fluorescence excitation wavelength is 315 nm, and the emission wavelength is approximately 423 nm. The cascade system can successfully detect ascorbic acid with good linearity. The system is further applied to a microfluidic chip to achieve detection in a lower concentration range. A more sensitive detection of ascorbic acid is achieved. The detection method of the present invention has the advantages of strong specificity and high sensitivity.

[0048] The present invention innovatively develops a cascade system that combines specific nitrogen-doped carbon nanocages (NCs) with oxidase activity and CuO with peroxidase activity. This system has excellent selectivity and sensitivity in ascorbic acid detection, with a high signal response only to ascorbic acid.

[0049] In this study, individual nanozyme reactions were cascaded, with the higher-priority product from reaction one serving as the substrate for reaction two. Experiments revealed that this cascade system exhibited excellent selectivity, with a strong response to ascorbic acid compared to several other similar substrates.

[0050] By cascading different types of nanozymes, the present invention enables selective detection of a substance, providing a selective detection strategy that addresses the problem of low nanozyme selectivity. Using an oxidase-peroxidase combination as an example, the present invention constructs a highly selective cascade system for ascorbic acid detection, and based on a fluorescence sensing method, effectively improves the sensitivity of the reaction.

[0051] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0052] (1) The present invention proposes a system and method for highly selective detection of substrates by constructing a cascade reaction, which has the advantage of being able to detect ascorbic acid with high selectivity. Compared with other substances (amino acids, urea, uric acid, etc.) as substrates, when AA is present in the system, the signal intensity is significantly superior to that of other interfering substances. Compared with other methods for achieving selective detection, achieving selective detection by constructing a cascade reaction is more convenient and more versatile.

[0053] (2) The present invention applies the proposed cascade reaction system to a microfluidic chip, which facilitates the transfer of substances through the confined space within the microfluidic chip. Furthermore, the flow reactor significantly eliminates any interference between the substrate and the nanozyme, further improving sensing sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is the UV detection image of NC oxidase activity;

[0055] Figure 2 The fluorescence detection diagram before and after adding a certain concentration of AA to the NC and CuO cascade system;

[0056] Figure 3 Fluorescence detection diagram of adding a certain concentration of AA to the NC and CuO cascade system under different temperature conditions

[0057] Figure 4 Fluorescence detection diagram of the NC and CuO cascade system with a certain concentration of AA added under different pH conditions;

[0058] Figure 5 Fluorescence detection diagram of the NC and CuO cascade system with the addition of a certain concentration of AA for different reaction times;

[0059] Figure 6 Fluorescence detection diagram of the NC and CuO cascade system with a certain concentration of AA and different concentrations of TA added;

[0060] Figure 7 The linear graph of the fluorescence intensity of TAOH at 423 nm and different concentrations of AA in an open system;

[0061] Figure 8 The linear graph of the fluorescence intensity of TAOH at 423 nm and different concentrations of AA in the microfluidic system;

[0062] Figure 9 Comparison of fluorescence signals when adding different reaction substrates at the same concentration for the NC and CuO cascade system;

[0063] Figure 10 A comparison chart of the AA detection effects of different cascade systems;

[0064] Figure 11 This is a rendering of the microfluidic device. DETAILED DESCRIPTION

[0065] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0066] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0067] The NC preparation method used in the embodiment of the present invention can be based on the literature (Zhao J, Lai H, Lyu Z, et al. Hydrophiliic Hierarchical Nitrogen-Doped Carbon Nanocages for Ultrahigh Supercapacitive Performance [J]. Advanced Materials, 2015, 27: 3541-3545.).

[0068] The preparation method of CuO can be referred to the literature (Zhu J, Li D, Chen H, et al. Highly dispersed CuO nanoparticles prepared by a novel quick-precipitation method [J]. Materials Letters, 2004, 58: 3324-3327.).

[0069] The structure and preparation method of the microfluidic chip control device can be according to the literature (Zhou, Q.; Yang, H.; Chen, X.; Xu, Y.; Han, D.; Zhou, S.; Liu, S.; Shen, Y.; Zhang, Y., Cascaded Nanozyme System with High Reaction Selectivity by Substrate Screening and Channeling in a Microfluidic Device. Angew. Chem., Int. Ed. 2022, 61, e202112453.).

[0070] Specifically, the preparation of NC and CuO is as follows:

[0071] (1) Weigh 4 g of basic magnesium carbonate and place it in a reaction vessel. After evacuating to vacuum, set a tube furnace under an inert gas argon atmosphere and heat it to 800 °C at a rate of 10 °C / min; then add 1 mL of precursor pyridine through a syringe pump and heat it at a rate of 1 mL·30 min using a syringe pump. -1 The material was fed at a rate of 100 μg / min and reacted at a high temperature. After 30 min, the injection pump was turned off and the heating was stopped. After the temperature of the tube furnace dropped, the inert gas was no longer introduced. The product was placed in a concentrated hydrochloric acid solution (6 M) and stirred for 45 h, then filtered, washed, and dried to obtain NC, which was dispersed in deionized water for later use.

[0072] (2) Add 150 mL of 0.02 M Cu(Ac)2 and 0.5 mL of HAc to a flask, heat and stir until boiling, quickly pour in 10 mL of 1 M NaOH, and terminate the reaction after obtaining a black product. Centrifuge and wash three times with ethanol, dry overnight to obtain CuO, and disperse in deionized water for later use.

[0073] Example 1

[0074] Take 10uL of 2.5mg / mL NC and add it to 1mL of 50μM ascorbic acid PBS buffer (pH 6, 0.2M). After the reaction is kept at 45℃ for 3 minutes, centrifuge and remove the supernatant to remove the NC. Then add TA (concentration 3mM) and 10uL of 5mg / mL CuO to the supernatant. After the reaction is continued at 45℃ for 25 minutes, collect the reaction solution and use fluorescence spectroscopy to detect the fluorescence signal around 423nm under 315nm excitation. Figure 2 It can be seen that by observing the peak intensity of the fluorescence signal at 423 nm, it is found that the fluorescence signal at 423 nm can be presented only when ascorbic acid exists in the system, otherwise no obvious signal can be observed.

[0075] Example 2

[0076] Take 10uL of 2.5mg / mL NC and add it to 1mL of 50μM ascorbic acid PBS buffer (pH 6, 0.2M). After constant temperature reaction at different temperatures (25℃-65℃) for 3 minutes, centrifuge to remove NC and take the supernatant. Add 10ul of 5mg / mL CuO and TA (concentration 3mM) to the supernatant. Continue to react at the same reaction temperature as the first step for 25 minutes, collect the reaction solution, and use fluorescence spectroscopy to detect the fluorescence signal around 423nm under 315nm excitation. Figure 3 It can be seen that by observing the fluorescence signal intensity, it was found that when the reaction temperature was between 25℃-45℃, the fluorescence signal intensity gradually increased with the increase of temperature, and when the reaction temperature was between 55℃-65℃, the fluorescence signal intensity gradually decreased with the increase of temperature. Therefore, 45℃ is the optimal temperature for this reaction.

[0077] Example 4

[0078] Take 10uL of 2.5mg / mL NC and add it to 1mL of 50μM ascorbic acid PBS buffer (pH between 5-10). After constant temperature reaction at 45℃ for 3min, centrifuge to remove NC and take the supernatant. Then add 10ul of 5mg / mL CuO and TA (concentration 3mM) to the supernatant. After continuing to react at 45℃ for 25 minutes, collect the reaction solution and use fluorescence spectroscopy to detect the fluorescence signal around 423nm under 315nm excitation. Figure 4 It can be seen that by observing the fluorescence signal intensity, it was found that when the reaction pH was between 5 and 6, the fluorescence signal intensity gradually increased with the increase of pH, and when the reaction pH was between 6 and 10, the fluorescence signal intensity gradually decreased with the increase of pH, that is, the signal intensity reached a peak at pH 6. Therefore, pH = 6 is the optimal pH for this reaction.

[0079] Example 5

[0080] Take 10uL of 2.5mg / mL NC and add it to 1mL of 50μM ascorbic acid PBS buffer (pH 6, 0.2M). After isothermal reaction at 45℃ for 3 minutes, centrifuge to remove NC and take the supernatant. Then add 10uL of 5mg / mL CuO and TA (concentration 3mM) to the supernatant. After continuing to react at 45℃ for different time periods (10-30 minutes), collect the reaction solution and use fluorescence spectroscopy to detect the fluorescence signal around 423nm under 315nm excitation. Figure 5 It can be seen that by observing the fluorescence signal intensity, it was found that when the reaction time was between 10 minutes and 25 minutes, the fluorescence signal intensity gradually increased with time, and when the reaction time was between 25 minutes and 30 minutes, the fluorescence signal intensity gradually decreased with time. Therefore, 25 minutes is the optimal reaction time for this reaction.

[0081] Example 6

[0082] Take 10uL of 2.5mg / mL NC and add it to 1mL of 50μM ascorbic acid PBS buffer (pH 6, 0.2M). After isothermal reaction at 45℃ for 3min, centrifuge to remove NC and take the supernatant. Then add 10ul of 5mg / mL CuO and 25uL of TA of different concentrations (0.5mM-4mM) to the supernatant. After continuing to react at 45℃ for 25 minutes, collect the reaction solution and use fluorescence spectroscopy to detect the fluorescence signal around 423nm under 315nm excitation. Figure 6As can be seen, observing the fluorescence signal intensity, it was found that when the added TA concentration was between 0.5mM and 3mM, the fluorescence signal intensity gradually increased with the increase of TA concentration. When the TA concentration was between 3mM and 4mM, the fluorescence signal intensity gradually decreased with the increase of TA concentration. Therefore, the reaction system with a TA concentration of 3mM was finally selected.

[0083] Example 7

[0084] 10 μL of the prepared NC solution (2.5 mg mL -1 ) was added to 1 mL of PBS buffer (pH 6, 0.2 M) containing varying concentrations of AA (50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, 3 μM, 1 μM, 0.5 μM, and 0.1 μM). The mixture was incubated at 45°C for 3 minutes, and the supernatant was collected by centrifugation. 10 μL of 5 mg / mL CuO and 3 mM TA were added to the supernatant. After a further 25 minutes of reaction at 45°C, the reaction solution was transferred to a cuvette and monitored by fluorescence spectroscopy. An excitation wavelength of 315 nm was used, and the fluorescence signal at approximately 423 nm was measured to quantify the concentrations of AA. Figure 7 This is the linear fit plot for AA detection using this assay, demonstrating the successful detection of ascorbic acid in the system. Experiments revealed good linearity between AA concentration and signal intensity over the range of 0.1 μM to 50 μM, with a detection limit of 85 nM. The linear fit equation is Flintensity = 4140 C (μM) + 21674.

[0085] Example 8

[0086] use Figure 11 The microfluidic chip includes two identical channels, one of which is channel 1 (the first channel) and the other is channel 2 (the second reaction channel), an inlet 1, an inlet 2 and an outlet.

[0087] Dow Corning 184 polydimethylsiloxane (consisting of a base component and a curing agent, thoroughly mixed in a 10:1 weight ratio) was poured into a silicon wafer template. Cured at 80°C and cooled to room temperature, the chip containing two channels (the first and second channels) was peeled from the template and holes were punched at the inlet and outlet of each channel using a hole punch. All channels were approximately 70 μm deep, 40 mm long, and 4 mm wide. The chip was plasma-treated and then bonded to a coverslip. Finally, polypropylene tubing was used to connect the inlets of the microfluidic chip to a syringe pump. NC and CuO were applied to the polydimethylsiloxane (PDMS) walls of the first channel (NC) and the second channel (CuO), respectively, until they no longer adhered. The PDMS was then sealed with a glass slide. Polypropylene tubing was used to connect the inlets of the microfluidic chip to a syringe pump.

[0088] PBS buffer containing varying concentrations of AA (2.5nM, 5nM, 10nM, 20nM, 30nM, 50nM, and 100nM) was pumped into the first channel of the microfluidic chip. The reaction was allowed to react at 45°C for 3 minutes. The reaction solution in the first channel was then pumped into the second channel, where TA was added to a final concentration of 3mM. The reaction continued at 45°C for 25 minutes. After the reaction was complete, the solution was pumped out of the chip and the reaction solution was collected and analyzed by fluorescence spectroscopy. An excitation wavelength of 315nm was used, and the fluorescence signal at approximately 423nm was measured to quantify the different concentrations of AA. Figure 8 The linear fit plot for AA detection using this method demonstrates the successful detection of ascorbic acid in the system. Experiments revealed that the fluorescence peak signal intensity gradually increased with increasing AA concentration, exhibiting excellent linearity. The detection limit was 0.77 nM, and the linear fit equation was Flintensity = 4366C (nM) + 124262. By using a microfluidic device, the sensitivity of the reaction was further improved, enabling the detection of lower concentrations of ascorbic acid.

[0089] Example 9

[0090] 10 μL of the prepared NC solution (2.5 mg mL -1 ) were added to a PBS buffer solution containing 50 μM of different substrates, incubated at 45°C for 3 minutes, and the supernatant was centrifuged. 10 μL of 5 mg / mL CuO and 25 μL of 120 mM TA were added to the filtrate, and the reaction was continued at 45°C for 25 minutes. The reaction solution was then detected by fluorescence spectroscopy. The excitation wavelength was 315 nm, and the fluorescence signal at around 423 nm was measured to detect whether the constructed NC and CuO nanozyme cascade reaction could catalyze the corresponding substrate. The test results are as follows: Figure 9As shown in the figure, it can be found that under the condition of the same substrate concentration, the method can successfully detect ascorbic acid in the system, and the signal intensity is significantly better than that of other substrates. This proves that the cascade reaction provided by the present invention can indeed achieve highly selective detection of substrates, and the effect is significant.

[0091] Example 10

[0092] Comparing the oxidase-peroxidase cascade system of Example 8 of the present invention with the existing ascorbic acid detection, the results are shown in Table 1 below. It can be seen that the oxidase-peroxidase cascade system of the present invention is significantly better in detection range and detection limit.

[0093] Table 1

[0094]

[0095] The references for the materials in Table 1 above are as follows:

[0096] [3]Zhou Z, Liao Y, Wu H, et al. Zeolitic imidazolate framework-8cappedgold nanoclusters as an “on-off-on” fluorescent nanoprobe for sensitive detection of MnO4-and ascorbic acid[J].Microporous and Mesoporous Materials, 2022,338:

[0097] [4]Zhang Z,Long D,Yang M,et al.A ratiometric fluorescence sensor forascorbic acid determination based on an AND-NAND logic pair[J].MicrochimicaActa,2021,188:

[0098] [5]Sun M, Zhong Z, Wang Y, et al.Dual-functional lanthanide-MOF probenanocomposite based on hydroxyapatite nanowires as fluorescent sensor forascorbic acid[J].Microchimica Acta, 2023,190:

[0099] [6]Abo-bakr AM,Abd-Elsabour M,Abou-Krisha M M.An Efficient NovelElectrochemical Sensor for Simultaneous Determination of Vitamin C andAspirin Based on a PMR / Zn-Al LDH / GCE[J].Electroanalysis,2021,33:2476-2489.

[0100] [7] Zhu Y, Deng X, Chen J, et al. Coffee grounds-derived carbon quantumdots as peroxidase mimetics for colorimetric and fluorometric detection of ascorbic acid[J]. Food Chemistry, 2023,429:

[0101] [8]Wan J, Zou JM, Zhou SJ, et al. A bimetallic(Ni / Co)metal-organicframework with excellent oxidase-like activity for colorimetric sensing of ascorbic acid[J]. Analytical Methods, 2023,15:1819-1825.

[0102] Comparative Example 1

[0103] Fe-NC was prepared using conventional methods. 10 μL of Fe-NC solution (2.5 mg mL -1 ) was added to 1 mL of PBS buffer (pH 6, 0.2 M) containing AA (50 μM), incubated at 45°C for 3 minutes, and centrifuged to obtain the supernatant. 10 μL of 5 mg / mL CuO and TA (3 mM) were added to the filtrate, and the reaction was continued at 45°C for 25 minutes. The reaction solution was then placed in a cuvette and detected by fluorescence spectroscopy. The excitation wavelength was selected to be 315 nm, and the system was optimized by measuring the fluorescence signal at around 423 nm. Figure 10 As shown, the NC-CuO cascade system was found to be more effective.

Claims

1. An oxidase-peroxidase cascade system, characterized in that: The system includes nitrogen-doped carbon nanocages (NCs) with oxidase activity and CuO with peroxidase activity.

2. The oxidase-peroxidase cascade system according to claim 1, characterized in that: The nitrogen-doped carbon nanocage with oxidase activity is obtained by calcining pyridine as a precursor at high temperature using a magnesium oxide template method.

3. A method for constructing the oxidase-peroxidase cascade system according to claim 1, characterized in that: The steps include: (1) Weighing basic magnesium carbonate and placing it in a reaction vessel, evacuating it to a vacuum and then heating it under an inert gas atmosphere; adding a precursor pyridine and reacting it at a high temperature; after the reaction is completed, placing the product in a concentrated acid solution, stirring it, filtering it, washing it, and drying it to obtain nitrogen-doped carbon nanocages NC with oxidase activity; (2) Add acetic acid and deionized water to copper acetate, stir and heat until boiling, then pour concentrated sodium hydroxide solution into the solution. Stop the reaction immediately after a black product is generated. After cooling, centrifuge, wash, and dry to obtain CuO with peroxidase activity: (3) NC in step (1) and CuO in step (2) are used to form an oxidase-peroxidase cascade system.

4. The construction method according to claim 3, characterized in that In step (1), basic magnesium carbonate is weighed and placed in a reaction vessel, which is evacuated to a vacuum and then heated under an inert gas atmosphere; a precursor pyridine is added, and heating is stopped after the reaction at a high temperature. After the temperature is lowered, the product is placed in a concentrated hydrochloric acid solution, stirred for 40 h to 50 h, filtered, washed, and dried.

5. Use of the oxidase-peroxidase cascade system according to claim 1 in the detection of ascorbic acid.

6. The use according to claim 5, characterized in that NC was added to an ascorbic acid solution, incubated, and centrifuged to obtain a supernatant. Terephthalic acid (TA) and CuO were dropped into the supernatant after centrifugation, and then incubated continuously. Finally, the obtained reaction solution was characterized by a fluorescence spectrophotometer.

7. The use according to claim 6, characterized in that The first step incubation time is 3-10 minutes, the second step incubation time is 20-30 minutes; the incubation temperature is 35-65°C.

8. Use of the oxidase-peroxidase cascade system according to claim 1 in detecting ascorbic acid in a microfluidic system.

9. The use according to claim 8, characterized in that So the application preferences include: (1) NC and CuO nanoparticles were coated on PDMS and then covered with smooth glass. (2) pumping the reaction solution added with ascorbic acid solution into the first channel to carry out the reaction; (3) Pump the reaction solution into the second reaction channel and simultaneously pump in TA to continue the reaction; (4) Detecting the collected reaction solution and detecting its fluorescence signal.

10. The use according to claim 9, characterized in that Therefore, the reaction time of step (2) is 3-10 minutes, the reaction time of step (3) is 20-30 minutes, and the reaction temperature is 35-65°C.

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