A heterogeneous photocatalytic superoxide radical absolute quantitative detection method based on biological enzyme calibration and interface elution

Abstract

This invention relates to a heterogeneous photocatalytic method for the absolute quantitative detection of superoxide radicals based on bioenzyme labeling and interfacial elution. The method is an interdisciplinary approach combining analytical chemistry, environmental functional materials, and heterogeneous photocatalysis. It addresses the technical shortcomings of heterogeneous photocatalytic systems in detecting superoxide radicals, including interfacial adsorption shielding, easy disproportionation loss of signal molecules, and the lack of absolute quantitative standards. The method involves constructing a standard curve using a xanthine / xanthine oxidase system; adjusting the pH of the reaction system; introducing a highly polar organic solvent for interfacial elution; and achieving complete capture of the signal molecules adsorbed at the solid-liquid interface through physical separation. This invention combines physical separation methods such as filtration and centrifugation to eliminate optical scattering interference, fills the gap in the lack of unified quantitative standards for long-wavelength photocatalytic activity, and provides a precise quantitative means for the photocatalytic ROS production rate. This invention is used for the quantitative detection of heterogeneous photocatalytic superoxide radicals.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry, environmental functional materials and heterogeneous photocatalysis, specifically to an absolute quantitative detection method for reactive oxygen species at solid-liquid heterogeneous interfaces under ultraviolet-visible-near-infrared broadband excitation, and particularly to a quantitative analysis method for superoxide radicals based on bioenzyme labeling and interface elution technology. Background Technology

[0002] With the increasing severity of the energy crisis and environmental pollution, multi-wavelength photocatalysis technology based on semiconductor materials has received widespread attention from the global academic and industrial communities in areas such as water purification, degradation of organic pollutants (e.g., deep oxidation of typical microplastics), and energy conversion. In heterogeneous photocatalytic systems, reactive oxygen species (ROS)—especially superoxide radicals (O2)—are particularly prominent. ·- Bismuth dioxide (BiO2) is widely recognized as a key intermediate driving redox reactions. Its production rate, steady-state concentration, and existing form not only directly determine the macroscopic activity of the catalyst but are also core parameters for elucidating catalytic reaction mechanisms and optimizing photoreactor design. With the development of semiconductor materials science, semiconductor materials such as titanium dioxide (TiO2), zinc oxide (ZnO), graphitic carbon nitride (g-C3N4), metal-organic frameworks (MOFs), and bismuth oxide (Bi2O3), due to their unique band structures and photochemical properties, have become a research focus in the field of environmental governance. Photocatalysis research has also expanded from traditional ultraviolet light response to visible light and the near-infrared (NIR) band, which accounts for approximately 50% of the solar spectrum. Photocatalytic reactions exhibit significant band-dependent characteristics depending on the energy excitation band: ultraviolet light (10–400 nm) primarily drives interband transitions in wide-bandgap semiconductors through high-energy photons; visible light (400–760 nm) matches the absorption edge of most narrow-bandgap materials; while near-infrared light (760–2500 nm) typically relies on sub-level transitions induced by narrow-bandgap materials or defect states (such as oxygen vacancies and atomic doping). However, in heterogeneous systems, especially for weak signal bands driven by low-energy photons, achieving precise quantitative capture of superoxide radicals has become a technological bottleneck restricting the development of this field.

[0003] Currently, laboratory testing for O2 ·- Commonly used methods each have limitations and are difficult to meet the complex requirements of heterogeneous interfaces:

[0004] (1) Electron paramagnetic resonance (EPR / ESR): Although it is considered the "gold standard" for detecting free radicals, its equipment is expensive and maintenance costs are extremely high. In heterogeneous powder suspension systems, it is difficult to provide standardized absolute molar concentration data due to the diffusion efficiency and solvent effect of the capture agent, such as DMPO.

[0005] (2) Fluorescent probe method: It has high sensitivity, but the heterogeneous catalyst solid powder produces strong background fluorescence and light scattering interference, and the fluorescence signal is easily affected by environmental quenching, resulting in poor stability.

[0006] (3) Nitrogen blue tetrazolium (NBT) reduction method: This method is the most widely used due to its simple operation and low cost. Its principle is based on O2... ·- NBT is reduced to a water-insoluble blue formazan precipitate. However, when applied to multi-band heterogeneous systems (such as bismuth oxide, MOFs, polymer semiconductors, etc.), three unresolved technical challenges exist: ① Interfacial adsorption shielding effect: In solid-liquid heterogeneous reactions, O2... ·- The superoxide radical reacts immediately with adsorbed NBT after being generated on the catalyst surface. The generated formazan molecules are strongly hydrophobic and readily anchor tightly to the pores or active sites of the catalyst. Traditional detection methods only measure the supernatant, ignoring the "signal loss" adsorbed on the solid surface, resulting in detection results that are often 1-2 orders of magnitude lower than the actual yield. ② Thermodynamic instability and self-disproportionation of signal molecules: Superoxide radicals readily undergo self-disproportionation reactions in aqueous solutions (especially under localized acidic environments). Current technologies lack effective signal protection methods after the reaction, leading to the loss of some of the target signal due to disproportionation reactions rather than normal loss during the ex-situ detection process, making it impossible to accurately trace the radical yield at the reaction time. ③ Lack of a unified "absolute quantitative" scale: Currently, the industry mostly uses "absorbance change value (ΔA)" or relative scavenging rate for expression. Due to the lack of a standardized calibration system, data from different catalytic systems, different laboratories, and different excitation bands cannot be compared horizontally, resulting in a lack of unified quantitative standards for evaluating "photocatalytic activity".

[0007] In summary, developing a detection method that can overcome adsorption shielding at heterogeneous interfaces, suppress signal disproportionation loss, and achieve a transition from qualitative colorimetry to absolute molar concentration is not only an urgent need for evaluating weak signal catalytic systems such as near-infrared catalysis, but also a key step in promoting the standardization and industrialization of photocatalysis technology. Summary of the Invention

[0008] The purpose of this invention is to overcome the technical defects of existing heterogeneous photocatalytic systems (especially powder suspension systems) in detecting superoxide radicals, such as interfacial adsorption shielding, easy disproportionation loss of signal molecules, and lack of absolute quantitative standards, and to provide a heterogeneous photocatalytic absolute quantitative detection method for superoxide radicals based on bioenzyme labeling and interfacial elution.

[0009] This invention addresses the technical bottlenecks in heterogeneous photocatalytic suspension systems, where product signal molecules are subject to strong adsorption shielding at the solid catalyst interface and interference from particle background scattering. It proposes an absolute quantitative detection method that couples interfacial elution extraction with multi-stage physical interception. This method overcomes the limitation of capturing trace superoxide radical signals driven by low-energy photons (such as near-infrared light), achieving full recovery and accurate measurement of signal molecules at complex solid-liquid interfaces. It provides a universal solution for constructing a unified quantitative standard for the generation rate of heterogeneous photocatalytic active species, filling the technical gap in the lack of a unified quantitative standard for photocatalytic activity evaluation.

[0010] To completely solve the technical challenges of interfacial adsorption and signal loss mentioned above, this invention constructs a coupled strategy of "in-situ capture-interfacial elution-physical separation". First, the high solubility of highly polar organic solvents such as dimethyl sulfoxide (DMSO), anhydrous ethanol, and methanol is utilized to break the van der Waals forces and hydrogen bonds between formazan and the catalyst surface. In particular, formazan molecules generated by near-infrared excitation are often anchored to active sites on the surface of catalysts (such as semiconductor materials with strong adsorption effects such as TiO2, ZnO, g-C3N4, MOFs, and Bi2O3) through van der Waals forces or electrostatic interactions. The introduction of a high proportion of highly polar organic solvents not only provides extremely high solubility but also competes for adsorption sites through solvation, effectively breaking the hydrogen bonds between formazan and the catalyst surface and releasing the adsorbed signal into the liquid phase. Second, filtration and centrifugation processes are introduced to eliminate the influence of submicron particles on the optical path through physical methods. Finally, a standardized bioenzyme calibration curve is constructed using a xanthine oxidase system to convert qualitative color changes into molar concentration values.

[0011] A heterogeneous photocatalytic absolute quantitative detection method for superoxide radicals based on bioenzyme labeling and interfacial elution is specifically performed according to the following steps:

[0012] (1) Establishing a standard curve by enzyme labeling: A known molar amount of superoxide radicals were generated using a biological enzyme-catalyzed reaction system. The superoxide radicals were reacted with nitrotetrazole blue probe to generate formazan. A highly polar organic solvent was added to extract the formazan, and the absorbance of the extract was measured at a characteristic wavelength. A standard regression equation between the absolute molar concentration of superoxide radicals and absorbance was established.

[0013] (2) Multi-band heterogeneous photocatalytic reaction: Add chlorinated nitrotetrazole blue probe to a suspension containing heterogeneous catalyst, adjust the pH value of the system, and carry out photocatalytic reaction under light irradiation of a preset wavelength.

[0014] (3) Termination and complete dissolution: After the reaction in step (2) is completed, a quencher is added to terminate the reaction and a reaction solution is obtained. A highly polar organic solvent is added, shaken, and eluted to obtain a mixture.

[0015] (4) Physical separation and detection: The mixture obtained in step (3) is subjected to solid-liquid separation, the clear liquid is collected and the absorbance at the characteristic wavelength is measured, and the absolute molar concentration of superoxide radicals is calculated by combining the standard regression equation in step (1).

[0016] Furthermore, the strongly polar organic solvent mentioned in steps (1) and (3) is one or a mixture of several of dimethyl sulfoxide, anhydrous ethanol and methanol; the volume ratio of the strongly polar organic solvent to the reaction solution is 2:(10~1).

[0017] Furthermore, after eluting with a strongly polar organic solvent in steps (1) and (3), deionized water is added for dilution, with the dilution ratio being 0.5 to 2 times the total volume of the mixture.

[0018] Furthermore, the bio-enzymatic reaction system described in step (1) is a xanthine / xanthine oxidase system; wherein the concentration of xanthine oxidase is 0.01~0.1 U / mL and the concentration of xanthine is 0.1~1 mmol / L.

[0019] Furthermore, in step (1), the concentration of the chlorinated nitrotetrazole blue probe is 0.1~1 mM.

[0020] Furthermore, in step (2), the concentration of the chlorinated nitrotetrazole blue probe is 0.1~1 mM.

[0021] Furthermore, the pH value of the system in step (2) is adjusted to 11.0~12.0 by adding an alkaline solution, which is an inorganic strong alkaline solution.

[0022] Furthermore, the illumination band in step (2) includes any single or multi-band composite light among ultraviolet light with wavelengths of 10~400 nm, near-infrared light with wavelengths of 400~760 nm and 760~2500 nm; the heterogeneous catalyst is one or a mixture of several of metal oxides, metal sulfides, polymer semiconductors and metal-organic framework materials.

[0023] Furthermore, the quencher mentioned in step (3) is superoxide dismutase, and the amount added is 6000~12000 U / mL.

[0024] Furthermore, the solid-liquid separation in step (4) is performed by vacuum filtration using a 0.22~0.80 μm organic filter membrane or by centrifugation. When vacuum filtration is used, a filter membrane with a pore size of 0.22 μm is used when the heterogeneous catalyst is made of nano-sized particles, and a filter membrane with a pore size of 0.45 μm is used when the heterogeneous catalyst is made of micron-sized particles. When centrifugation is used, the separation is performed at a speed of 8000~12000 rpm for 3~5 minutes.

[0025] The multi-band quantitative detection principle described in this invention aims to construct an activity evaluation system covering the entire spectral range of ultraviolet (10~400 nm), visible (400~760 nm), and near-infrared (760~2500 nm). For heterogeneous catalysts with different band structures, this method utilizes photons with different energy gradients to drive electronic transitions. For the ultraviolet and visible bands, high-energy electrons are mainly generated through interband transitions in semiconductors; for the near-infrared band, excitation is mainly achieved by capturing low-energy photons using narrow bandgap materials or defect state energy levels (such as oxygen vacancies). Although the energy of near-infrared photons is relatively low, the excited electron potential is still sufficient to reduce dissolved oxygen to generate superoxide radicals (O2). ·- Because the yield of reactive oxygen species generated by near-infrared excitation is extremely low and the signal is extremely weak, they are easily adsorbed and shielded by the complex pores and active sites of heterogeneous interfaces. This invention, through the competitive adsorption of a high proportion of strongly polar organic solvents, ensures that products adsorbed at the solid-liquid interface can be eluted to the liquid phase without damage, regardless of whether the system has a high yield in the UV / Vis region or a low yield in the near-infrared "weak signal" system. Furthermore, by introducing a bioenzyme calibration system as a unified "molecular ruler," the detection bias caused by differences in light intensity and photon energy across different wavelengths is eliminated, enabling quantitative comparison of catalytic activity at different wavelengths. This provides standardized data support for elucidating the full-spectrum catalytic mechanism of materials.

[0026] In particular, this method effectively eliminates the detection bias caused by the "solvent-induced color change effect" by performing bioenzyme calibration in the same strongly polar organic solvent system. Since strongly polar solvents (such as DMSO) can change the electron cloud distribution and energy level gap of formazan molecules, causing its characteristic absorption peak to shift relative to the aqueous phase, this scheme ensures a high degree of consistency between the standard curve and the test sample in terms of optical properties through "consistent solvent calibration".

[0027] This method is particularly suitable for the accurate assessment of the generation rate of trace reactive oxygen species under full-spectrum excitation in the ultraviolet-visible-near-infrared range (10~2500 nm).

[0028] This invention employs a quantitative detection technique based on improved bio-enzyme labeling and interface elution. The specific technical solution is as follows:

[0029] 1. Preparation of reaction system: Add 0.1-1 mM NBT solution and 0.1-1 mM xanthine solution to phosphate buffered saline (PBS) (pH=7~8, concentration 0.01~0.1 mM).

[0030] 2. Free radical excitation: Add xanthine oxidase (XOD) with known activity units (0.01~0.1 U / mL) to the above system. According to biochemical theory, in this system, during the oxidation of xanthine catalyzed by xanthine oxidase, O2 is generated. ·- The molar amount is linearly proportional to the enzyme activity and reaction time.

[0031] 3. Gradient calibration: By varying the volume of xanthine oxidase added (preferably within the range of 0 μL to 200 μL), and reacting in a constant temperature (20 to 30°C) water bath for 4 to 40 minutes, a gradient of O2 is generated in the system. ·- (The corresponding production amount is 0~0.08 μmol).

[0032] 4. Reaction Termination: Add an excess of superoxide radical quencher—6000~12000 U / mL superoxide dismutase (SOD)—at a volume of 0.5%~5% of the reaction solution volume. Utilize SOD to quench O2. ·- The extremely high disproportionation rate (close to the diffusion limit) forcibly terminates further reduction of NBT, ensuring controlled reaction time.

[0033] 5. Signal Extraction: Add a highly polar organic solvent, such as dimethyl sulfoxide (DMSO), anhydrous ethanol, or methanol, to the reacted solution system. The volume ratio of the highly polar organic solvent to the original reaction solution is 2:(10~1) to completely dissolve the blue formazan precipitate. To prevent the formation of flocculent precipitates that interfere with detection in a high-concentration organic solvent system, dilute with a certain proportion of deionized water after extraction. The preferred dilution ratio is 0.5 to 2 times the total volume of the mixture.

[0034] 6. Linear Regression: Measure the absorbance value at 560 nm to establish a linear regression relationship between absorbance A and O2. ·- The standard regression equation for molar quantity n: .

[0035] This invention is used for sampling and detection of multi-band heterogeneous photocatalytic systems, and the specific scheme is as follows:

[0036] 1. Catalytic reaction: The heterogeneous catalyst to be tested is added to a solution containing NBT (0.1~1 mM), and the reaction is performed at the isoelectric point (pH). pzc Heterogeneous catalysts with lower efficiency tend to cause localized interfacial acidification in aqueous solutions, thereby accelerating the growth of superoxide radicals (O2). ·- The protonation disproportionation reaction (2O2) ·- +2H +→H2O2+O2), causing abnormal loss of the detection signal. To suppress this disproportionation process and improve detection sensitivity, this invention adjusts the pH of the system to a strongly alkaline environment (preferably pH 11.0~12.0) before the reaction begins, thereby suppressing O2 by reducing the proton concentration. ·- The protonation disproportionation reaction, thereby prolonging the O2 reaction. ·- The catalyst's lifetime at complex heterogeneous interfaces ensures its ability to fully react with NBT to generate formazan signal molecules. Under various light irradiation conditions, the catalyst is excited to generate electrons and reduces dissolved oxygen to produce O2. ·- .

[0037] 2. In-situ termination: When the reaction reaches the set time point, immediately add 6000~12000 U / mL of SOD quencher to terminate the experiment.

[0038] 3. Interface elution and solubilization: Since the reaction occurs on the solid surface, the generated blue formazan will be rapidly adsorbed onto the catalyst particles. According to the volume ratio of organic solvent to original reaction liquid of 2:(10~1), a strong polar organic solvent such as dimethyl sulfoxide (DMSO), anhydrous ethanol, methanol, etc. is added to the suspension containing the heterogeneous catalyst. The high solvation energy of the strong polar solvent generates a "competitive adsorption" effect, which breaks the van der Waals forces or hydrogen bonds between the hydrophobic formazan molecules and the active sites on the catalyst surface, so that the adsorbed signal molecules are completely desorbed and enter the liquid phase. The volume ratio of strong polar organic solvent to original reaction liquid is 2:(10~1), so that the blue formazan precipitate is completely dissolved in the solvent.

[0039] 4. Precision Separation: Various separation methods exist. The mixture can be filtered using a 0.22–0.80 μm (preferably 0.45 μm) organic filter membrane, or centrifuged at 8000–12000 rpm to obtain a clear, transparent blue filtrate. Example 3 demonstrates that vacuum filtration yields better results. This step aims to thoroughly remove suspended catalyst micro / nano particles and eliminate background scattering interference in the optical path, thereby obtaining a clear analyte for absolute quantitative colorimetric analysis.

[0040] 5. Quantitative Calculation: By measuring the absorbance of the filtrate at 560 nm and substituting it into the regression equation obtained in the previous stage, the O2 produced by this heterogeneous system per unit time can be accurately calculated. ·- Absolute concentration.

[0041] The beneficial effects of this invention are that, through multi-dimensional technical coupling, the following breakthroughs have been achieved in the field of multi-band heterogeneous photocatalytic reactive oxygen species detection:

[0042] ① To address the limitations of the NBT method, which lacks quantitative standards and can only perform semi-quantitative colorimetric analysis, this invention introduces a xanthine / xanthine oxidase (XOD) bio-enzymatic standard reaction as a "molecular ruler," establishing the relationship between absorbance and known O2. ·- The standard regression equation for molar amounts makes the activity assessments of different catalytic systems comparable.

[0043] ② The advanced nature of pH control technology: Traditional detection methods are mostly performed in near-neutral environments, ignoring signal loss caused by local acidification at heterogeneous interfaces. This invention, by locking the pH at 11.0~12.0, increases the detected free radical yield by more than 30 times compared to neutral conditions (see Example 4), fundamentally solving the serious problem of missed detection caused by signal protonation disproportionation reactions.

[0044] ③ Overcoming the blind spot in interface detection: Addressing the challenge of blue formazan signal molecules, generated in multi-band heterogeneous photocatalytic reactions, readily adsorbing onto the surface of heterogeneous catalysts and leading to low liquid-phase detection values, this invention employs an in-situ interface elution technique using a high-proportion, highly polar organic solvent at a volume ratio of 2:(10~1) to the original reaction liquid. Unlike previous methods that only detected residual signals in the liquid phase, this technique powerfully extracts and removes the active products adsorbed at the solid-liquid interface using a highly polar organic solvent. Due to the abundant active sites and complex pore structure on the surface of heterogeneous catalysts, the generated formazan molecules are often anchored by van der Waals forces or electrostatic interactions. The introduction of a high proportion of highly polar solvent not only provides extremely high solubility but also competes for adsorption sites through solvation, thereby achieving complete removal of the "adsorbed" signal. This allows the detection results to more accurately reflect the total amount of superoxide radicals generated in the system.

[0045] ④ The advanced nature of the coupling between interface elution and physical separation: This combined technology resolves the contradiction between "signal capture" and "optical interference." The high proportion of highly polar solvent not only achieves full recovery of the adsorbed signal (see Example 5), but also, combined with physical filtration, completely eliminates the optical scattering effect of solid particles, allowing for the recovery of trace O2 in weak signal bands such as near-infrared. ·- It can also be accurately detected, filling the gap in the quantitative evaluation of long-wavelength activity.

[0046] ⑤ To address the significant differences in dispersibility and adsorption performance among various heterogeneous materials, this invention establishes a standardized detection procedure. This method is not only applicable to bismuth oxide (Bi2O3) and its composites, but can also be widely extended to various heterogeneous photocatalytic systems such as titanium dioxide (TiO2), graphitic carbon nitride (g-C3N4), and metal-organic frameworks (MOFs), solving the common technical challenge of quantifying powder suspension systems.

[0047] ⑥ To ensure high repeatability of experimental results, compared with the shortcomings of traditional methods such as poor control of reaction time and severe interference from heterogeneous catalyst scattering, this invention constructs a triple guarantee system of "precise SOD termination + stable color development with strongly polar organic solvent + physical separation". By forcibly terminating the reaction with SOD to ensure time accuracy, and by utilizing physical separation to completely eliminate background scattering interference from micro- and nano-particles, experimental errors are controlled within a low range, significantly improving the accuracy and reliability of detection.

[0048] ⑦ It possesses extremely strong optical self-correction capabilities. Traditional NBT methods, if simply using the aqueous extinction coefficient, will result in significant errors due to absorption peak shifts after the addition of organic solvents. This invention, through in-situ solvent calibration technology, transforms the solvent-induced color change effect from an "interference factor" into a "system constant," significantly improving the universality and accuracy of the method under different solvent ratios.

[0049] This invention is used for the quantitative detection of heterogeneous photocatalytic superoxide radicals. Attached Figure Description

[0050] Figure 1 This is a flowchart of an experimental setup for the absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution, using a vacuum filtration method for separation.

[0051] Figure 2 This is a flowchart of the experimental setup for a heterogeneous photocatalytic absolute quantitative detection method for superoxide radicals based on bioenzyme labeling and interface elution, using centrifugation for separation. Detailed Implementation

[0052] Specific Implementation Method 1: This implementation method is a heterogeneous photocatalytic absolute quantitative detection method for superoxide radicals based on bioenzyme labeling and interfacial elution, which is carried out according to the following steps:

[0053] (1) Establishing a standard curve by enzyme labeling: A known molar amount of superoxide radicals were generated using a biological enzyme-catalyzed reaction system. The superoxide radicals were reacted with nitrotetrazolium chloride (NBT) probe to generate formazan. The formazan was extracted with a highly polar organic solvent, and the absorbance of the extract was measured at a characteristic wavelength. A standard regression equation between the absolute molar concentration of superoxide radicals and absorbance was established.

[0054] (2) Multi-band heterogeneous photocatalytic reaction: Add chlorinated nitrotetrazole blue probe to a suspension containing heterogeneous catalyst, adjust the pH value of the system, and carry out photocatalytic reaction under light irradiation of a preset wavelength.

[0055] (3) Termination and complete dissolution: After the reaction in step (2) is completed, a quencher is added to terminate the reaction and a reaction solution is obtained. A highly polar organic solvent is added, shaken, and eluted to obtain a mixture.

[0056] (4) Physical separation and detection: The mixture obtained in step (3) is subjected to solid-liquid separation, the clear liquid is collected and the absorbance at the characteristic wavelength is measured, and the absolute molar concentration of superoxide radicals is calculated by combining the standard regression equation in step (1).

[0057] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the highly polar organic solvent mentioned in steps (1) and (3) is one or a mixture of several of dimethyl sulfoxide, anhydrous ethanol, and methanol; the volume ratio of the highly polar organic solvent to the reaction solution is 2:(10~1). Everything else is the same as in Specific Implementation Method One.

[0058] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 1 or 2 in that: after elution with a strongly polar organic solvent in steps (1) and (3), deionized water is added for dilution, with a dilution ratio of 0.5 to 2 times the total volume of the mixture. Everything else is the same as in Specific Implementation Method 1 or 2.

[0059] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the bio-enzyme-catalyzed reaction system in step (1) is a xanthine / xanthine oxidase system; wherein the concentration of xanthine oxidase is 0.01~0.1 U / mL, and the concentration of xanthine is 0.1~1 mmol / L. Everything else is the same as in Specific Implementation Methods One to Three.

[0060] Specific Implementation Method 5: This implementation method differs from Specific Implementation Methods 1 to 4 in that the concentration of the chlorinated nitrotetrazolium blue probe in step (1) is 0.1~1 mM. Everything else is the same as in Specific Implementation Methods 1 to 4.

[0061] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that the concentration of the chlorinated nitrotetrazole blue probe in step (2) is 0.1~1 mM. Everything else is the same as in Specific Implementation Methods One to Five.

[0062] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that the pH value of the system in step (2) is adjusted to 11.0~12.0 by adding an alkaline solution, which is an inorganic strong alkaline solution. Everything else is the same as in Specific Implementation Methods One to Six.

[0063] The alkaline solution includes, but is not limited to, inorganic strong alkaline solutions such as sodium hydroxide, potassium hydroxide, or sodium carbonate.

[0064] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in that: the illumination band mentioned in step (2) includes any single or multi-band composite light from ultraviolet light with wavelengths of 10~400 nm, near-infrared light with wavelengths of 400~760 nm, and near-infrared light with wavelengths of 760~2500 nm; the heterogeneous catalyst is one or a mixture of several of metal oxides, metal sulfides, polymer semiconductors, and metal-organic framework materials. Everything else is the same as in Specific Implementation Methods One to Seven.

[0065] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One to Eight in that the quencher mentioned in step (3) is superoxide dismutase, and the amount added is 6000~12000 U / mL. Everything else is the same as in Specific Implementation Methods One to Eight.

[0066] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Methods One to Nine in that: the solid-liquid separation in step (4) uses a 0.22~0.80 μm organic filter membrane for vacuum filtration or a centrifuge for centrifugation; when using vacuum filtration, if the heterogeneous catalyst is nano-sized particles, a filter membrane with a pore size of 0.22 μm is used; if the heterogeneous catalyst is micron-sized particles, a filter membrane with a pore size of 0.45 μm is used; when using centrifugation, the separation is performed at a speed of 8000~12000 rpm for 3~5 minutes. The rest is the same as in Specific Implementation Methods One to Nine.

[0067] The scope of this invention is not limited to the above-described embodiments; a combination of one or more specific embodiments can also achieve the purpose of the invention.

[0068] Example 1:

[0069] Combined with appendix Figure 1 , 2 This embodiment will be described in detail. Figure 1 and Figure 2 The complete processes of two physical separation methods, namely vacuum filtration and centrifugation, are demonstrated.

[0070] like Figure 1The apparatus shown includes a xenon lamp constant current power supply 1, a xenon lamp light source 2, a cutoff filter 3, a temperature sensor 4, a glass beaker 5, and a magnetic stirrer 6. The magnetic stirrer 6 ensures that the catalyst is in a fully suspended state. Next is the in-situ termination operation for the multi-band heterogeneous photocatalytic system, where a quencher 7 is added to the glass beaker 5 to terminate the experiment. The next step is the interfacial elution and solubilization of the multi-band heterogeneous photocatalytic system. An extractant 8 is added to the glass beaker 5 and the mixture is thoroughly shaken to extract the formazan completely from the surface of the heterogeneous catalyst particles. The next step is the vacuum filtration operation for precise separation of the multi-band heterogeneous photocatalytic system. The vacuum filtration device includes a circulating water vacuum pump 9, a rubber tube 10, a filter flask 11, a sintered core connector 12, a filter membrane 13, a filter cup 14, a duckbill clamp 15, and the glass beaker 5. The filtrate is collected in the filter flask 11 for subsequent colorimetric detection.

[0071] like Figure 2 The apparatus shown includes a xenon lamp constant current power supply 1, a xenon lamp light source 2, a cutoff filter 3, a temperature sensor 4, a glass beaker 5, and a magnetic stirrer 6. The magnetic stirrer 6 ensures that the catalyst is in a fully suspended state. Next is the in-situ termination operation for the multi-band heterogeneous photocatalytic system, where a quencher 7 is added to the glass beaker 5 to terminate the experiment. The next step is the interfacial elution and solubilization of the multi-band heterogeneous photocatalytic system, where an extractant 8 is added to the glass beaker 5 and the mixture is thoroughly shaken to extract the formazan completely from the surface of the heterogeneous catalyst particles. The next step is the centrifugation operation for precise separation of the multi-band heterogeneous photocatalytic system, including transferring the sample from the glass beaker 5 to a centrifuge tube 17, centrifuging a centrifuge 16, and slowly pouring out the clear liquid from the centrifuge tube as the required filtrate for subsequent colorimetric detection.

[0072] Both methods include three core operating units: ① reaction unit (multi-band light excitation + full suspension stirring); ② in-situ termination and interface elution unit (SOD quenching + high proportion of highly polar organic solvent extraction); ③ physical separation unit (vacuum filtration or centrifugation).

[0073] All in-situ quantitative superoxide radical experiments were conducted in a 250 mL glass beaker 5 containing 30 mg of catalyst and 100 mL of mixed solution (20 mL NBT + 80 mL pure water), placed on a magnetic stirrer 6. The mixture was stirred in the dark for 5 min to ensure uniform dispersion. Then, power source 1 (light source 2) was turned on for irradiation. The light was filtered by a cutoff filter 3 for wavelength selection. Temperature sensor 4 monitored the reaction temperature in real time. The xenon lamp light source 2 had a power of 300 W and a current of 20 A. Two types of cutoff filters 3 were used: UVCUT800 (near-infrared band) and UVCUT420 (visible band). The cutoff filter 3 was positioned 15 cm from the bottom of the reaction beaker 5, meaning the light source was 15 cm from the reaction vessel. After 1 h of irradiation, quencher (SOD) 7 was added. After 2 min of reaction, extractant 8 (125 mL DMSO) was added, and extraction was carried out with thorough shaking for 1.5 h. Physical separation: ① Using a vacuum filtration device, turn on the circulating water vacuum pump 9, connect the 1000 mL filter flask 11 to the sintered glass connector 12, place the filter membrane 13 between the sintered glass connector 12 and the 300 mL filter cup 14, fix the sintered glass connector 12 and the 300 mL filter cup 14 with a duckbill clamp 15, and perform vacuum filtration using a rubber tube 10. Obtain the final filtrate in the 1000 mL filter flask 11, and measure the photometric value using a UV spectrophotometer. ② Using a centrifuge device, transfer the solution from the glass beaker 5 to centrifuge tubes 17, balance them, and place them in a benchtop high-speed refrigerated centrifuge 16 for centrifugation at 8000 rpm for 3 min. After centrifugation, collect the clear liquid and measure the photometric value using a UV spectrophotometer.

[0074] Example 2:

[0075] Add 0.8 mL of 0.5 mM NBT, 0.8 mL of 0.5 mM xanthine, and 2 mL of 0.5 mM PBS to nine 10 mL centrifuge tubes; place them in a 25°C water bath and add 0.04 U / mL of xanthine oxidase (XOD) sequentially.

[0076] As shown in Table 1:

[0077] Table 1

[0078]

[0079] The reaction was stopped after a fixed reaction time of 10 min by adding 50 μL of 12000 U / mL SOD. 1 mL of DMSO was added for extraction, and after 1.5 h of extraction, 4 mL of pure water was added. The absorbance was measured at 500 nm to 700 nm using a UV spectrophotometer. Slight fluctuations in wavelength within the detection range were observed, which is attributed to the solvation effect of the signal molecules at different concentrations. As shown in Table 2, the characteristic wavelength fluctuated slightly between 547 nm and 674 nm as the concentration of the signal molecules changed. This phenomenon is attributed to the solvent-induced color change effect and the difference in the degree of solvation at high concentrations. Since the regression equation constructed in this invention was measured in a completely consistent DMSO / water composite solvent system, this wavelength drift was automatically corrected during calibration, thus ensuring an excellent linear correlation (R0) between the absorbance at 560 nm (or near the characteristic peak) and the molar amount of superoxide radicals. 2 > 0.96).

[0080] The obtained marking data are shown in Table 2 below:

[0081] Table 2

[0082]

[0083] The regression equation obtained by the classic enzyme labeling method is linear within a controllable error range.

[0084] Example 3:

[0085] Following the solvent ratio in Example 2, add 20 mL of 0.5 mM NBT and 80 mL of pure water to each of the three beakers, add two drops of 2 mol / L NaOH to adjust the pH of the system to 11.20 ~ 11.30, add 30 mg of catalyst (Maclean / TCI), and stir in the dark for 5 min;

[0086] Beaker (M0): Dark reaction for 1 h, using Bi2O3 / BiO synthesized from materials from McLean Company. 2-x catalyst;

[0087] Beaker (M1): Visible light for 1 h; Bi2O3 / BiO synthesized using materials from McLean Corporation. 2-x catalyst;

[0088] Beaker (M2): Near-infrared light for 1 h; Bi2O3 / BiO synthesized using materials from McLean Corporation. 2-x catalyst;

[0089] Beaker (T0): Dark reaction for 1 h, using BiO synthesized from materials from TCI Corporation. 2-x catalyst;

[0090] Beaker (T1): Visible light for 1 h; BiO synthesized using materials from TCI Corporation. 2-x catalyst;

[0091] Beaker (T2): Near-infrared light for 1 h; BiO synthesized using materials from TCI. 2-x catalyst;

[0092] Turn off the light, add 0.5 mL of 12000 U / mL SOD to terminate the reaction and continue; add 125 mL of DMSO for extraction, and extract for 1.5 h; obtain a clear solution by filtration, and measure the absorbance at 500 nm to 700 nm using a UV spectrophotometer.

[0093] The obtained in-situ experimental data are shown in Table 3 below:

[0094] Table 3

[0095]

[0096] By quantitatively calculating the absorbance at characteristic wavelengths, the Bi₂O₃ / BiO₃ ratio can be quantitatively determined. 2-x The catalyst exhibits an absorbance of 0.008 under dark conditions, generating approximately 0.00940 μmol of superoxide radicals; an absorbance of 0.017 under visible light, generating approximately 0.03428 μmol of superoxide radicals; and an absorbance of 0.005 under near-infrared light, generating approximately 0.00111 μmol of superoxide radicals.

[0097] The absorbance of the TCI material under dark reaction was 0.005, which could generate approximately 0.00111 μmol of superoxide radicals; the absorbance under visible light was 0.003, which could not generate superoxide radicals; and the absorbance under near-infrared light was 0.005, which could generate approximately 0.00111 μmol of superoxide radicals.

[0098] Because the photon energy in the near-infrared band is low, the number of free radicals generated is small. However, under the extraction and separation system described in this invention, the signal can still be detected clearly and stably, and the detection limit meets the requirements for quantitative analysis.

[0099] Example 4:

[0100] Following the solvent ratio in Example 3, 20 mL of 0.5 mM NBT and 80 mL of pure water were added to each of two beakers. Two drops of 2 mol / L NaOH were added to one beaker to adjust the pH to 11.20–11.30. The pH of the other beaker was not adjusted, and the pH was measured to be 7–8. 30 mg of catalyst (Bi₂O₃ / BiO₂) was added to each beaker. 2-x(Catalyst), stir in the dark for 5 min; turn on the light source and irradiate with visible light for 1 h, turn off the light, add 0.5 mL of 12000 U / mL SOD to terminate the reaction and continue; add 125 mL of DMSO for extraction, and extract for 1.5 h; obtain a clear liquid by filtration, and detect the absorbance at 500 nm~600 nm using a UV spectrophotometer.

[0101] The obtained in-situ experimental data are shown in Table 4 below:

[0102] Table 4

[0103]

[0104] Under neutral conditions without pH adjustment, the detected superoxide radical yield was extremely low (0.0011 μmol), accounting for only 3% of the results detected by the method of this invention. This is mainly because superoxide radicals are extremely unstable in aqueous solution, especially prone to protonation disproportionation reaction (2O2) in locally acidic microenvironments. ·- +2H + →H2O2+O2), resulting in a large number of free radicals being abnormally consumed before detection.

[0105] The method of this invention significantly improves the stability of superoxide radicals by adjusting the pH of the reaction system to a strongly alkaline environment of 11.2-11.3. Under high pH conditions, the proton concentration in the system is extremely low, effectively inhibiting the disproportionation reaction, prolonging the lifetime of superoxide radicals, and ensuring that they can fully react with the NBT probe to generate formazan signal molecules. Detection results show that the superoxide radical yield is increased to 0.0398 μmol.

[0106] The above results show that the pH control technology (11.0 ~ 12.0) described in this invention can effectively suppress the self-disproportionation consumption of superoxide radicals during the treatment process. If pH control is not performed, the detection results will be seriously low, with an error of more than 95%.

[0107] Example 5:

[0108] Following the solvent ratio in Example 3, add 20 mL of 0.5 mM NBT and 80 mL of pure water to each of two beakers. Add two drops of 2 mol / L NaOH to adjust the pH of the system to 11.20-11.30, and add 30 mg of catalyst (Bi₂O₃ / BiO₂). 2-x Stir in the dark for 5 min; turn on the light source and irradiate with visible light for 1 h, turn off the light, add 0.5 mL of 12000 U / mL SOD to terminate the reaction and continue the process;

[0109] One beaker was extracted with 125 mL of DMSO for 1.5 h, while the other beaker was not added with any extractant. To maintain a consistent volume, 125 mL of pure water was added for mixing. The resulting clear liquid was obtained by filtration, and the absorbance was measured at 500 nm–600 nm using a UV spectrophotometer.

[0110] The obtained in-situ experimental data are shown in Table 5 below:

[0111] Table 5

[0112]

[0113] Traditional aqueous phase detection methods can only capture a very small amount of free formazan signal in the liquid phase, with absorbance values ​​below the detection limit and almost no superoxide radicals generated. This is mainly because the superoxide radicals generated by the heterogeneous photocatalytic reaction and the formazan molecules generated by the NBT reaction are strongly hydrophobic, readily adsorbing onto the active sites and pore structures of the catalyst surface through van der Waals forces and hydrogen bonds, making it difficult for them to desorb into the liquid phase. The method of this invention introduces a high proportion of the highly polar organic solvent DMSO for interfacial elution, successfully releasing all formazan signal molecules adsorbed on the catalyst surface into the liquid phase, increasing the absorbance value to 0.018, corresponding to a superoxide radical yield of 0.0371 μmol.

[0114] The above results demonstrate that the highly polar organic solvent interface elution technology described in this invention can effectively break the interaction between formazan molecules and the catalyst surface through solvation to compete for adsorption sites, thereby achieving full capture of the "adsorbed" signal molecules at the solid-liquid interface. Using only traditional aqueous phase detection methods would severely underestimate the true yield of superoxide radicals.

Claims

1. A heterogeneous photocatalytic absolute quantitative detection method for superoxide radicals based on bioenzyme labeling and interfacial elution, characterized in that... This method is specifically carried out in the following steps: (1) Establishing a standard curve by enzyme labeling: A known molar amount of superoxide radicals were generated using a biological enzyme-catalyzed reaction system. The superoxide radicals were reacted with nitrotetrazole blue probe to generate formazan. A highly polar organic solvent was added to extract the formazan, and the absorbance of the extract was measured at a characteristic wavelength. A standard regression equation between the absolute molar concentration of superoxide radicals and absorbance was established. (2) Multi-band heterogeneous photocatalytic reaction: Add chlorinated nitrotetrazole blue probe to a suspension containing heterogeneous catalyst, adjust the pH value of the system, and carry out photocatalytic reaction under light irradiation of a preset wavelength. (3) Termination and complete dissolution: After the reaction in step (2) is completed, a quencher is added to terminate the reaction and a reaction solution is obtained. A highly polar organic solvent is added, shaken, and eluted to obtain a mixture. (4) Physical separation and detection: The mixture obtained in step (3) is subjected to solid-liquid separation, the clear liquid is collected and the absorbance at the characteristic wavelength is measured, and the absolute molar concentration of superoxide radicals is calculated by combining the standard regression equation in step (1).

2. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... The strongly polar organic solvent mentioned in steps (1) and (3) is one or a mixture of several of dimethyl sulfoxide, anhydrous ethanol and methanol; the volume ratio of the strongly polar organic solvent to the reaction solution is 2:(10~1).

3. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... After eluting with a strongly polar organic solvent in steps (1) and (3), deionized water is added for dilution. The dilution ratio is 0.5 to 2 times the total volume of the mixture.

4. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... The bio-enzymatic reaction system described in step (1) is a xanthine / xanthine oxidase system; wherein the concentration of xanthine oxidase is 0.01~0.1 U / mL and the concentration of xanthine is 0.1~1 mmol / L.

5. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... Step (1) The concentration of the chlorinated nitrotetrazole blue probe is 0.1~1 mM.

6. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... In step (2), the concentration of the chlorinated nitrotetrazole blue probe is 0.1~1 mM.

7. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... The pH value of the system mentioned in step (2) is adjusted to 11.0~12.0 by adding an alkaline solution, which is an inorganic strong alkaline solution.

8. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... The illumination bands mentioned in step (2) include any single or multi-band composite light from ultraviolet light with wavelengths of 10~400 nm, near-infrared light with wavelengths of 400~760 nm and 760~2500 nm; the heterogeneous catalyst is one or a mixture of several of metal oxides, metal sulfides, polymer semiconductors and metal-organic framework materials.

9. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... The quencher mentioned in step (3) is superoxide dismutase, and the amount added is 6000~12000 U / mL.

10. The method for absolute quantitative detection of heterogeneous photocatalytic superoxide radicals based on bioenzyme labeling and interfacial elution according to claim 1, characterized in that... The solid-liquid separation in step (4) is performed by vacuum filtration using a 0.22~0.80 μm organic filter membrane or by centrifugation. When vacuum filtration is used, a filter membrane with a pore size of 0.22 μm is used when the heterogeneous catalyst is made of nano-sized particles, and a filter membrane with a pore size of 0.45 μm is used when the heterogeneous catalyst is made of micron-sized particles. When centrifugation is used, the separation is performed at a speed of 8000~12000 rpm for 3~5 minutes.

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