Bimolecular fluorescent probe method for distinguishing hydroxyl radical generation position in heterogeneous Fenton reaction system

Abstract

The invention provides a bimolecular fluorescent probe method for distinguishing hydroxyl radical generation positions in a heterogeneous Fenton reaction system, and relates to the field of environmental chemistry. Comprising the following steps: adding an outer ball adsorptive first probe and a non-adsorptive second probe into a to-be-detected heterogeneous Fenton reaction system, fully reacting, sampling, detecting and correcting to obtain a concentration ratio of a second oxidation product to a first oxidation product; and adding a pH regulator according to the concentration ratio so as to regulate and control the generation rate and the generation position of the hydroxyl free radicals. According to the method, a bimolecular probe system is creatively adopted, two probes are distributed on the surface of a mineral and in a solution after being combined at different concentrations and are used for representing distribution of hydroxyl radicals, the generation position of the hydroxyl radicals is defined, and the long-standing mechanism dispute is solved; by establishing and applying a precise correction model aiming at the coumarin fluorescence inner filter effect, key technical obstacles in double-probe fluorescence detection are successfully overcome, and the reliability of a quantitative result is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environmental chemistry, in particular, to a method for distinguishing the generation position of hydroxyl radicals in heterogeneous Fenton reaction system by using a double-molecule fluorescent probe. BACKGROUND

[0002] Heterogeneous Fenton reaction is crucial in environmental pollution remediation, soil organic matter transformation and biomedical applications, and the core active species is hydroxyl radical (HO•). However, for a long time, there has been scientific controversy about whether HO• is generated on the surface of catalysts (such as iron oxides) and oxidizes adsorbates, or is generated on the surface of catalysts and diffuses into the solution to oxidize dissolved substances. In addition, how the adsorption mode (inner sphere and outer sphere adsorption) of adsorbates affects the oxidation vulnerability of adsorbates has been left unresolved.

[0003] The prior art has the following main defects: 1. Lack of spatial resolution: traditional detection methods (such as using single probe molecules such as terephthalic acid, coumarin, organic pollutants, antibiotics, etc.) cannot distinguish whether HO• is generated on the surface of the catalyst or in the bulk solution, leading to ambiguous or even contradictory interpretations of the reaction mechanism; 2. Unclear probe behavior: In most studies, the interaction mode of the probe molecule with the catalyst surface (such as inner sphere and outer sphere adsorption) is unclear, which directly affects the judgment of its ability to capture HO• and the interpretation of the oxidation mechanism; 3. Analysis interference is not resolved: When multiple probes are used, the fluorescence spectra of their oxidation products may overlap, and some molecular probes (such as coumarin) have "fluorescence inner filter effect", leading to distorted detection signals and inaccurate quantification. In addition, the newly generated complex products after the oxidation of some probes interact with the mineral interface, affecting the progress of the reaction, or the generation of a variety of pollutants after the oxidation of complex compounds (organic pollutants, antibiotics, etc.), making it difficult to accurately identify the reaction mechanism (the generation and oxidation sites of HO•); 4. Poor detection sensitivity and complex detection method: The detection of oxidation products in previous Fenton reactions was mainly through liquid chromatography or liquid chromatography-mass spectrometry, which requires the development of a high-sensitivity, stable and simple method for scientific research; In summary, due to the inability of the prior art to clearly determine the generation position of HO•, it is difficult to actively and accurately regulate the reaction path based on the properties of the target pollutants (such as adsorption strength) to achieve selective and efficient degradation. SUMMARY

[0004] The application aims to provide a double-molecule fluorescent probe method for distinguishing the generation position of hydroxyl radicals in a heterogeneous Fenton reaction system, which creatively adopts a double-molecule probe system, and two probes are combined at different concentrations and are respectively distributed on the mineral surface (external sphere adsorption) and in the solution for characterizing the distribution of hydroxyl radicals. The application determines the generation position of hydroxyl radicals in a classical heterogeneous Fenton system (driven by ferrihydrite and goethite), solves the long-standing mechanism controversy, overcomes the key technical obstacles in the double-probe fluorescence detection by establishing and applying a precise correction model for the fluorescence inner filter effect of coumarin, and ensures the reliability of the quantitative results.

[0005] The application solves the technical problems by adopting the following technical solutions.

[0006] In one aspect, the application provides a double-molecule fluorescent probe method for distinguishing the generation position of hydroxyl radicals in a heterogeneous Fenton reaction system, including the following steps: adding an external sphere adsorption first probe and a non-adsorption second probe into the heterogeneous Fenton reaction system to be measured, adding H2O2 to start the heterogeneous Fenton reaction after the first probe is adsorbed and stabilized, and then sampling, detecting and correcting to obtain the concentration ratio of the second oxidation product and the first oxidation product; and determining the generation position of the hydroxyl radicals according to the concentration ratio.

[0007] Further, the external sphere adsorption first probe is terephthalate, and the non-adsorption second probe is coumarin.

[0008] Further, the sampling detection includes detecting the fluorescence intensity of the mixed solution of the first oxidation product and the second oxidation product at 315 nm / 425 nm and 325 nm / 455 nm, respectively. The fluorescence intensity of the mixed solution of the first oxidation product and the second oxidation product at 315 nm / 425 nm and 325 nm / 455 nm is detected.

[0009] Further, the correction includes correcting the fluorescence intensity and the concentration of the first oxidation product and the second oxidation product, and the correction method is as follows: ; ;

[0010]

[0011] Among them, and respectively represent the fluorescence intensity of the mixed solution of the first oxidation product and the second oxidation product at 315 nm / 425 nm and 325 nm / 455 nm. The fluorescence intensity of the mixed solution of the first oxidation product and the second oxidation product at 315 nm / 425 nm and 325 nm / 455 nm is detected. and These represent the concentrations of the first and second oxidation products after correction, respectively. , This represents the corresponding standard curve parameters; This represents the correction coefficient for the fluorescence internal filtration effect.

[0012] Furthermore, the concentration ratio is calculated as follows:

[0013] ;like A value less than 0.1 indicates that hydroxyl radicals are mainly generated on the catalyst surface; if... A value of 0.1-0.6 indicates that hydroxyl radicals can be generated in both the bulk solution phase and the mineral interface.

[0014] Furthermore, it also includes adding a pH adjuster to the heterogeneous Fenton reaction system with ferrohydride as a catalyst to regulate the generation rate and reaction position of hydroxyl radicals. The pH adjuster is HCl and / or NaOH, and the pH value does not exceed 4.5 or 5.5-6.5.

[0015] On the other hand, embodiments of the present invention also provide a bimolecular fluorescent probe system for a heterogeneous Fenton reaction system, which employs the above-described bimolecular fluorescent probe method and includes: a reaction catalysis module, a detection module, and a control module; the reaction catalysis module includes a reaction vessel and a heterogeneous Fenton catalyst and a target pollutant loaded therein; the detection module includes a dual-probe feeding unit for feeding materials into the reaction catalysis module, a fluorescence detection unit for monitoring the concentrations of the first oxidation product and the second oxidation product, and a signal unit for determining the generation location of hydroxyl radicals; the control module includes a central controller for receiving signals from the signal unit and a pH adjustment unit for controlling the pH value within the reaction catalysis module.

[0016] Furthermore, the catalyst is ferrohydrate.

[0017] Furthermore, the signal unit also incorporates a correction algorithm, and outputs a signal indicating the location of hydroxyl radical generation based on the concentration and ratio of the first and second oxidation products after correction.

[0018] Furthermore, the pH adjustment unit includes a pH meter and an automatic pH titration device, the automatic pH titration device containing a pH adjusting agent, and the pH adjustment unit and the central controller are wirelessly connected.

[0019] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. This invention is the first to use a bimolecular probe approach. Two probes are combined at different concentrations and distributed on the mineral surface and in the solution. This can directly distinguish whether HO• is generated on the “surface” or in the “solution” in the heterogeneous Fenton reaction, which solves the long-standing mechanistic controversy in this field and provides evidence that the formation location of HO• migrates with pH. 2. By establishing and applying a precise calibration model for the internal filtration effect of coumarin fluorescence, this invention successfully overcomes the key technical obstacles in dual-probe fluorescence detection and ensures the reliability of quantitative results. 3. This invention reveals the core regulatory chain of "pH → Fe(II) speciation → HO•generation location", providing a direct and operable regulatory strategy for the design of advanced oxidation processes; it can also guide the selection of the optimal reaction pH for different types of pollutants (adsorption vs. solubility), improve degradation efficiency, and reduce energy consumption. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the overall reaction principle of an embodiment of the present invention; Figure 2 This is a graph showing the fluorescence intensity changes of 2-hydroxyterephthalic acid and 7-hydroxycoumarin at different concentrations in Example 1 of the present invention; Figure 3 This is a schematic diagram of the infrared spectrum characterizing the adsorption of TPA and Coumarin in the ferrihydrite and goethite system in Example 2 of the present invention. Figure 4 This is an analysis diagram showing the effect of TPA and Coumarin adsorption on the zeta potential of the two minerals in the ferroalloy and goethite system in Example 2 of the present invention. Figure 5 This is a schematic diagram illustrating the effect of pH on probe oxidation and Fe(II) formation in the reaction of ferrohydrate with H2O2 in Example 3 of the present invention. Figure 6 The oxidation spectrum and analysis diagram of HO•pegyrite-bound terephthalic acid at pH 4.0 in Example 3 of the present invention are shown. Figure 7 The oxidation spectrum and analysis diagram of HO•pegyrite-bound terephthalic acid at pH 5.0 in Example 3 of the present invention; Figure 8This is a comparison of the adsorption and oxidation spectra of oxalate on the surface of ferroalloy in Example 4 of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0024] In all embodiments of the present invention, the English letters represent: TPA: Terephthalic acid; hTA: 2-hydroxyterephthalic acid; Coumarin: a type of coumarin; 7-Coumarin: 7-hydroxycoumarin.

[0025] Example 1 like Figure 1 As shown, this embodiment provides a detailed bimolecular fluorescent probe method for distinguishing the generation site of hydroxyl radicals in a heterogeneous Fenton reaction system based on this reaction principle, including the following steps: S1: Select the heterogeneous Fenton reaction system to be tested (including a solid catalyst, hydrogen peroxide, and the medium to be treated, etc.), and simultaneously add an externally adsorbed first probe and a non-adsorbed second probe. The externally adsorbed first probe is a charged organic molecule capable of forming an external coordination surface complex on the catalyst surface; in this embodiment, TPA is preferred. This probe adsorbs onto the catalyst surface through electrostatic interactions or hydrogen bonding, but does not form direct internal coordination bonds and resists non-radical electron transfer oxidation. The non-adsorbed second probe is a neutral organic molecule that does not significantly interact with the catalyst surface; in this embodiment, Coumarin is preferred. This probe is mainly distributed in the bulk solution phase. After a period of reaction, the characteristic oxidation products of both probes are simultaneously detected using fluorescence spectroscopy, including: The oxidation fluorescence product of the first probe with external spherical adsorption (such as TPA) is hTA, and the detection wavelength is... The wavelengths are 315nm and 425nm. The oxidation product of a non-adsorption second probe (such as Coumarin) is 7-Coumarin, and the detection wavelength is... It is 325 nm / 455 nm.

[0026] In addition, since the non-adsorbent second probe Coumarin produces a significant "fluorescence internal filtering effect" on the fluorescence signals of itself and the oxidation products of the first probe, this embodiment designs a dedicated correction model to accurately correct the original fluorescence signal and obtain the true concentrations of the two oxidation products based on the Beer-Lambert law.

[0027] The correction method includes the following steps: Known concentrations (0-250 nM) of hTA and (0-50 nM) of 7-Coumarin were mixed with Coumarin at increasingly higher concentrations, with Coumarin concentrations set at 0, 0.5, 2.5, 10, 25, 50, 100, and 200 μM. Then, [the mixtures were] prepared at [a specific concentration range]. The fluorescence intensity was measured at wavelengths of 315 nm / 425 nm and 325 nm / 455 nm, and denoted by the labels "-C" and "-T", respectively. The experimental results are as follows: Figure 2 As shown in (a)-(d), the parameters for each calibration curve (y=ax+b) are detailed in Table 1. The slopes of each calibration curve at different Coumarin concentrations in Table 1 are then used. An exponential relationship was established with coumarin concentration; see details. Figure 2 (a) - Figure 2 The illustration in (d) is used to correct for the known coumarin concentration on the fluorescence internal filtering effect of the fluorescent products hTA and 7-coumarin at two measurement wavelengths.

[0028] Table 1

[0029] Based on the results shown in the figure and Table 1, we derive the correction coefficient for the fluorescence internal filtering effect. - :

[0030]

[0031]

[0032]

[0033] In the formula, This represents the slope coefficient of the standard curve when Coumarin is absent (i.e., the Coumarin concentration is 0 μM). This indicates the use of the corresponding fitting exponential equation (based on) Figure 2 (a) - Figure 2 (d) The slope coefficients of the curves calculated from the data in the illustrations. These equations are derived by inputting the concentration of Coumarin present in the sample (…). ) can be calculated :

[0034]

[0035]

[0036] 9994). Then, according to Beer-Lambert's law, the fluorescence intensity of the sample in the mixed solution can be expressed as: ; ; in, and They represent in The fluorescence intensity of the mixture of the first and second oxidation products was measured at wavelengths of 315 nm / 425 nm and 325 nm / 455 nm. - , - The values ​​used are those from Table 1 when the Coumarin concentration is 0 nM. Based on this formula, we further derive the hTA ( ) and 7-Coumarin ( Concentration of )

[0037] Then, based on the established fluorescence intensity correction model and fluorescence product concentration calculation formula, we compared the correlation between the corrected measured values ​​and actual values ​​of the fluorescence products hTA and 7-coumarin. The results are shown in Table 2:

[0038] It can be seen that, under different concentrations of Coumarin, the difference between the corrected result and the actual concentration is extremely low, confirming the accuracy of the correction model.

[0039] S2: Based on the concentration ratio of the second oxidation product to the first oxidation product:

[0040] A pH adjuster is added to the heterogeneous Fenton reaction system (catalyst is ferrous hydroxide). The pH adjuster consists of HCl and NaOH. A value less than 0.1 indicates that hydroxyl radicals are mainly generated on the catalyst surface and oxidize the surface-adsorbed probes. In this case, adjusting the pH to maintain it within the range of 5.5-6.5 will promote the generation of hydroxyl radicals, thereby selectively oxidizing the surface-adsorbed pollutants and protecting the unadsorbed substances (dissolved substances) from oxidation. A value of 0.1-0.6 indicates that hydroxyl radicals can be generated in both the bulk solution phase and the mineral interface. In this case, adjusting the pH to maintain it within the range of no more than 4.5 is beneficial for Fe(II) to dissociate and enter the solution, triggering a homogeneous Fenton reaction, promoting the generation of hydroxyl radicals, and thus enhancing the oxidation of dissolved substances.

[0041] Example 2 This embodiment further validates the adsorption sites of TPA and Coumarin on Ferrihydrite and Goethite, including the following experimental steps: First, a suspension of nano-sized ferrihydrite or goethite was dried on the surface of an ATR device with ZnSe crystals at the bottom. Then, 10 mM NaCl was added, and the mixture was heated at 400-4000 cm⁻¹. -1 At the specified wavelength, spectra were acquired at a frequency of 1 minute per spectrum. After baseline stabilization, TPA (200 μM) was added, and infrared spectra were continuously recorded at minute intervals for 60 minutes. Experimental results are as follows: Figure 3 As shown. Among them, Figure 3 (a) shows the adsorption dynamics of TPA on the surfaces of ferrihydrite and goethite films at pH 4.5, studied using FTIR-ATR infrared spectroscopy. The displayed spectra are baseline-corrected infrared spectra. The upward arrows indicate the increase in infrared intensity during adsorption. The infrared spectrum (blue) of TPA in aqueous solution at pH 5.8 is also shown. The adsorption spectra of TPA on the iron mineral surfaces are consistent with those in solution (no red or blue shift), indicating that its adsorption mode is exospheric adsorption.

[0042] Figure 3 (b) Based on 1379cm -1 Adsorption kinetics of TPA at infrared peak intensity on ferrihydrite and goethite.

[0043] Figure 3 (c) Batch experiments show the percentage of TPA adsorption on both minerals after 30 minutes with increasing TPA concentration in the range of 10 mM NaCl and pH 3.5 to 6.5. Error bars represent the standard error of the mean (n = 3).

[0044] Figure 3(d) Baseline-corrected infrared spectra of Coumarin adsorbed on the surfaces of ferrihydrite and goethite at pH 4.5 under the same experimental conditions. The adsorption spectra coincide with the baseline, indicating that the adsorption amount is negligible.

[0045] Furthermore, the effects of TPA and Coumarin on the zeta potential of ferrihydrite and goethite were analyzed to determine whether adsorption between the two probes and the mineral surfaces existed. A 10 mM Coumarin or TPA solution was automatically titrated with a 50 m² / L ferrihydrite or goethite suspension at a rate of 10 μL / min, with the titration concentration increasing incrementally until a concentration of 4 μmol / m² (equivalent to 200 μM) was reached. The experiment was conducted at pH 4.5, with the pH manually adjusted using 1.0 M HCl or NaOH. The experimental results are as follows: Figure 4 As shown, Figure 4 The dashed lines in a and b represent the concentrations of Coumarin and TPA used in the experiments (0, 20, 100, and 200 μM, corresponding to 0, 0.2, 1.0, and 2.0 μmol / m²). The zeta potential of the iron oxide particles was determined using a Stabino II analyzer (Colloid Metrix GmbH, Meerbusch, Germany) based on flow potential and the Smoukhovsky model. Each reported value is the average of three consecutive measurements. It was found that Coumarin had no effect on the zeta potential of the two iron mineral surfaces, while TPA neutralized the positive charge on the surfaces of both iron minerals through adsorption.

[0046] The above experimental results show that Cousarin does not adsorb but is distributed in the solution, while TPA adsorbs on the catalyst surface in an exospheric adsorption mode. This further corroborates the adsorption sites determined in Example 1. This proves that the probe of the present invention can characterize the generation sites of hydroxyl radicals in both ferrihydrite and goethite catalysts.

[0047] Example 3 This embodiment further verifies the effect of pH on the oxidation products of two probes in a heterogeneous Fenton reaction system driven by ferrohydrate and H2O2, including the following steps: TPA (0-200 μM) was adsorbed onto 100 m² / L ferrohydrate, Coumarin (0-200 μM) was added, and a heterogeneous Fenton reaction was initiated by adding H2O2 (100 μM). After reacting for 1 hour, the reaction was quenched by adding 10% (v / v) methanol, and hTA was desorbed into the solution using 1 mM NaH2PO4. The solution was then filtered through a 0.22 μm filter membrane, diluted with acetate buffer at pH 5.1, and then... Fluorescence intensities were measured at two wavelengths: 315 nm / 425 nm and 325 nm / 455 nm. The internal filtration effect was then corrected using an established method, and the concentrations of 7-Coumarin and hTA were calculated.

[0048] Experimental results are as follows Figure 5 As shown. For the sake of brevity, Figure 5 (a) Only the changes in oxidation product yield with pH are shown in the experiments using 100 μM TPA and 100 μM Coumarin. It can be seen that as pH increases, the yield of 7-Coumarin decreases, while hTA first increases and then decreases, with the highest yield of hTA at pH 5.5 and the lowest yield of 7-Coumarin at pH 6.5.

[0049] Figure 5 (b) shows the change in the 7-coumarin / hTA yield ratio as a function of pH in experiments using 100 μM TPA and 100 μM Coumarin. The dashed line represents the average ratio of 7-coumarin to hTA generated in a homogeneous Fenton reaction containing 100 μM TPA and 100 μM Coumarin. This indicates that under pH conditions greater than 4.5, hydroxyl radicals are mainly generated on the mineral surface; while under pH conditions less than 4.5, hydroxyl radicals are generated both on the mineral surface and in the solution.

[0050] Figure 5 (c) shows the change in hTA yield with increasing Coumarin concentration in the presence of 100 μM TPA. This indicates that as pH increases, the amount of hydroxyl radicals that Coumarin can competitively react with decreases (competing with TPA for hydroxyl radicals), meaning that under high pH conditions, hydroxyl radicals mainly react with adsorbed TPA.

[0051] Figure 5 (d) represents the formation of Fe(II) during the reaction of ferrohydrate with different concentrations of H₂O₂ at pH 3.5 to 6.5 for 60 minutes. The measured Fe(II) includes surface-bound (≡Fe(II)) and soluble (Fe(II)) states. aq This confirms that the reduction and dissolution of ferrohydrate by H2O2, i.e., in the ferrohydrate-H2O2 heterogeneous Fenton reaction system, under low pH conditions, the reduction and dissolution of Fe(II) and the resulting homogeneous Fenton reaction are the main causes of Coumarin oxidation in solution.

[0052] Furthermore, this embodiment also utilizes the MCR-ALS method to analyze and investigate the oxidation spectral changes of HO• on TPA bound to ferrihydrite at pH 4.0, to confirm the process of hydroxylation by hydroxyl radicals generated on the mineral surface under the adsorption mode of the outer sphere. First, ferrihydrite nanoparticles were dried on the surface of an ATR device with ZnSe crystals at the bottom to form a ferrihydrite film. Then, the ferrihydrite film was cultured in 10 mM NaCl at pH 4.0 or 5.0, and the spectrum was acquired at a frequency of 1.83 minutes / spectrum until the baseline stabilized. Then, a small amount of TPA was adsorbed on the surface of the ferrihydrite, and the excess TPA in the solution was removed. After the spectrum stabilized again, H2O2 (100 μM) was added to stimulate the heterogeneous Fenton reaction, and the dynamic changes of TPA spectrum during the oxidation process were continuously collected.

[0053] Experimental results are as follows Figure 6 As shown, where, Figure 6 (a) is the infrared spectrum of the reaction between TPA in its bound state with HO•. Figure 6 (b) - Figure 6 (d) shows the multivariate curve-resolved alternating least squares (MCR-ALS) analysis of the infrared spectrum of HO• oxidized ferrophosphate-bound TPA; the resolved MCR-ALS spectra (components C1, C2, and C3) and their corresponding component contributions are indicated by the same color. The spectra of ferrophosphate-bound TPA and ferrophosphate-bound hTA are also displayed. The results indicate that HO• is generated on the surface of ferrophosphate and oxidizes the TPA adsorbed on the surface of the catalyst ferrophosphate through external coordination via a hydroxylation reaction, generating hTA.

[0054] In addition, this embodiment investigated the oxidation of ferrophosphate-bound TPA by HO• at pH 5.0 in the same manner. The experimental results are as follows: Figure 7 As shown, it can be seen that Figure 6 and Figure 7 The same trend is observed, which also proves that HO• is generated on the surface of ferrihydrite and oxidizes the TPA adsorbed on the surface of the catalyst ferrihydrite through external coordination reaction to generate hTA.

[0055] Example 4 This embodiment further verifies the influence of adsorption mode on molecular oxidative vulnerability. By comparing the adsorption spectrum of oxalate (a typical inner-sphere adsorbed molecule) on the surface of ferrihydrite with its spectrum in solution, a significant shift in the infrared spectrum of the mineral surface was found, confirming its inner-sphere adsorption mode. Furthermore, this embodiment also investigates the oxidative spectral changes of HO• on ferrihydrite-bound oxalate at pH 5.0 to confirm the hydroxylation process caused by hydroxyl radicals generated on the mineral surface under the inner-sphere adsorption mode. First, nano-ferrihydrite was dried on the surface of an ATR device with ZnSe crystals at the bottom to form a ferrihydrite film. The ferrihydrite film was then incubated in 10 mM NaCl at pH 5.0, with spectral acquisition performed at a frequency of 1.83 minutes per spectrum until baseline stability was achieved. A small amount of oxalate was then adsorbed onto the ferrihydrite surface, and excess oxalate in the solution was removed. After the spectrum stabilized again, a higher concentration of H2O2 (400 μM) was added to stimulate a heterogeneous Fenton reaction, and the dynamic changes in the oxalate spectrum during the oxidation process were continuously collected.

[0056] Experimental results are as follows Figure 8 As shown. Among them, Figure 8 (a) By comparing the infrared spectra of 100 mM free oxalate in an aqueous solution at pH 5.0 with those of 10 μM oxalate adsorbed on ferroalloy at pH 5.0, a significant shift in the infrared spectrum of the mineral surface was found, confirming its internal spherical adsorption mode.

[0057] Figure 8 (b) illustrates the oxidation of oxalate bound to ferrihydrite by HO•. The infrared spectrum of the reaction between oxalate adsorbed on the ferrihydrite surface and HO• is shown at pH 5.0. HO• is generated by adding 400 μM H₂O₂ to the ferrihydrite ATR device containing adsorbed oxalate. Infrared spectra were continuously acquired for 12 hours at a temporal resolution of 1.83 minutes per spectrum. The results indicate that hydroxyl radicals generated at the mineral interface did not cause changes in the oxalate spectrum, suggesting that substances adsorbed on inner spheres are difficult to oxidize by hydroxyl radicals generated at the mineral interface. Therefore, the adsorption mode (inner or outer sphere adsorption) plays a decisive role in the oxidative vulnerability of the adsorbed substance.

[0058] Example 5 This embodiment provides a bimolecular fluorescent probe system for a heterogeneous Fenton reaction system, employing the aforementioned bimolecular fluorescent probe method, comprising: a reaction catalysis module, a detection module, and a control module. The reaction catalysis module includes a reaction vessel and a heterogeneous Fenton catalyst and a target pollutant loaded therein. The detection module includes a dual-probe feeding unit for feeding materials into the reaction catalysis module, a fluorescence detection unit for monitoring the concentrations of the first and second oxidation products, and a signal unit for determining the generation location of hydroxyl radicals. The signal unit also incorporates a correction algorithm and outputs a signal indicating the generation location of hydroxyl radicals based on the corrected concentrations and ratio of the first and second oxidation products. The control module includes a central controller for receiving signals from the signal unit and a pH adjustment unit for controlling the pH value within the reaction catalysis module. Specifically, it can be represented as follows: The reaction vessel can be a reactor made of high-temperature and corrosion-resistant materials. The vessel opening is pre-installed with a dual-probe feeding port and a pH feeding port. An automatic pH titration device is installed above the pH feeding port, and a pH meter is installed inside the vessel to ensure real-time feeding during the reaction. The dual-probe feeding unit can be a dual-channel injection pump, with two probe solutions loaded in the two channels respectively (e.g., TPA and Coumarin in this embodiment). The heterogeneous Fenton catalyst is ferrohydrate.

[0059] Furthermore, a sampling port is provided at the bottom of the reactor, which is connected to a fluorescence detection unit (a fluorescence spectrophotometer can be selected). The fluorescence detection unit operates at a set wavelength (selected in this embodiment). The fluorescence intensity of the two oxidation products was detected at 315 nm / 425 nm and 325 nm / 455 nm, respectively, and the detected data was fed back to the central controller in the signal unit via electrical signal or wireless signal transmission.

[0060] In addition, the automatic pH titration device contains pH adjusters (HCl and NaOH in separate containers), and the pH adjustment unit and the central controller are wirelessly connected.

[0061] The specific workflow of this system is as follows: First, a heterogeneous Fenton catalyst and contaminants are added to the reactor, followed by two probes in a preset ratio. After a period of reaction, samples are collected through a sampling port at the bottom of the reactor and transmitted to a fluorescence detection unit for detection. The fluorescence detection unit transmits the detected sample fluorescence intensity data to a central controller, which uses a pre-set correction algorithm (refer to Example 1) to correct the concentrations of the first and second oxidation products in the reaction system. Then, based on the corrected concentrations of the two oxidation products and their ratio, a signal indicating the location of hydroxyl radical generation is output: when the ratio of the concentration of the first oxidation product to the concentration of the second oxidation product... A value less than 0.1 indicates that hydroxyl radicals are mainly generated on the catalyst surface, outputting a "surface generation" signal; if... A value of 0.1-0.6 indicates that hydroxyl radicals can be generated in both the bulk solution phase and the mineral interface, and outputs a "bulk generation" signal.

[0062] Then, based on the output signal and the pH value transmitted by the pH meter, the central controller autonomously controls the automatic pH titration equipment to add HCl or NaOH to the reaction vessel, so that the pH value in the reaction vessel reaches the preset standard (set to 5.5-6.5 or below 4.5 depending on the type of contaminant). Once the pH value stabilizes, the titration stops to ensure that the heterogeneous Fenton reaction proceeds under optimal pH conditions.

[0063] In summary, the embodiments of the present invention provide a bimolecular fluorescent probe method for distinguishing the generation site of hydroxyl radicals in a heterogeneous Fenton reaction system. It is the first to use a bimolecular probe method, in which two probes are combined at different concentrations and distributed on the mineral surface and in the solution. This method can directly distinguish whether HO• is generated on the "surface" or in the "solution" in the heterogeneous Fenton reaction, which solves the long-standing mechanistic controversy in this field and provides evidence that the generation site of HO• migrates with pH. This invention successfully overcomes key technical obstacles in dual-probe fluorescence detection by establishing and applying a precise calibration model for the Coumarin fluorescence internal filtration effect, thus ensuring the reliability of quantitative results. This invention reveals the core regulatory chain of "pH → Fe(II) speciation → HO•generation location", providing a direct and operable regulatory strategy for the design of advanced oxidation processes; it can also guide the selection of the optimal reaction pH for different types of pollutants (adsorption vs. solubility), improve degradation efficiency and reduce energy consumption.

[0064] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A bimolecular fluorescent probe method for distinguishing the generation site of hydroxyl radicals in a heterogeneous Fenton reaction system, characterized in that, The process includes the following steps: adding an outer spherical adsorbent first probe and a non-adsorbent second probe to the heterogeneous Fenton reaction system to be tested; after the reaction is complete, sampling and testing are performed to obtain the concentration ratio of the second oxidation product to the first oxidation product; and based on the concentration ratio, the generation location of the hydroxyl radical is determined.

2. The bimolecular fluorescent probe method according to claim 1, characterized in that, The first adsorbent probe on the outer sphere is terephthalate, and the second non-adsorbent probe is coumarin.

3. The bimolecular fluorescent probe method according to claim 2, characterized in that, The sampling and detection includes respectively in The fluorescence intensity of the mixture of the first oxidation product and the second oxidation product was detected at wavelengths of 315 nm / 425 nm and 325 nm / 455 nm.

4. The bimolecular fluorescent probe method according to claim 3, characterized in that, The correction includes correcting for fluorescence intensity and the concentrations of the first and second oxidation products, in the following manner: ; ; in, and The mixture of the first oxidation product and the second oxidation product respectively represents the solution in The fluorescence intensity of the mixture of the first and second oxidation products at wavelengths of 315 nm / 425 nm and 325 nm / 455 nm; and These represent the concentrations of the first and second oxidation products after correction, respectively. , This represents the corresponding standard curve parameters; This represents the correction coefficient for the fluorescence internal filtration effect.

5. The bimolecular fluorescent probe method according to claim 4, characterized in that, The concentration ratio is calculated as follows: like A value less than 0.1 indicates that hydroxyl radicals are mainly generated on the catalyst surface; if... A value of 0.1-0.6 indicates that hydroxyl radicals can be generated in both the bulk solution phase and the mineral interface.

6. The bimolecular fluorescent probe method according to claim 1, characterized in that, Also includes: A pH adjuster is added to a heterogeneous Fenton reaction system using ferrohydrate as a catalyst to regulate the generation rate and reaction site of hydroxyl radicals. The pH adjuster is HCl and / or NaOH, and the pH value does not exceed 4.5 or 5.5-6.

5.

7. A bimolecular fluorescent probe system for heterogeneous Fenton reaction systems, employing the bimolecular fluorescent probe method as described in any one of claims 1 or 6, characterized in that, include: The reaction catalysis module, detection module, and control module; the reaction catalysis module includes a reaction vessel and a heterogeneous Fenton catalyst and target pollutant loaded therein; The detection module includes a dual-probe feeding unit for feeding materials into the reaction catalysis module, a fluorescence detection unit for monitoring the concentrations of the first oxidation product and the second oxidation product, and a signal unit for determining the generation location of hydroxyl radicals; the regulation module includes a central controller for receiving signals from the signal unit and a pH adjustment unit for regulating the pH value within the reaction catalysis module.

8. The bimolecular fluorescent probe system according to claim 7, characterized in that, The heterogeneous Fenton catalyst is ferrohydrate.

9. The bimolecular fluorescent probe system according to claim 8, characterized in that, The signal unit also incorporates a correction algorithm, and outputs a signal indicating the location of hydroxyl radical generation based on the concentration and ratio of the first and second oxidation products after correction.

10. The bimolecular fluorescent probe system according to claim 9, characterized in that, The pH adjustment unit includes a pH meter and an automatic pH titration device. The automatic pH titration device contains the pH adjusting agent. The pH adjustment unit and the central controller are wirelessly connected.

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