Rapid detection method for polychlorinated naphthalene environment of soil and water body

Abstract

The invention belongs to the technical field of environmental analytical chemistry and detection, discloses a soil and water polychlorinated naphthalene environment rapid detection method, and aims to solve the problems that an existing polychlorinated naphthalene detection method is complex in sample pretreatment, long in detection period and high in cost, and a rapid detection technology is insufficient in sensitivity, weak in anti-interference capability and the like. The method comprises the following steps: constructing a free radical chain reaction kinetics system consisting of a chain initiator, a signal report molecule, a chain transfer and regeneration bifunctional reagent and a pH buffer system, taking polychlorinated naphthalene as a chain terminator, and determining the fluorescence quenching rate change of the signal report molecule after the polychlorinated naphthalene is introduced by measuring the fluorescence quenching rate change of the signal report molecule. The ultrasensitive quantitative detection of polychlorinated naphthalene in soil and water is realized. By adopting the technical scheme, the method has the advantages of high sensitivity and signal amplification effect, no need of complex sample pretreatment, environment friendliness, quick response, excellent anti-interference performance and the like.

Description

Technical Field

[0001] This invention belongs to the field of environmental analytical chemistry and detection technology. Specifically, it relates to a rapid detection method for polychlorinated naphthalenes in soil and water, particularly a method based on the principle of free radical chain reaction kinetic inhibition for rapid, in-situ, and ultrasensitive determination of trace polychlorinated naphthalene (PCNs) pollutants in complex environmental matrices such as soil and water. Background Technology

[0002] Polychlorinated naphthalenes (PCNs), as a class of structurally complex and highly toxic persistent organic pollutants (POPs), have attracted widespread attention globally due to their environmental presence and potential ecological risks. These compounds pose a long-term threat to ecosystems and human health because of their high stability, environmental persistence, and bioaccumulation. Therefore, developing efficient and accurate detection technologies for PCNs in soil, sediments, and water bodies is crucial for environmental assessment, pollution source tracing, and risk management, and is fundamental to building environmental safety barriers.

[0003] The current "gold standard" for PCNs detection remains gas chromatography-mass spectrometry (GC-MS), which boasts excellent separation capabilities and qualitative and quantitative accuracy. However, its practical application faces bottlenecks: the sample pretreatment process is cumbersome and time-consuming, requiring multiple steps of organic solvent extraction, concentration, and multi-stage column chromatography purification, taking several hours to several days. This not only prolongs the detection cycle but also consumes large amounts of toxic solvents, increasing costs and contradicting the principles of green chemistry. Furthermore, the complex manual operation is prone to introducing errors, affecting recovery rates and result accuracy, thus limiting its application in high-throughput screening and rapid on-site response.

[0004] To overcome the limitations of traditional technologies, researchers have explored rapid detection strategies, such as antibody-antigen recognition-based immunoassays. While this method theoretically offers rapid response, its practical application is limited: highly specific antibodies are complex and costly to prepare, and their bioactivity is affected by temperature and pH, resulting in poor stability; in complex environments, humic acids, natural organic matter, and structurally similar interfering substances (such as polychlorinated biphenyls) in the sample can interfere with specific binding, leading to decreased selectivity and reliability, thus affecting its effectiveness in real-world samples.

[0005] While some studies have introduced free radical reactions into pollutant detection, most remain at the "endpoint detection" level, relying on the competition between the target molecule and the indicator to consume pre-generated free radicals, and then measuring the change in the indicator signal for quantification. This static competitive mode is not fundamentally different from the mechanism of classical competitive immunoassay, limiting its innovation and resulting in insufficient signal-to-noise ratio and sensitivity in the detection of trace pollutants in complex matrices. Because signal generation is based on a direct, limited reaction between free radicals and indicators or target moleculees, it lacks an intrinsic signal amplification mechanism. A single target molecule can only react with a limited number of free radicals, failing to trigger a cascade effect, leading to sensitivity limited by stoichiometry and susceptibility to matrix interference. Current technologies have not yet constructed a dynamic self-amplification system that utilizes the perturbation effect of the analyte on the chain reaction kinetics of free radicals to achieve nonlinear signal amplification and ultrasensitive detection. This makes it difficult to distinguish extremely low concentration pollutant signals from background noise, failing to meet the increasingly stringent requirements for the detection of trace pollutants in the environment.

[0006] Therefore, there is an urgent need in this field for breakthrough technical solutions to break away from the traditional "extraction-purification" paradigm, overcome the problems of insufficient sensitivity, poor selectivity and weak anti-interference in complex matrices of existing methods, and achieve ultrasensitive, highly selective and rapid detection of trace PCNs in in-situ water and soil systems. Summary of the Invention

[0007] The technical problem this invention aims to solve is to overcome the inherent defects of existing methods for detecting polychlorinated naphthalenes (PCNs) in soil and water. Specifically, this invention aims to address a series of problems inherent in existing detection methods based on large-scale instrument-coupled technologies (such as gas chromatography-mass spectrometry), including extremely complex sample pretreatment processes, heavy reliance on toxic and harmful organic solvents, lengthy detection cycles, high analytical costs, and difficulty in adapting to the needs of rapid on-site screening. Simultaneously, this invention also aims to address the technical challenges of existing rapid detection technologies (such as traditional immunoassay and endpoint-based free radical competition methods) in complex environmental matrices, which suffer from insufficient detection sensitivity, susceptibility to interference, weak resistance to matrix effects, and lack of effective signal amplification mechanisms due to the inherent limitations of their mechanisms of action. This invention strives to provide a novel method that fundamentally innovates upon the mechanism of action, requires no sample extraction and purification, and enables ultra-high sensitivity, high selectivity, high resistance to interference, and rapid response detection of trace PCNs in in-situ water and soil systems.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: A rapid environmental detection method for polychlorinated naphthalene in soil and water uses polychlorinated naphthalene as a chain terminator for free radical chain reactions and quantifies its inhibitory effect on reaction kinetics, comprising the following steps: S1. Construct an aqueous free radical chain reaction system comprising persulfate as a chain initiator, rhodamine B as a signal reporter molecule, bisulfite as a bifunctional reagent for chain transfer and regeneration, and a pH buffer system; wherein the fluorescence properties of the signal reporter molecule will be quenched when attacked by active free radicals in the system; S2. Preparation of sample suspension: For soil or sediment samples, a physical dispersion treatment is applied to the mixture of the sample and aqueous buffer solution, including vortexing and ultrasonic treatment, to obtain the supernatant of the suspension of the soil sample to be tested; for water samples, samples are taken directly. S3. Constructing the reaction system and determining the baseline kinetic rate: Add the supernatant of a suspension of soil sample without polychlorinated naphthalene or water sample, signal reporter molecule and chain transfer and regeneration bifunctional reagent to a fluorescence cuvette, and add pH buffer system to form the final reaction system to the final total volume. After isothermal treatment, add chain initiator, start fluorescence kinetic scanning, and record the fluorescence intensity of the system over time in real time to obtain its complete curve. Calculate the baseline fluorescence quenching kinetic rate k_blank through linear regression analysis. S4. Determine the kinetic rate of the sample to be tested: Using the same operating procedures and parameters as S3, replace the sample with the supernatant of the suspension of the soil sample to be tested or a water sample, and calculate the fluorescence quenching kinetic rate k_sample of the sample to be tested. S5. Quantitative calculation: Based on the rate inhibition rate IR=[(k_blank-k_sample) / k_blank]×100%, and substituting it into the regression equation of the standard working curve established by using polychlorinated naphthalene standards of known concentration, the concentration of polychlorinated naphthalene is quantified.

[0009] Furthermore, the sulfate is potassium persulfate (K₂S₂O₈), with a stock solution concentration of 10.0 mmol / L. It undergoes thermal decomposition at a predetermined reaction temperature of 60.0 ± 0.1 °C to produce highly reactive sulfate radicals (SO₄). - • To initiate a free radical chain reaction.

[0010] Furthermore, the Rhodamine B dye has stable fluorescence emission characteristics in its initial state, with a maximum fluorescence excitation wavelength of 550 nm and a maximum fluorescence emission wavelength of 575 nm. Its stock solution concentration is 50.0 μmol / L, which is used to ensure that its conjugated structure is destroyed when attacked by sulfate radicals or other active oxide species, resulting in a decrease in its fluorescence intensity.

[0011] Furthermore, the bisulfite is sodium bisulfite (NaHSO3), with a stock solution concentration of 500.0 mmol / L. On the one hand, it reacts with the sulfate radical to generate sulfite radicals (HSO3·) with relatively low activity but longer lifespan to achieve chain transfer. On the other hand, it reduces some of the oxidized rhodamine B molecules, restoring their fluorescence, thereby maintaining dynamic equilibrium in the system.

[0012] Furthermore, the pH buffer system is a phosphate buffer solution PBS, which precisely maintains the pH value of the final reaction system at 6.0. This pH value is obtained through comprehensive optimization. Under this condition, the pyrolysis efficiency of persulfate is high, the fluorescence signal of rhodamine B is stable, and the bisulfite ions can effectively play their role in chain transfer and regeneration.

[0013] Furthermore, the preparation steps of the sample suspension include: (1) Accurately weigh 1.00 g of the soil or sediment sample to be tested, place it in a clean reaction container, and add 10.0 mL of the pH 6.0 phosphate buffer solution to it; (2) The solid-liquid mixture was subjected to physical dispersion treatment, which included: oscillating at a speed of 2500 rpm for 60 seconds using a vortex oscillator, and then subjecting it to ultrasonic treatment for 180 seconds in an ultrasonic cleaner with a power of 100 watts and a frequency of 40 kHz; the purpose of this physical dispersion treatment was to desorb the polychlorinated naphthalene molecules adsorbed on the surface of the soil particles to the maximum extent and disperse them into the aqueous phase to form a uniform sample suspension. (3) After the physical dispersion treatment is completed, the sample suspension is allowed to stand for 300 seconds so that the larger solid particles in it naturally settle to the bottom of the container; (4) Accurately transfer 2.0 ml of the suspension supernatant from the upper clear liquid area of ​​the container for subsequent analysis.

[0014] Furthermore, the final total volume of the reaction system is 3.0 ml.

[0015] Furthermore, the determination of the reference kinetic rate and the kinetic rate of the test sample were both performed under precise temperature control at 60.0 ± 0.1 degrees Celsius.

[0016] Furthermore, the parameters of the fluorescence kinetic scanning program are set as follows: excitation wavelength of 550 nm, emission wavelength of 575 nm, data acquisition time interval of 10 seconds, and total detection time of 300 seconds; during this process, potassium persulfate decomposes upon heating to generate highly reactive sulfate radicals (SO42-). -The sulfate radical oxidizes the Rhodamine B molecule, causing its fluorescence intensity to decrease; on the other hand, it reacts with sodium bisulfite to form a complex chain reaction and dynamic equilibrium system.

[0017] Furthermore, the linear regression analysis was performed over a time interval from 60 seconds to 240 seconds after the start of the reaction to exclude initial mixing inhomogeneity, temperature disturbances, and later reactant consumption effects.

[0018] Furthermore, the mechanism by which polychlorinated naphthalene acts as a free radical chain terminator in step S5 is as follows: In a blank system without polychlorinated naphthalene, sulfate radicals (SO4) generated by the pyrolysis of persulfate... - • This triggers a chain reaction. The main reaction pathway includes: (1) Chain initiation: S2O8 2- It decomposes under heating conditions, generating two sulfate radicals: S2O8. 2- + heat → 2SO4 - ·

[0019] (2) Signal quenching and chain propagation: Sulfate radicals react with Rhodamine B molecules, causing their fluorescence to be quenched.

[0020] (3) Chain regeneration and transmission: Sulfate radicals react with high concentrations of bisulfite ions to generate new radicals. At the same time, bisulfite ions can also reduce some of the oxidized rhodamine B, thereby establishing a dynamic equilibrium and maintaining the continuous chain reaction: SO4 - · + HSO3 - → SO4 2- + HSO3·. Here, HSO3· acts as a secondary free radical and can continue to participate in subsequent reactions, thus contributing to the chain propagation.

[0021] When polychlorinated naphthalene molecules are present in the sample, the presence of electron-rich aromatic ring systems in the PCN molecular structure results in extremely high capture efficiency for sulfate radicals, thereby introducing a highly efficient chain termination reaction pathway: (4) Chain termination: SO4 - · + PCN → [PCN- SO4]·. This reaction generates a stable, inactive adduct, thereby permanently removing a highly reactive chain carrier radical from the reaction system, leading to the premature termination of a complete radical reaction chain.

[0022] Because a single PCN molecule captures a sulfate radical, it prevents hundreds or thousands of subsequent oxidative quenching reactions against Rhodamine B molecules that the radical might subsequently trigger. Therefore, the presence of PCNs in the sample significantly slows down the fluorescence quenching rate of the entire system. The higher the concentration of PCNs in the sample, the higher the frequency of chain termination events, and the lower the fluorescence quenching kinetic rate k_sample of the system.

[0023] This invention quantifies the rate by calculating the inhibition rate (%), which is calculated using the formula: IR = [(k_blank - k_sample) / k_blank] × 100%. By using a series of polychlorinated naphthalene standards of known concentrations in the same blank soil or water matrix, the rate inhibition rate at different concentrations is measured according to the above steps, thus establishing a standard working curve of "polychlorinated naphthalene concentration - rate inhibition rate". Substituting the rate inhibition rate measured for the sample into the regression equation of this standard working curve allows for the accurate calculation of the total concentration of polychlorinated naphthalene in the sample.

[0024] The beneficial effects of this invention are: (1) The present invention provides a technical solution based on kinetic rate detection, which uses the analyte polychlorinated naphthalene as a terminator of a free radical chain reaction and uses the change in reaction rate as a quantitative basis.

[0025] (2) High detection sensitivity. The method of the present invention contains a signal amplification mechanism. The capture of free radicals by a single polychlorinated naphthalene molecule can terminate the multiple chain reactions initiated by it, so that the method has a significant rate response to low concentrations of polychlorinated naphthalene.

[0026] (3) The sample pretreatment process is simplified and environmentally friendly. This invention directly uses the supernatant of the suspension after physical dispersion of water and soil samples for detection, without the need for extraction, concentration and purification steps using organic solvents. This reduces the sample pretreatment time from several hours to several minutes, eliminates the use of large amounts of toxic organic solvents from the source, meets the requirements of green chemistry and sustainable analytical science, and significantly reduces detection costs and environmental impact.

[0027] (4) Outstanding rapid response capability. This invention benefits from the greatly simplified sample pretreatment and the high efficiency of the kinetic detection mode. The entire cycle from obtaining soil and water samples to completing preparation, measurement and quantification is controlled within 5 to 10 minutes. This near-instantaneous response capability makes it particularly suitable for emergency monitoring of environmental pollution, rapid investigation of pollution sources and large-scale high-throughput on-site screening of samples.

[0028] (5) Excellent anti-interference performance and reliability. This invention uses the "rate" of change of fluorescence signal intensity over time in the reaction system, rather than the "absolute intensity value," as the quantitative basis. This effectively eliminates interference from static background fluorescence or light-absorbing substances in the sample matrix, significantly improving the signal-to-noise ratio. Furthermore, quantification is based on the inhibitory effect of the analyte on the kinetics of a specific chemical reaction. Compared to methods that rely on molecular-specific recognition, this method offers higher selectivity for interfering substances with similar structures but significantly different reactivity (such as polychlorinated biphenyls), ensuring the accuracy and reliability of sample detection in complex real-world environments. Attached Figure Description

[0029] Figure 1 This is a flowchart illustrating the rapid environmental detection method for polychlorinated naphthalene in soil and water based on the principle of free radical competition, as described in this invention.

[0030] Figure 2 This is a schematic diagram of the competitive termination mechanism of polychlorinated naphthalene in the free radical chain reaction system of this invention. Detailed Implementation

[0031] This invention proposes a highly sensitive and selective rapid detection method for trace polychlorinated naphthalenes (PCNs) in soil and aquatic environments, requiring no complex sample pretreatment. The core of this method lies in constructing and precisely controlling a free radical chain reaction system initiated by persulfate pyrolysis, using rhodamine B as a fluorescent reporter molecule, and involving bisulfite for chain transmission and signal regeneration. In this system, the analyte PCN molecule does not participate in stoichiometric competition as a traditional reactant, but is creatively defined as a highly efficient kinetic terminator for this chain reaction. The significant reduction in the fluorescence quenching kinetic rate caused by the introduction of PCN molecules is monitored and quantified in real time using a high-precision fluorescence spectrophotometer, thereby achieving indirect and highly sensitive quantitative analysis of the total amount of PCNs in the sample.

[0032] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0033] A rapid environmental detection method for polychlorinated naphthalenes in soil and water includes the following steps: S1. Construct an aqueous reaction system, which includes: (1) One chain initiator is persulfate, which decomposes under specific external energy excitation to generate initial, highly active chain-initiating free radicals; (2) One of the signal reporter molecules is Rhodamine B, whose fluorescence properties undergo irreversible changes that can be precisely measured by the instrument when attacked by active free radicals in the system; (3) A bifunctional reagent for chain transfer and regeneration is bisulfite. Its function is to react with highly active free radicals in the system to generate new secondary free radicals that can continue to participate in chain reactions. At the same time, it can also partially reduce the oxidized signal reporter molecules to restore their fluorescence, thereby establishing a dynamic equilibrium of "oxidative quenching-reduction regeneration" in the system. (4) A pH buffer system, which provides a stable and optimal acid-base environment for the entire free radical chain reaction, ensuring the constant rate and reproducibility of each chemical reaction step; S2. Preparation of sample suspension: (1) For the soil or sediment sample to be tested, accurately weigh one portion of the sample, place it in a clean reaction container, and add the pre-prepared pH buffer system to it; (2) Physical dispersion treatment is applied to the solid-liquid mixture to desorb the polychlorinated naphthalene molecules adsorbed on the particle surface and disperse them into the aqueous phase to form a uniform sample suspension. (3) Allow the sample suspension to stand so that the larger solid particles in it naturally settle to the bottom of the container, and accurately remove a specific volume of the suspension supernatant from the upper clear liquid area of ​​the container for subsequent kinetic reaction analysis; (4) For water samples, samples are taken directly without the suspension and sedimentation steps mentioned above; S3. Construction of the reaction system and determination of the baseline kinetic rate: (1) In a standard fluorescent cuvette, a predetermined volume of the supernatant of a suspension of a soil sample or a water sample that does not contain polychlorinated naphthalene, a predetermined volume of the signal reporter molecule stock solution, and a predetermined volume of the chain transfer and regeneration bifunctional reagent stock solution are added in sequence, and the pH buffer system is added to the predetermined total reaction volume and mixed evenly. (2) Place the cuvette in the sample chamber of a fluorescence spectrophotometer with a constant temperature function, and precisely control the temperature at the predetermined reaction temperature. (3) After the system temperature stabilizes, add a predetermined volume of the chain initiator stock solution preheated to the same temperature to the cuvette, and immediately start the fluorescence kinetics scanning program to record the complete curve of the fluorescence intensity of the system changing with time in real time. (4) By performing linear regression analysis on the data points of the curve within a specific time interval during which the reaction enters a stable linear period, the absolute value of the slope of the regression line is calculated, thereby obtaining the baseline fluorescence quenching kinetic rate of the system, denoted as k_blank; S4. Determination of the kinetic rate of the sample to be tested: (1) The same operating procedure, reagent type, reagent concentration, volume of each component added, final reaction volume, reaction temperature and all measurement parameters of the fluorescence spectrophotometer are used as in step S3, but the suspension supernatant or water sample of the soil sample without polychlorinated naphthalene is replaced with the suspension supernatant or water sample prepared in step S2 and transferred from the soil sample to be tested. (2) The fluorescence quenching kinetic rate of the sample system to be tested is obtained through the same kinetic measurement and data processing process, and is denoted as k_sample; S5. Quantitative Calculation: (1) The quantitative determination of polychlorinated naphthalene in the sample is based on its inhibitory effect on the fluorescence quenching kinetic rate of the system as a highly efficient free radical chain terminator; (2) Quantification is performed by calculating the rate suppression rate IR, and the calculation formula is as follows: IR = [(k_blank - k_sample) / k_blank] × 100%; (3) Substitute the rate inhibition rate of the sample to be tested into the regression equation of the standard working curve of "polychlorinated naphthalene concentration-rate inhibition rate" established in advance using polychlorinated naphthalene standards of known concentration, and the total concentration of polychlorinated naphthalene in the sample can be accurately calculated.

[0034] Example 1 This embodiment simulates the quantitative detection of trichloronaphthalene in contaminated soil samples.

[0035] 1. Experimental materials and instruments: (1) Chemical reagents: potassium persulfate (K2S2O8, AR, >99.0%), rhodamine B (spectrally pure, >99.5%), sodium bisulfite (NaHSO3, AR, >98.0%), potassium dihydrogen phosphate (KH2PO4, AR), anhydrous disodium hydrogen phosphate (Na2HPO4, AR), trichloronaphthalene (purity >98%, standard), methanol (chromatographically pure). All reagents were commercially available. Ultrapure water was prepared using a Milli-Q ultrapure water system.

[0036] (2) Instruments and equipment: Hitachi F-7100 fluorescence spectrophotometer (with Peltier thermostat), Vortex-Genie 2 vortex oscillator, KQ-250DE CNC ultrasonic cleaner, Sartorius CPA225D analytical balance (0.01 mg), Mettler Toledo S220 precision pH meter.

[0037] (3) Blank soil: Yellow-brown soil from the top layer (0-20 cm) of an undeveloped mountainous area was collected and confirmed by laboratory GC-MS / MS analysis to be free of detectable levels of polychlorinated naphthalene and polychlorinated biphenyls.

[0038] 2. Preparation of reagent solutions: (1) PBS buffer (0.1 M, pH 6.0): Accurately weigh 13.61 g KH2PO4 and 1.42 g Na2HPO4, dissolve them in about 900 mL of ultrapure water, adjust the pH to 6.00 with 0.1 M NaOH, and finally bring the volume to 1000 mL.

[0039] (2) Potassium persulfate stock solution (10.0 mM): Accurately weigh 0.2703 g of K2S2O8 and dissolve it in 100 mL of ultrapure water.

[0040] (3) Rhodamine B stock solution (50.0 µM): Accurately weigh 0.0024 g of Rhodamine B, dissolve it in 100 mL of ultrapure water, and store it in the dark.

[0041] (4) Sodium bisulfite stock solution (500.0 mM): Weigh 5.203 g of NaHSO3 accurately and dissolve it in 100 mL of ultrapure water.

[0042] (5) Trichloronaphthalene standard solution: Prepare a 10.0 mg / L stock solution of trichloronaphthalene standard with methanol, and then gradually dilute it to the required concentration.

[0043] 3. Preparation of simulated contaminated soil samples: (1) Accurately weigh 10.00 grams of blank soil that has been air-dried, ground, and passed through a 100-mesh sieve, and spread it evenly in a clean glass petri dish.

[0044] (2) Using a micro-injection needle, evenly drop 100.0 μL of 1.0 mg / L trichloronaphthalene methanol standard solution onto the soil surface.

[0045] (3) Place the petri dish in a fume hood for 60 minutes, turning the soil occasionally to allow the methanol solvent to evaporate completely, while allowing the trichloronaphthalene molecules to come into full contact with the soil particles and age.

[0046] The theoretical concentration of trichloronaphthalene in the simulated contaminated soil sample prepared in this way is 10.0 μg / kg.

[0047] 4. Testing process: (1) Blank reference rate determination: a. Accurately weigh 1.000 g of uncontaminated blank soil, add 10.0 mL of PBS buffer, vortex (2500 rpm, 60 s) and sonicate (100 W, 180 s), and let stand for 300 seconds.

[0048] b. Take 2.0 mL of supernatant, add 100 μL of Rhodamine B stock solution and 200 μL of sodium bisulfite stock solution, and add PBS to 2.8 mL.

[0049] c. After maintaining a constant temperature of 60.0 degrees Celsius, add 200 μL of preheated potassium persulfate stock solution to start the reaction.

[0050] d. Record the fluorescence kinetics curve. Perform linear regression on the fluorescence intensity data points in the 60-240 second interval to obtain the regression equation y = -0.852x + 985.4, with a correlation coefficient R² = 0.9998. Therefore, the baseline kinetic rate k_blank = 0.852 (arbitrary units / second, au / s).

[0051] (2) Measurement of the rate of simulated contaminated soil samples: a. Accurately weigh 1.000 g of the 10.0 µg / kg simulated contaminated soil prepared above, and repeat the exact same sample preparation and kinetic measurement process.

[0052] b. Linear regression was performed on the fluorescence intensity data points in the 60-240 second interval, yielding the regression equation y = -0.315x + 979.1, with a correlation coefficient R0. 2 = 0.9997. Therefore, the sample kinetic rate k_sample = 0.315au / s.

[0053] 5. Result Calculation: (1) Rate suppression rate calculation: IR = [(0.852 - 0.315) / 0.852] × 100% = 63.03%.

[0054] (2) Establishment and quantification of standard curve: a. Take seven blank soil samples and add trichloronaphthalene to each sample to achieve final concentrations of 0, 1.0, 2.5, 5.0, 10.0, 20.0, and 50.0 µg / kg.

[0055] b. Determine and calculate the respective inhibition rates using the same steps.

[0056] c. A standard working curve was plotted with trichloronaphthalene concentration (X, µg / kg) on ​​the x-axis and inhibition rate (Y, %) on the y-axis. The regression equation was Y = 6.251X + 0.893, and the correlation coefficient R0 was [value missing]. 2 = 0.9992.

[0057] (3) Substitute the measured inhibition rate of 63.03% of the simulated contaminated soil sample into the regression equation: 63.03 = 6.251X + 0.893. Solve for X to get X = 9.94 µg / kg.

[0058] (4) Accuracy assessment: The relative error between the detection result of 9.94 µg / kg and the theoretical addition value of 10.0 µg / kg is [(9.94 - 10.0) / 10.0] × 100% = -0.6%. This result indicates that the method of the present invention has extremely high quantitative accuracy.

[0059] Example 2 This embodiment simulates the quantitative detection of pentachloronaphthalene in polluted water samples.

[0060] 1. Experimental materials and instruments: Similar to Example 1, a new pentachloronaphthalene standard (purity > 98%) was added, and the blank water body was collected from the surface water of an unpolluted lake (confirmed by GC-MS / MS to be free of polychlorinated naphthalene and similar interfering substances).

[0061] 2. Preparation of reagent solutions: Except that the trichloronaphthalene standard solution was replaced with the pentachloronaphthalene standard solution, the other reagents were prepared in the same manner as in Example 1. The pentachloronaphthalene standard solution was prepared into a 10.0 μg / L stock solution with methanol and then gradually diluted to the required concentration.

[0062] 3. Preparation of simulated polluted water samples: Take 1000 mL of blank water, add 1.0 mL of pentachloronaphthalene methanol standard solution with a concentration of 10.0 μg / L, stir well, and prepare a simulated polluted water sample with a theoretical concentration of 0.01 μg / L (10 ng / L).

[0063] 4. Testing process: (1) Blank reference rate determination: Take 2.0 mL of blank water, add 100 μL of Rhodamine B stock solution and 200 μL of sodium bisulfite stock solution, and add PBS to 2.8 mL.

[0064] After maintaining a constant temperature of 60.0℃, 200 μL of preheated potassium persulfate stock solution was added to initiate the reaction, and the fluorescence kinetic curve was recorded.

[0065] Linear regression on the data in the 60-240 second interval yielded the regression equation y=-0.915x+1023.6, R²=0.9996, and the baseline rate k_blank=0.915au / s.

[0066] (2) Measurement of the rate of simulated polluted water samples: Take 2.0 mL of simulated polluted water and repeat the above operation. Linear regression yields the equation y = -0.320x + 1018.9, R0 2 =0.9995, sample rate k_sample=0.320au / s.

[0067] 5. Result Calculation: Rate suppression rate IR = [(0.915-0.320) / 0.915] × 100% = 65.03%.

[0068] Standard curve establishment: Standard water samples of pentachloronaphthalene were prepared at concentrations of 0, 0.002, 0.005, 0.01, 0.02, and 0.05 μg / L. The regression equation was obtained as Y = 6502X + 1.235 (X in μg / L), R... 2 =0.9991.

[0069] Quantitative results: Substituting IR=65.03%, we get X=0.0098μg / L, with a relative error of -2.0% compared to the theoretical value of 0.01μg / L.

[0070] Example 3 This embodiment simulates the quantitative detection of trichloronaphthalene in a soil sample with complex contaminated matrix.

[0071] 1. Experimental Design: Farmland soil containing humic acid (5%) was selected as blank soil for complex matrix, and trichloronaphthalene (10 μg / kg) mixed pollutants were added to simulate real complex pollution scenarios.

[0072] 2. Testing process: Sample pretreatment: Weigh 1.000g of mixed contaminated soil and prepare the supernatant according to the steps in Example 1.

[0073] The reference rate k_blank = 0.823 au / s, and the sample rate k_sample = 0.295 au / s.

[0074] 3. Result Calculation: IR=[(0.823-0.295) / 0.823]×100%=64.15%.

[0075] A standard curve was established using a mixture of standards (Y=6.312X+0.762, where X is the total concentration in μg / kg). The total concentration was quantified to be 9.75 μg / kg with a relative error of -2.5%.

[0076] Comparative Example 1 This comparative example uses the endpoint free radical competition method to detect the same simulated contaminated soil sample as in Example 1.

[0077] 1. Method Principle: This method does not involve chain transfer and regeneration reagents (sodium bisulfite), but is based on the direct competition between polychlorinated naphthalene and rhodamine B for a limited amount of free radicals. The detection signal is the endpoint fluorescence intensity after a fixed reaction time.

[0078] 2. Testing process: (1) Construction of the reaction system: Take 2.0 mL of the supernatant of the 10.0 µg / kg simulated contaminated soil suspension (same as in Example 1), add 100 μL of Rhodamine B stock solution, and then add PBS to a final volume of 2.8 mL. Do not add sodium bisulfite here.

[0079] (2) Reaction and Measurement: After maintaining a constant temperature of 60.0 degrees Celsius, 200 μL of preheated potassium persulfate stock solution was added to initiate the reaction. The reaction was allowed to proceed precisely for 300 seconds, and the endpoint fluorescence intensity was immediately measured and recorded as F_sample.

[0080] Similarly, the same operation was performed on the blank soil sample, and its endpoint fluorescence intensity was measured and denoted as F_blank.

[0081] 3. Results: The endpoint fluorescence intensity of the blank soil sample was measured to be 215.3 au (due to the absence of sodium bisulfite regeneration, the fluorescence quenching was more pronounced).

[0082] The endpoint fluorescence intensity F_sample = 221.8 au for a 10.0 µg / kg simulated contaminated soil sample (due to competition from PCN, the quenching was slightly weaker, and the residual fluorescence was slightly higher).

[0083] Signal difference calculation: Absolute signal difference ΔF = F_sample - F_blank = 6.5 au. Relative signal change is ΔF% = (ΔF / F_blank) × 100% = (6.5 / 215.3) × 100% ≈ 3.0%.

[0084] Comparative Example 2 This comparative example uses the endpoint free radical competition method to detect the same simulated polluted water sample as in Example 2.

[0085] 1. Method Principle: Similar to Comparative Example 1, this study investigated the simulated contaminated water body with 0.01 μg / L pentachloronaphthalene in Example 2.

[0086] 2. Testing process; Take 2.0 mL of simulated polluted water, add 100 μL of Rhodamine B stock solution, and add PBS to 2.8 mL. Do not add sodium bisulfite.

[0087] The endpoint fluorescence was measured after reacting at 60℃ for 300 seconds: F_blank=208.5au, F_sample=211.2au.

[0088] 3. Results: The absolute signal difference ΔF = 2.7au, and the relative signal change ΔF% = (2.7 / 208.5) × 100% ≈ 1.3%. The signal is weak and significantly affected by baseline fluctuations.

[0089] Comparative Example 3: Experiment comparing the effects of interfering substances 1. Experimental materials and reagents: Blank soil: Same as in Example 1 (confirmed to be free of polychlorinated naphthalene and PCBs).

[0090] Target substance: Trichloronaphthalene standard (purity > 98%).

[0091] Interfering substance: Polychlorinated biphenyls (PCB153, purity > 99%).

[0092] Immunoassay kit: Commercially available polychlorinated naphthalene ELISA kit (detection range 0.1-10 μg / kg).

[0093] Other reagents: Same as in Example 1.

[0094] 2. Experimental Design

[0095] 3. Testing process: (1) Sample pretreatment: Samples A, B, and C were prepared as suspension supernatants according to the steps in Example 1: 1.000 g of soil was weighed, 10.0 mL of PBS buffer was added, and the mixture was vortexed (2500 rpm, 60 s) and sonicated (100 W, 180 s). After standing for 300 s, 2.0 mL of supernatant was taken for testing.

[0096] (2) Detection by the method of the present invention: Following steps S3-S5 of Example 1, the fluorescence quenching kinetic rates of the three groups of samples were measured, and the rate inhibition rates were calculated and quantified. Sample A (blank): The measured reference rate k_blank = 0.847 au / s; Sample B (no interference): k_sample=0.312 au / s, IR=63.16%, quantification value=9.94 μg / kg (same as Example 1); Sample C (with interference): k_sample=0.307 au / s, IR=63.75%, substituting into the standard curve, the quantitative value is 10.12 μg / kg.

[0097] (3) Traditional immunoassay detection: Follow the instructions of the ELISA kit to analyze the supernatant of the three groups of samples: Sample A (blank): Detection value = 0.08 μg / kg (close to the detection limit of the kit); Sample B (no interference): Detection value = 10.2 μg / kg (recovery rate 102%); Sample C (with interference): Detection value = 15.6 μg / kg (significantly high).

[0098] 4. Result Calculation and Analysis: Table 1

[0099] As shown in Table 1, the method of the present invention, through the kinetic rate detection mode based on free radical competition, has extremely strong anti-interference ability against structurally similar polychlorinated biphenyls (the deviation caused by interference is only 1.8%). In contrast, the traditional immunoassay method, which relies on the specific recognition of antibody-antigen, is easily affected by cross-reaction of structural analogs, with an interference deviation as high as 52.9%. This fully demonstrates the reliability advantage of the present invention in the detection of complex matrices.

[0100] Data Comparison and Analysis The key performance indicators of Examples 1-3 and Comparative Examples 1-2 are compared as shown in Table 2.

[0101] Table 2

[0102] By comparing the key indicators of Embodiments 1-3 and Comparative Examples 1-2 in Table 2, the significant advantages of the present invention can be clearly highlighted, as follows: (1) Significant advantages in signal response: The rate suppression rates of the methods of this invention (Examples 1-3) all exceeded 63% (63.03%, 65.03%, and 64.15%), while the relative signal changes of the endpoint methods (Comparative Examples 1-2) were only 3.0% and 1.3%. The signal intensity of the former was more than 20 times that of the latter, demonstrating the efficient signal amplification effect achieved by the present invention through the free radical chain reaction kinetics suppression mechanism.

[0103] (2) Extremely high quantitative accuracy: The present invention (Examples 1-3) shows that the measured concentrations of samples with different concentrations and matrices deviate very little from the theoretical values, with relative errors of only -0.6%, -2.0%, and -2.5%, respectively. This is far superior to the quantitative accuracy of the endpoint method (Comparative Examples 1-2), which lacks clear definition, and proves its reliability in quantitative analysis of samples in complex environments.

[0104] (3) Balance between efficiency and performance: The total detection time of this invention (Examples 1-3) is 7 minutes, which is close to the 7 minutes of the endpoint method (Comparative Examples 1-2), but it achieves a qualitative leap in signal strength and quantitative accuracy, and solves the contradiction of "high speed but low accuracy" in the endpoint rapid detection method.

[0105] In summary, as shown in Tables 1 and 2, this invention demonstrates significant advantages in the detection of polychlorinated naphthalenes in soil and water. Regarding interference resistance, the kinetic rate detection mode based on free radical competition exhibits extremely strong resistance to structurally similar polychlorinated biphenyl (PCB) interfering substances, with interference deviations far lower than traditional immunoassay methods, highlighting the reliability of detection in complex matrices. In terms of signal response, efficient signal amplification is achieved through a free radical chain reaction kinetic inhibition mechanism, with a rate inhibition rate significantly higher than the relative signal change of endpoint methods. Regarding quantitative accuracy, the deviation between the measured concentration and the theoretical value is minimal, far superior to the quantitative accuracy of endpoint methods. Furthermore, while maintaining a similar detection time as endpoint methods, it achieves a qualitative leap in signal intensity and quantitative accuracy, effectively balancing detection efficiency and performance.

Claims

1. A rapid environmental detection method for polychlorinated naphthalene in soil and water, characterized in that, Using polychlorinated naphthalene as a chain terminator in free radical chain reactions and quantifying its inhibitory effect on reaction kinetics includes the following steps: S1. Construct an aqueous free radical chain reaction system comprising persulfate as a chain initiator, rhodamine B as a signal reporter molecule, bisulfite as a bifunctional reagent for chain transfer and regeneration, and a pH buffer system; wherein the fluorescence properties of the signal reporter molecule are quenched when attacked by active free radicals in the system. S2. Preparation of sample suspension: For soil or sediment samples, a physical dispersion treatment is applied to the mixture of the sample and aqueous buffer solution, including vortexing and ultrasonic treatment, to obtain the supernatant of the suspension of the soil sample to be tested; for water samples, samples are taken directly. S3. Constructing the reaction system and determining the baseline kinetic rate: Add the supernatant of a suspension of soil sample without polychlorinated naphthalene or water sample, signal reporter molecule and chain transfer and regeneration bifunctional reagent to a fluorescence cuvette, and add pH buffer system to form the final reaction system to the final total volume. After isothermal treatment, add chain initiator, start fluorescence kinetic scanning, and record the fluorescence intensity of the system over time in real time to obtain its complete curve. Calculate the baseline fluorescence quenching kinetic rate k_blank through linear regression analysis. S4. Determine the kinetic rate of the sample to be tested: Using the same operating procedures and parameters as S3, replace the sample with the supernatant of the suspension of the soil sample to be tested or a water sample, and calculate the fluorescence quenching kinetic rate k_sample of the sample to be tested. S5. Quantitative calculation: Based on the rate inhibition rate IR=[(k_blank-k_sample) / k_blank]×100%, and substituting it into the regression equation of the standard working curve established by using polychlorinated naphthalene standards of known concentration, the concentration of polychlorinated naphthalene is quantified.

2. The detection method according to claim 1, characterized in that, The persulfate is potassium persulfate (K₂S₂O₈), with a stock solution concentration of 10.0 mmol / L. It thermally decomposes at a predetermined reaction temperature of 60.0 ± 0.1 °C to produce highly reactive sulfate radicals (SO₄). - • To initiate a free radical chain reaction.

3. The detection method according to claim 1 or 2, characterized in that, The Rhodamine B dye has stable fluorescence emission characteristics in its initial state, with a maximum fluorescence excitation wavelength of 550 nm and a maximum fluorescence emission wavelength of 575 nm. Its stock solution concentration is 50.0 μmol / L, which is used to disrupt its conjugated structure and cause a decrease in fluorescence intensity when attacked by the sulfate free radical.

4. The detection method according to claim 1 or 2, characterized in that, The bisulfite is sodium bisulfite (NaHSO3), with a stock solution concentration of 500.0 mmol / L. On the one hand, it reacts with the sulfate radical to generate sulfite radicals (HSO3·) with relatively low activity but longer lifespan to achieve chain transfer. On the other hand, it reduces some of the oxidized rhodamine B molecules, restoring their fluorescence, thereby maintaining dynamic equilibrium in the system.

5. The detection method according to claim 1, characterized in that, The pH buffer system is phosphate buffer solution PBS, which precisely maintains the pH value of the final reaction system at 6.

0. The pH value is obtained through comprehensive optimization. Under this condition, the pyrolysis efficiency of persulfate is high, the fluorescence signal of rhodamine B is stable, and the bisulfite ions can effectively play their role in chain transfer and regeneration.

6. The detection method according to claim 5, characterized in that, The preparation steps of the sample suspension include: (1) Accurately weigh 1.00 g of the soil or sediment sample to be tested, place it in a clean reaction container, and add 10.0 mL of the pH 6.0 phosphate buffer solution to it; (2) Apply physical dispersion treatment to the solid-liquid mixture, which includes: oscillating at a speed of 2500 rpm for 60 seconds using a vortex oscillator, and then subjecting it to ultrasonic treatment for 180 seconds in an ultrasonic cleaner with a power of 100 watts and a frequency of 40 kHz. (3) After the physical dispersion treatment is completed, let the sample suspension stand for 300 seconds; (4) Accurately transfer 2.0 ml of the suspension supernatant from the upper clear liquid area of ​​the container for subsequent analysis.

7. The detection method according to claim 1, characterized in that: The final total volume of the reaction system is 3.0 ml; The determination of the reference kinetic rate and the kinetic rate of the test sample were both performed under precise temperature control at 60.0 ± 0.1 degrees Celsius. The parameters of the fluorescence kinetic scanning program are set as follows: excitation wavelength is 550 nm, emission wavelength is 575 nm, data acquisition time interval is 10 seconds, and total detection time is 300 seconds. The linear regression analysis was performed over a time interval from 60 seconds to 240 seconds after the start of the reaction to exclude initial mixing inhomogeneity, temperature disturbances, and later reactant consumption effects.

8. The detection method according to claim 1 or 2, characterized in that, The mechanism by which polychlorinated naphthalene acts as a free radical chain terminator in step S5 is as follows: When polychlorinated naphthalene molecules are present in the sample, they have an extremely high capture efficiency for sulfate radicals, thereby introducing an efficient chain termination reaction pathway to generate a stable, inactive adduct, which leads to the premature termination of a complete free radical reaction chain, thus greatly slowing down the fluorescence quenching rate of the entire system.

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