A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

Abstract

The present invention belongs to the field of environmental and food pollution detection, and relates to a trimodal detection method for perfluorooctane sulfonic acid based on cobalt-doped carbon dot nanozymes. The present invention uses citric acid and ethylenediamine as synthetic raw materials, and synthesizes a blue fluorescent nanozyme with peroxidase activity in one step by a one-pot hydrothermal method. After washing, centrifugation, freeze-drying, and finally dissolving in deionized water, a cobalt-doped carbon dot fluorescent nanozyme with peroxidase activity is obtained. The cobalt-doped carbon dot fluorescent nanozyme synthesized by this method has good water solubility, extremely small nanometer size and excellent blue fluorescence performance, and its peroxidase activity can be used to establish a method for rapid simultaneous determination of PFOS by fluorescence, colorimetry and photothermal trimodality, with a detection limit as low as 0.028μmol / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental and food pollution detection, and relates to a trimodal detection method for perfluorooctane sulfonic acid based on cobalt-doped carbon dot nanozymes. Background Art

[0002] Perfluorooctane sulfonic acid (PFOS) is one of the most prevalent perfluoroalkyl compounds in the environment. It is highly resistant to natural degradation and is found worldwide. Due to its hydrophobic and fatty properties, surface activity, and chemical stability, it has been manufactured and used in various industrial and household products for decades. It is also widely used in the synthesis of fluorinated polymers, aqueous film-forming foams, and antifouling and water-repellent agents. It is a toxic, persistent organic pollutant that can persist in the environment for a long time and has been frequently detected in various environmental media, including air, water, soil, animals, plants, drinking water, food, and even in human tissues such as hair and nails. Although some methods have effectively reduced PFOS concentrations in drinking water using physical, chemical, and microbiological techniques, these methods are rarely applicable to reducing PFOS in the environment, especially in agricultural soils and food. In this context, the development of a rapid and effective PFOS detection method is of great significance for protecting the ecological environment, food safety, and human health.

[0003] In recent years, various analytical methods based on different analytical techniques have been developed to detect PFOS, including liquid chromatography-mass spectrometry, electrochemical analysis, fluorescence technology, and colorimetric analysis. Due to the differences in pollution sources, the environmental levels of PFOS in different regions vary greatly. The currently commonly used PFOS detection method is mainly related to liquid chromatography-mass spectrometry (Liquid chromatographic tandem mass spectrometric (LC-MS / MS) determination of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in the Yolk of Poultry Eggs in Malaysia, DOI: 10.3390 / molecules25102335). Although its sensitivity is very high, its operation is time-consuming, complicated, and costly, and often requires a complicated pretreatment process and has the limitation of needing professional operation. Therefore, there is an urgent need to develop an analytical method that is simple to operate, low in cost, highly sensitive, and has good selectivity to detect the content of PFOS. Recently, researchers have discovered that nanozymes, as functional nanomaterials with enzyme-mimicking properties, can not only overcome the inherent limitations of natural enzymes in terms of stability and preparation cost, but also have the designability, versatility, operability and applicability of nanomaterials. They are one of the most popular candidate materials in environmental monitoring, risk warning assessment and pollution source analysis, and therefore have received widespread attention.

[0004] Due to their surface modification, heteroatom doping, and complexation with nanoparticles, carbon dots are ideal for these composite materials due to their unique physical and chemical properties, such as excellent water dispersibility, stable chemical inertness, high resistance to photobleaching, and superior surface engineering. They have a wide range of applications, including immunosensors, colorimetric sensors, fluorescence sensors, and electrochemical sensors. For example, Zhuo et al. demonstrated that manganese-doped carbon dots have catalytic activity similar to that of oxidases. Based on the principle of the "TMB color change reaction," they can be used to quantitatively determine ascorbic acid within a certain concentration range (Manganese(II)-doped carbon dots as effective oxidase mimics for sensitive colorimetric determination of ascorbic acid, DOI: 10.1007 / s00604-019-3887-6). Summary of the Invention

[0005] The present invention aims to provide an efficient and accurate fluorescence, colorimetric, and photothermal trimodal detection method for detecting PFOS based on cobalt-doped carbon dots (Co@CDs) nanozymes, which is very important for protecting environmental health and people's life safety. The Co@CDs nanozyme in the present invention has peroxidase activity and can catalyze H2O2 to produce hydroxyl radicals, which in turn oxidize TMB to blue ox-TMB. However, the presence of PFOS will cause electron transfer with hydroxyl radicals, thereby blocking the formation of ox-TMB, showing a PFOS concentration-dependent colorimetric signal decrease, thereby developing a PFOS detection method based on TMB color development. At the same time, since the ultraviolet maximum absorption peak of ox-TMB is around 652nm, it can absorb 660nm near-infrared laser light, converting light energy into heat energy and raising the temperature. However, the presence of PFOS inhibits the formation of ox-TMB, thus showing a PFOS concentration-dependent temperature decrease, thus, a detection method based on the change of PFOS concentration and temperature can be developed. In addition, the oxidation product ox-TMB can absorb the energy of the fluorescence emission of the Co@CDs nanozyme itself at 460nm, resulting in its fluorescence quenching, which is attributed to the fluorescence resonance energy transfer (FRET) effect between the donor (Co@CDs nanozyme) and the acceptor (ox-TMB). However, the presence of PFOS inhibits the formation of ox-TMB, thereby blocking the FRET between the donor (Co@CDs nanozyme) and the acceptor (ox-TMB), restoring the fluorescence emission of the Co@CDs nanozyme itself at 460nm, and showing a PFOS concentration-dependent fluorescence signal enhancement. This allows the development of a PFOS fluorescence detection method based on the fluorescence changes of the Co@CDs nanozyme.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes, comprising the following steps:

[0007] Step 1: Determine the fluorescence intensity F1 of PFOS standards at different concentrations at 460 nm under 365 nm excitation:

[0008] The Co@CDs nanozyme solution was placed in a centrifuge tube, and 3,3',5,5'-tetramethylbenzidine solution and H2O2 were added. Then, different concentrations of perfluorooctane sulfonate standard with a pH value of 3.9-4.1 were added. After mixing evenly, the mixture was reacted for 15-20 minutes and transferred to a cuvette. The fluorescence intensity at 460 nm was measured using a fluorescence spectrometer and recorded as the fluorescence value F1.

[0009] Step 2: and / or measure the absorbance value A1 of PFOS standards of different concentrations at 652 nm:

[0010] The Co@CDs nanozyme solution was placed in a centrifuge tube, 3,3',5,5'-tetramethylbenzidine and H2O2 were added, and then different concentrations of perfluorooctane sulfonate standards with a pH value of 3.9-4.1 were added. After mixing evenly, the mixture was reacted for 15-20 minutes and transferred to a cuvette. The absorbance at 652 nm was measured using a UV spectrophotometer and recorded as absorbance A1.

[0011] Step 3: and / or measure the photothermal signal value of PFOS standards of different concentrations under 660nm laser irradiation

[0012] The Co@CDs nanozyme solution was placed in a centrifuge tube, 3,3',5,5'-tetramethylbenzidine and H2O2 were added, and then different concentrations of perfluorooctane sulfonate standards with a pH value of 3.9-4.1 were added. After mixing evenly, the mixture was reacted for 15-20 minutes and transferred to a centrifuge tube. The temperature value was measured with a portable photothermal imager and recorded as temperature T1.

[0013] Step 4: Construct a linear regression equation for the standard

[0014] The fluorescence intensity F1 value measured by the PFOS standard at different concentrations and the concentration of the standard were used to construct the linear regression equation of the fluorescence spectrometer.

[0015] and / or constructing a UV spectrophotometer linear regression equation using the absorbance A1 values ​​measured for perfluorooctane sulfonic acid standards of different concentrations and the concentration of the standards;

[0016] and / or constructing a linear regression equation for the photothermal signal using T1 values ​​calculated from tests of perfluorooctane sulfonic acid standards at different concentrations and the concentration of the standards;

[0017] Step 5: Use the sample to be tested instead of the standard to measure the fluorescence intensity value F of the sample to be tested by fluorescence spectrometer n ; and / or use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested by UV spectrophotometer n ; and / or use the sample to be tested instead of the standard to perform portable photothermal imaging to measure and calculate the temperature value T of the sample to be tested n ;

[0018] Step 6: Convert the fluorescence intensity value F n Substitute the linear regression equation of the fluorescence spectrometer to calculate the concentration value of the sample to be tested, that is, the PFOS residual value; and / or convert the absorbance value A n Substitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the PFOS residual value; and / or substitute the temperature value T nSubstitute the temperature change into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOS residual value.

[0019] The preferred technical solution is: the preparation method of the Co@CDs nanozyme comprises the following steps:

[0020] S1: Chitosan, citric acid, cobalt chloride hexahydrate, and ethylenediamine were added to deionized water containing glacial acetic acid; after sonication for 25-35 minutes, the mixture was transferred to a reactor with a polytetrafluoroethylene-lined reaction chamber; the reaction mixture was kept at 175-185°C for 5-8 hours, and then naturally cooled to room temperature;

[0021] S2: The reactants were taken out and centrifuged at 10,000-14,000 rpm for 8-15 minutes. The supernatant was filtered through a 0.22 μm filter membrane to remove impurities. The obtained product was then placed in a dialysis bag and dialyzed for 24 hours, with the deionized water for dialysis replaced every 5-7 hours. After drying, the Co@CDs nanozyme was obtained.

[0022] The preferred technical solution is: the mass ratio of chitosan, citric acid and cobalt chloride hexahydrate is: 0.04-0.06:0.04-0.06:0.2-0.3.

[0023] The preferred technical solution is: the concentration of the Co@CDs nanozyme solution is 0.2 mg / mL; the concentration of the TMB solution is 2.0 mmol / L; the concentration of the H2O2 solution is 2.0 mmol / L; the volume ratio between the Co@CDs nanozyme solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 is: 8-12:9-11:10.

[0024] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a kit for detecting perfluorooctane sulfonic acid residues, comprising Co@CDs nanozyme, H2O2 and 3,3',5,5'-tetramethylbenzidine solution.

[0025] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0026] 1. The Co@CDs nanozyme synthesis method of the present invention has the advantages of a wide range of raw materials and low cost, which is economical and affordable. Compared with other existing methods for synthesizing nanozymes, it has simple materials, a fixed method, and can be synthesized in large quantities.

[0027] 2. Compared with existing traditional methods for detecting PFOS, such as high-performance liquid chromatography-tandem mass spectrometry and gas chromatography-tandem mass spectrometry, the present invention does not require cumbersome sample pretreatment and professional operation, resulting in shorter detection time, lower cost, and wider application range. In particular, the Co@CDs nanozyme in the present invention has high peroxidase activity, which can catalyze H2O2 to produce hydroxyl radicals, thereby oxidizing TMB to blue ox-TMB. However, the presence of PFOS will cause electron transfer with hydroxyl radicals, thereby blocking the formation of ox-TMB, showing a PFOS concentration-dependent colorimetric signal decrease, thereby developing a PFOS detection method based on TMB color development. At the same time, since the maximum absorption peak of the oxidation product ox-TMB is around 652nm, it can absorb 660nm near-infrared laser, converting light energy into heat energy, and increasing the temperature. However, the presence of PFOS inhibits the formation of ox-TMB, resulting in a PFOS concentration-dependent temperature decrease, thus, a PFOS photothermal detection method based on temperature decrease can be developed. In addition, the oxidation product ox-TMB can absorb the energy of the fluorescence emission of the Co@CDs nanozyme itself at 460nm, resulting in its fluorescence quenching, which is attributed to the fluorescence resonance energy transfer effect (FRET) between the donor (Co@CDs nanozyme) and the acceptor (ox-TMB); however, the presence of PFOS inhibits the formation of ox-TMB, thereby blocking the FRET between the donor (Co@CDs nanozyme) and the acceptor (ox-TMB), restoring the fluorescence emission of the Co@CDs nanozyme itself at 460nm, and showing a PFOS concentration-dependent fluorescence signal enhancement, thereby developing a PFOS fluorescence detection method based on the fluorescence change of the Co@CDs nanozyme. In summary, the present invention has developed a PFOS trimodal detection method based on the fluorescence signal enhancement, colorimetric and photothermal signal reduction driven by Co@CDs nanozyme.

[0028] 3. Based on the disclosed invention technology for detecting PFOS in the same field (a colorimetric detection platform based on cytidine-5' phosphate-terminated gold nanoclusters with peroxidase-like activity and its application, application number: 202310444869.4; it uses gold nanoclusters as synthetic raw materials, and the linear range of PFOS detection is: 2.0-50.0μmol / L, the detection limit is: 0.150μmol / L, and the detection time is 20.0-30.0min), this invention invents a PFOS trimodal detection method based on Co@CDs nanozyme, which obtains a wider detection linear range: 0.168-100μmol / L, a lower detection limit: 0.028μmol / L, and a shorter detection time: 18.0min. At the same time, this patent uses citric acid, cobalt chloride hexahydrate, etc. as synthetic raw materials. Compared with HAuCl4·3H2O as the Au source, the synthesis technology path is simplified, the method is more controllable, and it is easy to achieve large-scale synthesis. Moreover, this patent overcomes the problem of false positive results in a single colorimetric detection mode. Based on the colorimetric mode of PFOS inhibiting TMB oxidation, the fluorescence resonance energy transfer effect of ox-TMB on Co@CDs nanozyme is used to propose a linear relationship between PFOS concentration and fluorescence enhancement of Co@CDs nanozyme. In addition, the photothermal conversion effect of ox-TMB is used to convert 660nm near-infrared laser into effective thermal energy, and a linear relationship between PFOS concentration and temperature change is developed. Finally, the triple mode of fluorescence signal enhancement, colorimetric and photothermal signal reduction is used to enhance the reliability and practicality of PFOS detection results, avoiding misjudgment caused by false positive results.

[0029] 4. This invention proposes the first trimodal PFOS detection method based on Co@CDs nanozymes. Based on the relationships between PFOS concentration and the blue fluorescence change of Co@CDs nanozymes, PFOS concentration and TMB color intensity, and PFOS concentration and temperature change, a rapid PFOS determination method using fluorescence, colorimetry, and photothermal analysis can be established. Furthermore, through mutual confirmation between the different modalities, the trimodality provides built-in cross-reference correction, greatly improving the reliability and accuracy of PFOS detection in complex environmental samples and avoiding false positives. As described in Example 7, the average recovery rate in actual environmental samples ranged from 99.95% to 102.86%, demonstrating that trimodal detection provides more accurate results. Furthermore, it is worth noting that the photothermal response has a low background signal and is not restricted by time, location, or specialized operators, making it portable.

[0030] 5. The present invention proposes a trimodal detection method for PFOS based on Co@CDs nanozymes. Depending on the sensitivity and test scenario, trimodal detection can meet different test requirements. As described in Example 8, in the absence of a 660nm laser, the fluorescence method and colorimetry method are used with built-in cross-reference correction. After mutual confirmation between different modes, the numerical value of PFOS in the sample to be tested can be more conveniently obtained; as described in Example 9, when the PFOS concentration is very low, the two methods may have large errors in the colorimetric or photothermal mode, and the fluorescence method has the highest sensitivity. Therefore, the fluorescence mode can be used to quickly obtain the PFOS concentration of the sample to be tested.

[0031] 6. This invention was successfully applied to the detection of PFOS in real-world samples (including water and soil) in the environment, demonstrating excellent sensitivity and selectivity. Furthermore, the trimodal PFOS detection method based on Co@CDs nanozymes demonstrated greater adaptability in practical applications, meeting varying test requirements based on sensitivity and available instrumentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Transmission electron microscopy image of Co@CDs nanozyme.

[0033] Figure 2 This is the electron paramagnetic resonance spectrum of Co@CDs nanozyme, which proves that Co@CDs nanozyme can catalyze hydrogen peroxide to produce hydroxyl radicals and is a nanozyme with peroxidase catalytic activity.

[0034] Figure 3 Fluorescence spectra of (1) TMB, (2) H2O2, (3) TMB+H2O2, (4) Co@CDs, (5) Co@CDs+H2O2, (6) Co@CDs+TMB, (7) Co@CDs+TMB+H2O2, and (8) Co@CDs+TMB+H2O2+PFOS measured by fluorescence spectroscopy.

[0035] Figure 4 UV spectra of (1) TMB, (2) H2O2, (3) TMB+H2O2, (4) Co@CDs, (5) Co@CDs+H2O2, (6) Co@CDs+TMB, (7) Co@CDs+TMB+H2O2, and (8) Co@CDs+TMB+H2O2+PFOS measured by UV spectrophotometer.

[0036] Figure 5Photothermal signal responses of (1) TMB, (2) H2O2, (3) TMB+H2O2, (4) Co@CDs, (5) Co@CDs+H2O2, (6) Co@CDs+TMB, (7) Co@CDs+TMB+H2O2, and (8) Co@CDs+TMB+H2O2+PFOS measured by a portable photothermal imager.

[0037] Figure 6 Optimized conditions for PFOS detection using the Co@CDs+TMB+H2O2 system. Figure (A) shows the optimized TMB concentration, with the optimal concentration being 2.0 mmol / L; Figure (B) shows the optimized H2O2 concentration, with the optimal concentration being 2.0 mmol / L; Figure (C) shows the optimized Co@CDs concentration, with the optimal concentration being 200.0 μg / mL; Figure (D) shows the optimized pH, with the optimal concentration being pH 4.0.

[0038] Figure 7 The optimal reaction time for the detection of PFOS in the Co@CDs+TMB+H2O2 system was optimized. The results showed that the optimal reaction time for this system was 18.0 min.

[0039] Figure 8 Shown are the fluorescence spectra and standard curves of PFOS at different concentrations.

[0040] Figure 9 Shows the absorbance and standard curve of different concentrations of PFOS.

[0041] Figure 10 This is the standard curve of photothermal signal of different concentrations of PFOS.

[0042] Figure 11 This is a bar chart of the trimodal detection of interfering substances in a complex environment, proving that the fluorescence, colorimetric and photothermal trimodal system of Co@CDs+TMB+H2O2 has good selectivity for PFOS.

[0043] Figure 12 This is a process flow chart for trimodal detection of perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes. DETAILED DESCRIPTION

[0044] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in these embodiments.

[0045] See also Figure 1-12. It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no technical substantive significance. Any modification of the structure, change in the proportional relationship or adjustment of the size. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0046] Related reagents used in the embodiments of the present invention:

[0047] The raw materials used in this invention: chitosan (low viscosity, <200 mPa·s) and citric acid (99.5%) were purchased from Shanghai Aladdin Reagent Co., Ltd.; cobalt chloride hexahydrate (99.0%), ethylenediamine (99.0%), and TMB (3,3',5,5'-tetramethylbenzidine, 99.5%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; acetic acid-sodium acetate buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; H2O2 (hydrogen peroxide solution) was purchased from Sinopharm Group Co., Ltd. All chemicals were purchased directly from suppliers without further purification; all water used in the experiments was ultrapure water.

[0048] Acetic acid-sodium acetate buffer (pH 4.0) is prepared by taking 50.0 mL of acetic acid-sodium acetate buffer with a pH of 5.5, and adjusting the pH of the acetic acid-sodium acetate buffer with a pH of 5.5 to 4.0 using a pH meter and hydrochloric acid solution and sodium hydroxide solution.

[0049] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0050] Example 1: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0051] The preparation method of Co@CDs nanozyme includes the following technical steps.

[0052] (1) Synthesis of Co@CDs nanozymes

[0053] Co@CDs were synthesized using a one-pot hydrothermal method. Briefly, 0.05 g of chitosan, 0.05 g of citric acid, 0.24 g of cobalt chloride hexahydrate, and 100 μL of ethylenediamine were added to 5 mL of deionized water containing 1% (v / v) glacial acetic acid. After sonication for 30 min, the mixture was quickly transferred to a Teflon-lined reaction chamber and maintained at 180°C for 6 h. The reaction mixture was then cooled naturally to room temperature.

[0054] (2) Purification and preservation of Co@CDs nanozymes

[0055] The reaction mixture was centrifuged at 12,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter to remove impurities. The resulting product was then dialyzed in a dialysis bag for 24 hours, with the dialysis water replaced every 6 hours. The Co@CDs were freeze-dried in a vacuum freeze dryer and stored in a refrigerator at 4°C, protected from light.

[0056] The peroxidase activity of Co@CDs nanozyme catalyzes hydrogen peroxide to oxidize TMB to generate blue ox-TMB. When PFOS is present in the test substance, PFOS can inhibit the formation of blue ox-TMB. Moreover, the formed ox-TMB will quench the blue fluorescence of Co@CDs nanozyme at 460nm through the fluorescence resonance energy transfer effect. In addition, ox-TMB will convert light energy into heat energy under 660nm laser irradiation. The higher the concentration of ox-TMB, the higher the temperature. Figure 1 Shown is the TEM image of Co@CDs nanozyme. It can be clearly observed that Co@CDs nanozyme is spherical and its size is about 1-3nm, indicating that it has nanoscale size. Figure 2 This demonstrates that Co@CDs nanozymes can catalyze hydrogen peroxide to produce hydroxyl radicals, demonstrating that they are nanozymes with peroxidase catalytic activity. Combined with the above data, it is demonstrated that the synthesized Co@CDs are nanozymes.

[0057] Example 2: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0058] A trimodal detection method for PFOS based on Co@CDs nanozymes

[0059] 2.1 Optimization of the PFOS detection method conditions by Co@CDs nanozyme catalytic activity

[0060] 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution was transferred to a centrifuge tube, 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution were added, and then the sample to be tested (200.0 μL system) with the pH adjusted to 4.0 was added. After mixing evenly, the reaction was carried out for 18.0 min and the solution was transferred to a cuvette. The fluorescence spectrum under excitation at a wavelength of 365 nm was measured using a fluorescence spectrophotometer, and the fluorescence intensity value was recorded as F1; and / or the absorbance value at 652 nm was measured using an ultraviolet spectrophotometer, recorded as the absorbance value A1; and / or the temperature spectrum under 660 nm laser irradiation was measured using a portable photothermal imager, recorded as T1;

[0061] Figure 3Figure 2 shows the fluorescence spectrum measured using a fluorescence spectrometer. The Co@CDs nanozyme alone exhibits a distinct blue fluorescence emission. The generated ox-TMB quenches the fluorescence of the Co@CDs nanozyme (as shown in Group 7). However, in the presence of PFOS (as shown in Group 8), the fluorescence of the Co@CDs nanozyme is restored due to the inhibition of TMB oxidation. Figure 4 The UV spectra measured by UV spectrophotometer are shown. From the figure, we can clearly see that (7) Co@CDs+TMB+H2O2 clearly generates ox-TMB, and the absorbance at 652nm is enhanced. (8) The combination of Co@CDs+TMB+H2O2+PFOS inhibits the oxidation of TMB, and the absorbance is reduced. Figure 5 This is a temperature change diagram measured by a portable photothermal imager. The ox-TMB generated by (7) Co@CDs+TMB+H2O2 can rapidly increase its temperature under the irradiation of a near-infrared laser at 660nm, while the temperature of (8) changes slowly.

[0062] Example 3: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0063] In order to study the optimal reaction conditions for the TMB color development reaction catalyzed by Co@CDs nanozymes, the optimal conditions of the detection reagents were optimized using the control variable method in the UV absorption spectrum mode. Figure 6 , Figure 6 A is the optimal concentration of H2O2. As the concentration of H2O2 increases, the absorbance value gradually increases until the H2O2 concentration reaches 2.0mmol / L, when the absorbance no longer changes but decreases. Therefore, the optimal H2O2 concentration is 2.0mmol / L. Figure 6 B is the optimized reaction concentration of TMB. As the TMB concentration increases, the absorbance value gradually increases until the absorbance no longer changes when the TMB concentration reaches 2.0 mmol / L. Therefore, the optimal TMB concentration is 2.0 mmol / L. Figure 6 C is the optimized reaction concentration of Co@CDs nanozyme. As the concentration of Co@CDs nanozyme increases, the absorbance value gradually increases until the absorbance no longer changes when the concentration of Co@CDs nanozyme is 0.2 mg / mL. Therefore, the optimal concentration of Co@CDs nanozyme is 0.2 mg / mL. Figure 6 D is the optimized pH under the same conditions, with the optimal value being pH 4.0.

[0064] Example 4: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0065] After the above optimization conditions were completed, the optimal reaction time of the TMB color development reaction catalyzed by Co@CDs nanozyme was confirmed by UV absorption spectroscopy under the same conditions.

[0066] Specifically: 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution, 10.0 μL of 2.0 mmol / L H2O2 and 10.0 μL of 2.0 mmol / L TMB were added to 170 μL of acetic acid-sodium acetate buffer with a pH value of 4.0, and the change in absorbance at 652 nm was measured by UV absorption spectroscopy every 2.0 min. Figure 7 As shown in the figure, the absorption peak of oxidized TMB increases with increasing reaction time until reaching equilibrium at 18.0 min, when TMB is completely oxidized and the absorbance at 652 nm remains unchanged. Therefore, 18.0 min is confirmed to be the optimal reaction time for this detection system.

[0067] Example 5: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0068] Standard curve establishment and sample detection method

[0069] (1) Establish the linear equation for detection

[0070] 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution, 10.0 μL of 2.0 mmol / L H2O2, and 10.0 μL of 2.0 mmol / L TMB were added to 160 μL of acetic acid-sodium acetate buffer at pH 4.0, followed by the addition of 10.0 μL of PFOS standard solutions of different concentrations. After reacting for 18.0 min, the fluorescence intensity value F1 at 460 nm was measured using a fluorescence spectrometer; and / or the absorbance value A1 at 652 nm was measured using an ultraviolet spectrophotometer; and / or the temperature spectrum under 660 nm laser irradiation was measured using a portable photothermal imager and recorded as T1;

[0071] The measured fluorescence intensity, absorbance and temperature values ​​are as follows:

[0072]

[0073]

[0074] See also Figure 8 , Figure 9 and Figure 10 After fitting and settling with origin software, it was found that there was a good linear relationship between the change in fluorescence intensity at 460 nm and the PFOS concentration in the range of 0.168-100 μmol / L, and the regression equation was F=57.81C PFOS+26.0427, the correlation coefficient was 0.9978, and the detection limit was 0.028μmol / L; there was a good linear relationship between the change in absorption intensity at 652nm and the PFOS concentration in the range of 0.798-100μmol / L, and the regression equation was A=0.01697C PFOS +0.0835, the correlation coefficient was 0.9989, and the detection limit was 0.144μmol / L; there was a good linear relationship between the temperature change under 660nm laser irradiation and the PFOS concentration in the range of 1.707-100μmol / L, and the regression equation was T=0.26249C PFOS +0.0596, the correlation coefficient was 0.9969, and the detection limit was 0.325 μmol / L.

[0075] (2) Pretreatment of test samples

[0076] The detection object is liquid food or environmental water containing PFOS residues.

[0077] Complex environmental samples require simple pretreatment before testing to remove floating impurities and insoluble matter. Filter with filter paper or filter membrane with a pore size of 0.22 μm, collect the filtrate, and adjust the pH to 4.0 with acetic acid-sodium acetate buffer for testing.

[0078] (3) Testing of samples to be tested

[0079] Measure the fluorescence spectrum of the sample to be tested under 365nm wavelength excitation

[0080] 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution was transferred to a centrifuge tube, 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution were added, and then the sample to be tested (200.0 μL system) with the pH value adjusted to 4.0 was added. After mixing evenly, the reaction was carried out for 18.0 min and the sample was transferred to a cuvette. After the reaction was carried out for 18.0 min, the fluorescence spectrum was measured under 365 nm excitation using a fluorescence spectrophotometer, and the fluorescence intensity at 460 nm was recorded as F. n ;

[0081] And / or measure the absorbance value A of the sample to be tested at 652nm n

[0082] Pipette 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution into a centrifuge tube, add 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution, then add the sample to be tested (200.0 μL system) with the pH adjusted to 4.0, mix well, react for 18.0 min, transfer to a cuvette, and measure the absorbance at 652 nm using a UV spectrophotometer, which is recorded as the absorbance value A.n ;

[0083] And / or measure the photothermal response signal value T of the sample to be tested under 660nm laser irradiation n

[0084] 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution was transferred to a centrifuge tube, 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution were added, and then the sample to be tested (200.0 μL system) with the pH value adjusted to 4.0 was added. After mixing evenly, the mixture was reacted for 18.0 min and transferred to a centrifuge tube. After 18.0 min of reaction, the sample was irradiated with a 660 nm near-infrared laser, and the temperature change value T was observed using a portable temperature imager. n ;

[0085] (4) Calculate the PFOS concentration in the sample to be tested

[0086] The linear equation of fluorescence spectrum is ΔF=57.81C PFOS +26.0427; the linear equation of absorbance is ΔA=0.01697C PFOS +0.0835; the linear equation for temperature change is ΔT=0.26249C PFOS +0.0596; Substitute into (3) to get F n 、A n and T n , we can get the PFOS concentration C in the sample to be tested PFOA The value is the PFOS residue value.

[0087] Example 6: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0088] Effects of different environmental interfering substances on the PFOS trimodal detection method based on Co@CDs nanozymes

[0089] See also Figure 11 , showing the selectivity of the fluorescence, colorimetric and photothermal trimodal detection of the present invention in different environments tested using different samples. 1: blank control group; 2: pentafluorobenzoic acid (PFBA); 3: perfluorohexanoic acid (PFHxA); 4: perfluorononanoic acid (PFNA); 5: perfluoropentanoic acid (PFpeA); 6: perfluorobutanesulfonic acid (PFBS); 7: perfluoropropionic acid (PFPA); 8: perfluorooctanoic acid (PFOA); 9: K + ;10:Ca 2+ ;11:Mg 2+ ; 12: Fe 3+ ;13:Al 3+ ;14:Cl - ;15:SO42- ;16:NO3 - ; 17: PFOS. The results show that the present invention can specifically detect PFOS, while other interfering substances have no effect on the method of the present invention, demonstrating the anti-interference and high sensitivity of the present invention.

[0090] Example 7: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0091] A trimodal detection method for PFOS based on Co@CDs nanozymes for the detection of PFOS residues in environmental samples

[0092] (1) Sample pretreatment

[0093] Random environmental water samples: Environmental water samples were randomly collected from Feicui Lake in Shushan District, Hefei City, Anhui Province. The environmental water samples were filtered through 0.22 μm filter paper or membrane to remove floating impurities and insoluble precipitates. The filtrate was collected and added to acetic acid-sodium acetate buffer at pH 4.0 and stored for subsequent use.

[0094] Random tap water samples: Tap water samples were randomly collected from the tap water pipeline of Hefei University of Technology in Hefei, Anhui Province. Tap water samples were filtered through 0.22 μm filter paper or membrane to remove floating impurities and insoluble precipitates. The filtrate was collected and added to acetic acid-sodium acetate buffer at pH 4.0 and stored for subsequent use.

[0095] Random soil samples: Soil samples were randomly collected from streets near Shushan District, Hefei City, Anhui Province. The collected soil was thoroughly dissolved in ultrapure water and sonicated for 30 minutes. The supernatant was then centrifuged at 4000 rpm for 10 minutes. The supernatant was then filtered through 0.22 μm filter paper or membrane to remove floating impurities and insoluble precipitates. The filtrate was collected and added to acetic acid-sodium acetate buffer (pH 4.0) for subsequent use.

[0096] (2) Test samples

[0097] 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution was transferred to a centrifuge tube, 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution were added, and then the sample to be tested (200.0 μL system) with the pH value adjusted to 4.0 was added. After mixing evenly, the reaction was carried out for 18.0 min and the solution was transferred to a cuvette. The fluorescence spectrum under excitation at a wavelength of 365 nm was measured using a fluorescence spectrophotometer, and the fluorescence change value at 460 nm was recorded as F n ;

[0098] Determine the absorbance change value A of the sample to be tested at 652nm n

[0099] Pipette 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution into a centrifuge tube, add 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution, then add the sample to be tested (200.0 μL system) with the pH adjusted to 4.0, mix well, react for 18.0 min, transfer to a cuvette, and measure the absorbance at 652 nm using a UV spectrophotometer, which is recorded as the absorbance value A. n ;

[0100] Determine the photothermal response signal change value T of the sample to be tested under 660nm laser irradiation n

[0101] 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution was transferred to a centrifuge tube, 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution were added, and then the sample to be tested (200.0 μL system) with the pH value adjusted to 4.0 was added. After mixing evenly, the mixture was reacted for 18.0 min and transferred to a centrifuge tube. The solution was irradiated with a 660 nm near-infrared laser, and the temperature change value T was observed using a portable temperature imager. n ;

[0102] (3) Calculate the PFOS concentration in the sample to be tested

[0103] The linear equation of fluorescence spectrum is ΔF=57.81C PFOS +26.0427; the linear equation of absorbance is ΔA=0.01697C PFOS +0.0835; the linear equation for temperature change is ΔT=0.26249C PFOS +0.0596; Substitute into (3) to get F n 、A n and T n , we can get the PFOS concentration C in the sample to be tested PFOA The value is the PFOS residue value.

[0104] The results show:

[0105] Table 1 Trimodal detection of PFOS based on Co@CDs nanozymes in different environmental samples

[0106]

[0107]

[0108] Example 8: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0109] A dual-modality method based on Co@CDs nanozymes can simultaneously detect PFOS residues in different environments. For example, in the absence of a 660nm laser, the fluorescence method and the colorimetric method can more conveniently determine the PFOS value in the sample to be tested.

[0110] Test samples

[0111] Pipette 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution into a centrifuge tube, add 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution, and then add the sample to be tested (200.0 μL system) with the pH adjusted to 4.0. After mixing evenly, react for 18.0 minutes and transfer to a fluorescence cuvette. Use a fluorescence spectrophotometer to measure the fluorescence spectrum under excitation at a wavelength of 365 nm, and record the fluorescence change value at 460 nm as F1; and / or use an ultraviolet spectrophotometer to measure the absorbance value at 652 nm, which is recorded as absorbance value A1.

[0112] Calculate the PFOS concentration in the sample to be tested

[0113] The linear equation of fluorescence spectrum is ΔF=57.81C PFOS +26.0427; the linear equation of absorbance is ΔA=0.01697C PFOS +0.0835; Substituting F1 and A1 obtained in the above, we can get the PFOS concentration C in the sample to be tested. PFOA The value is the PF OS residual value.

[0114] The results show:

[0115] Table 2 Simultaneous detection of PFOS residues based on fluorescence and colorimetric dual modalities of Co@CDs nanozymes

[0116]

[0117]

[0118] Example 9: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0119] A method based on the fluorescence mode of Co@CDs nanozymes to detect PFOS residues in different environments. For example, when the PFOS concentration is extremely low, the colorimetric mode or photothermal mode is not applicable and may cause large errors. Therefore, the more sensitive fluorescence mode can be used to calculate the value of PFOS in the sample to be tested.

[0120] Test samples

[0121] Pipette 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution into a centrifuge tube, add 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution, then add the sample to be tested (200.0 μL system) with the pH adjusted to 4.0, mix well and react for 18.0 minutes, then transfer to a fluorescence cuvette, measure the fluorescence spectrum under 365 nm excitation wavelength using a fluorescence spectrophotometer, and record the fluorescence change value F1 at 460 nm;

[0122] Calculate the PFOS concentration in the sample to be tested

[0123] The linear equation of fluorescence spectrum is ΔF=57.81C PFOS +26.0427; Substitute the F1 obtained in the above equation to obtain the PFOS concentration C in the sample to be tested. PFOA The value is the PFOS residue value.

[0124] The results show:

[0125] Table 3 Detection of PFOS residues based on Co@CDs nanozyme fluorescence mode

[0126]

[0127] The present invention has developed a PFOS trimodal detection method based on Co@CDs nanozyme using fluorescence signal enhancement, colorimetric and photothermal signal reduction. In fluorescence mode, the higher the PFOS concentration, the stronger the fluorescence emission of Co@CDs nanozyme at 460nm; in colorimetric (visible to the naked eye) mode, the higher the PFOS concentration, the less blue ox-TMB, and the lighter the blue visible to the naked eye; in photothermal mode, the higher the PFOS concentration, the less ox-TMB, the lower the heat energy generated under 660nm laser irradiation, and the lower the temperature. Finally, a fluorescence, colorimetric and photothermal trimodal detection method for detecting PFOS based on Co@CDs nanozyme can be established. The results obtained by each of the three modes are used to verify the accuracy of the PFOS detection results, thereby avoiding the output of false positive results. In addition, in different environmental scenarios, the most convenient and appropriate method among the above three methods can be selected to detect PFOS concentrations under different environments, greatly improving its scope of application.

[0128] Example 2: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0129] A method for preparing Co@CDs nanozyme, comprising:

[0130] S1: Using a one-pot hydrothermal method, we directly obtain Co@CDs nanozymes with peroxidase activity from readily available raw materials without intermediate isolation.

[0131] Step S1 specifically involves adding 0.05 g of chitosan (low viscosity, <200 mPa·s), 0.05 g of citric acid (99.5%), 0.24 g of cobalt chloride hexahydrate (99.0%), and 100 μL of ethylenediamine (99.0%) to 5.0 mL of deionized water containing 1% (v / v) glacial acetic acid. After sonication for 30 min (100% power), the mixture is rapidly transferred to a polytetrafluoroethylene-lined reactor and maintained at 180°C for 6 h. The reaction mixture is then allowed to cool naturally to room temperature.

[0132] S2: Purification and preservation of Co@CDs nanozymes.

[0133] Step S2 specifically comprises: removing the solution from the reactor and centrifuging it at 12,000 rpm for 10 minutes. The supernatant is filtered through a 0.22 μm filter to remove impurities. The resulting product is then placed in a dialysis bag and dialyzed for 24 hours, with the dialysis water replaced with ultrapure water every 6 hours. The dialyzed Co@CDs nanozyme solution is freeze-dried in a vacuum freeze dryer, and the freeze-dried Co@CDs nanozyme powder is stored in a refrigerator at 4°C in the dark.

[0134] A trimodal detection method for PFOS based on Co@CDs nanozymes, with the following specific steps:

[0135] (1) Determine the fluorescence intensity value F1 of PFOS standards of different concentrations at 460 nm under excitation at a wavelength of 365 nm: (Note: The Co@CDs nanozyme has fluorescence properties, has an optimal emission wavelength under excitation at a wavelength of 365 nm, and has a maximum emission at 460 nm).

[0136] Pipette 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution into a centrifuge tube, add 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution, then add the sample to be tested (200.0 μL system) adjusted to pH 4.0, mix well, react for 18.0 minutes, transfer to a cuvette, and measure the fluorescence intensity at 460 nm using a fluorescence spectrometer, which is recorded as the fluorescence value F1; and / or

[0137] (2) Determine the absorbance value A1 of the standard at different concentrations at 652 nm: (Note: The maximum UV absorption peak of TMB oxidized to ox-TMB is around 652 nm);

[0138] Pipette 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution into a centrifuge tube, add 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution, then add the sample to be tested (200.0 μL system) adjusted to pH 4.0, mix well, react for 18.0 minutes, transfer to a cuvette, and measure the absorbance at 652 nm using a UV spectrophotometer, which is recorded as absorbance value A1; and / or

[0139] (3) Determine the photothermal signal values ​​of PFOS standards of different concentrations under 660 nm laser irradiation: (Note: ox-TMB can absorb light energy and convert it into heat energy under 600 nm near-infrared laser irradiation, thereby increasing the temperature);

[0140] Pipette 10.0 μL of 4.0 mg / mL Co@CDs nanozyme solution into a centrifuge tube, add 2.0 mmol / L TMB and 2.0 mmol / L H2O2 solution, then add the sample to be tested (200.0 μL system) with the pH adjusted to 4.0, mix well, react for 18.0 minutes, and transfer to a centrifuge tube. Use a portable photothermal imager to measure the temperature value, which is recorded as temperature T1;

[0141] (4) Constructing the linear regression equation of the standard

[0142] Constructing a fluorescence spectrometer linear regression equation using the fluorescence intensity values ​​F1 values ​​measured for different concentrations of the standard and the concentration of the standard; and / or

[0143] Constructing a UV spectrophotometer linear regression equation using the absorbance values ​​A1 values ​​measured for standards of different concentrations and the concentration of the standards; and / or

[0144] The linear regression equation of the photothermal signal was constructed using the T1 values ​​calculated from the tests of different concentrations of the standard and the concentration of the standard;

[0145] (5) Use the sample to be tested instead of the standard to measure the fluorescence intensity value F of the sample to be tested by fluorescence spectrometer n ; Use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested by UV spectrophotometer n Or use the sample to be tested instead of the standard to measure and calculate the temperature value T of the sample to be tested by the portable thermal imager n ;

[0146] (6) The fluorescence intensity value F n Substitute the fluorescence spectrum into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOS residual value; and / or convert the absorbance value A nSubstitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the PFOS residual value; and / or substitute the temperature value T n Substitute the temperature change into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOS residual value.

[0147] The concentration of the Co@CDs nanozyme solution is 0.2 mg / ml; the concentration of the TMB solution is 2.0 mmol / L; the concentration of the H2O2 solution is 2.0 mmol / L; acetic acid-sodium acetate buffer is used as the solvent, and the standard is dispersed in the acetic acid-sodium acetate buffer, pH 4.0; the volume ratio between the Co@CDs nanozyme solution, TMB solution, H2O2 and acetic acid-sodium acetate buffer is: 10:10:10:160-180; the optional buffer can also be a phosphate buffer or a citric acid-sodium citrate buffer.

[0148] After the mixture in step (1) is evenly mixed, the mixture is reacted for 18.0 minutes and then transferred to a cuvette for detection.

[0149] The test sample is liquid food or environmental water with PFOS residues. Before testing, the test sample is filtered with filter paper or filter membrane with a pore size of 0.22μm and the filtrate is collected.

[0150] The regression equation of fluorescence spectrum is F=57.81C PFOS +26.0427; the regression equation of the UV spectrophotometer is A=0.01697C PFOS +0.0835; the linear regression equation for temperature change is T = 0.26249C PFOS +0.0596.

[0151] Example 3: A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes

[0152] A trimodal detection method for PFOS based on Co@CDs nanozymes, the method comprising:

[0153] S11: 20.0 μL of 2.0 mg / mL Co@CDs nanozyme solution, 20.0 μL of 2.0 mmol / L H2O2, and 20.0 μL of 2.0 mmol / L TMB were added to 140 μL of acetic acid-sodium acetate buffer at pH 4.0, followed by the addition of 10.0 μL of PFOS standard solutions of different concentrations. After the reaction lasted for 18.0 min, the fluorescence intensity value F1 at 460 nm was measured using a fluorescence spectrometer; and / or the absorbance value A1 at 652 nm was measured using an ultraviolet spectrophotometer; and / or the temperature spectrum under 660 nm laser irradiation was measured using a portable photothermal imager and recorded as T1;

[0154] S12 constructing a linear regression equation for the fluorescence spectrum using the absorbance values ​​F1 values ​​measured for the standards of different concentrations and the concentrations of the standards; and / or

[0155] Constructing a UV spectrophotometer linear regression equation using the absorbance values ​​A1 values ​​measured for standards of different concentrations and the concentration of the standards; and / or

[0156] The temperature change linear regression equation was constructed using the T1 values ​​calculated from the tests of different concentrations of the standard and the concentration of the standard;

[0157] S13 uses the sample to be tested that has been pre-treated by filtration with 0.22 μm filter paper or filter membrane instead of the standard to perform fluorescence spectrum measurement to obtain the fluorescence intensity value F of the sample to be tested. n ; and / or use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested by UV spectrophotometer n ; and / or use the sample to be tested instead of the standard to perform portable photothermal imager measurement to obtain the temperature change value T of the sample to be tested n .

[0158] S14 converts the fluorescence intensity value F n Substitute the fluorescence spectrum into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOS residual value; and / or convert the absorbance value A n Substitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the PFOS residual value; and / or convert the ratio T n Substitute the temperature change into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOS residual value.

[0159] The present invention can use fluorescence, colorimetry, and photothermal detection of PFOS simultaneously, or can use fluorescence, colorimetry, or photothermal detection of PFOS independently. Relatively speaking, the fluorescence detection mode has the highest sensitivity for PFOS.

[0160] The present invention provides a PFOS residue detection kit, comprising a Co@CDs nanozyme, H2O2, a buffer, and a TMB colorimetric solution. The buffer is an acetic acid-sodium acetate buffer, a phosphate buffer, or a citric acid-sodium citrate buffer.

[0161] In one aspect of the present invention, the present invention also discloses the use of relevant detection reagents in trimodal detection of PFOS residues in complex environmental samples.

[0162] The method established in the present invention shows a good linear relationship between the change value of fluorescence intensity at 460nm and the PFOS concentration in the range of 0.168-100μmol / L, and the regression equation is F=57.81C PFOS+26.0427, the correlation coefficient was 0.9978, and the detection limit was 0.028μmol / L, which was obtained using the formula for the minimum detection limit (3σ / k, σ is the standard deviation of 11 blank samples); there was a good linear relationship between the change in absorption intensity at 652nm and the PFOS concentration in the range of 0.798-100μmol / L, and the regression equation was A=0.01697C PFOS +0.0835, the correlation coefficient was 0.9989, and the detection limit was 0.144μmol / L, which was obtained using the formula for the minimum detection limit (3σ / k, σ is the standard deviation of 11 blank samples); there was a good linear relationship between the temperature change under 660nm laser irradiation and the PFOS concentration in the range of 1.707-100μmol / L, and the regression equation was T=0.26249C PFOS +0.0596, the correlation coefficient was 0.9969, and the detection limit was 0.325 μmol / L, which was obtained using the formula for the minimum detection limit (3σ / k, where σ is the standard deviation of 11 blank samples).

[0163] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes, characterized by: The steps include: Step 1: Determine the fluorescence intensity F1 of PFOS standards at different concentrations at 460 nm under excitation at 365 nm: The Co@CDs nanozyme solution was placed in a centrifuge tube, and 3,3',5,5'-tetramethylbenzidine solution and H2O2 were added. Then, different concentrations of perfluorooctane sulfonate standard with a pH value of 3.9-4.1 were added. After mixing evenly, the reaction lasted for 15-20 minutes and the tube was transferred to a cuvette. The fluorescence intensity at 460 nm was measured using a fluorescence spectrometer and recorded as the fluorescence value F1. Step 2: and / or measure the absorbance of PFOS standards at different concentrations at 652 nm A1: The Co@CDs nanozyme solution was placed in a centrifuge tube, 3,3',5,5'-tetramethylbenzidine and H2O2 were added, and then different concentrations of perfluorooctane sulfonate standards with a pH value of 3.9-4.1 were added. After mixing evenly, the mixture was reacted for 15-20 minutes and transferred to a cuvette. The absorbance at 652 nm was measured using a UV spectrophotometer and recorded as absorbance A1. Step 3: and / or measure the photothermal signal value of PFOS standards with different concentrations under 660 nm laser irradiation The Co@CDs nanozyme solution was placed in a centrifuge tube, 3,3',5,5'-tetramethylbenzidine and H2O2 were added, and then different concentrations of perfluorooctane sulfonate standards with a pH value of 3.9-4.1 were added. After mixing evenly, the mixture was reacted for 15-20 minutes and transferred to a centrifuge tube. The temperature value was measured with a portable photothermal imager and recorded as temperature T1. Step 4: Construct a linear regression equation for the standard The fluorescence intensity F1 value measured by the PFOS standard at different concentrations and the concentration of the standard were used to construct the linear regression equation of the fluorescence spectrometer. and / or constructing a UV spectrophotometer linear regression equation using the absorbance A1 values ​​measured for perfluorooctane sulfonic acid standards of different concentrations and the concentration of the standards; and / or constructing a linear regression equation for the photothermal signal using T1 values ​​calculated from tests of perfluorooctane sulfonic acid standards at different concentrations and the concentration of the standards; Step 5: Use the sample to be tested instead of the standard to measure the fluorescence intensity value F of the sample to be tested by fluorescence spectrometer n ; and / or use the sample to be tested instead of the standard to measure the absorbance value A of the sample to be tested by UV spectrophotometer n ; and / or use the sample to be tested instead of the standard to perform portable photothermal imaging to measure and calculate the temperature value T of the sample to be tested n ; Step 6: Convert the fluorescence intensity value F n Substitute the linear regression equation of the fluorescence spectrometer to calculate the concentration value of the sample to be tested, that is, the PFOS residual value; and / or convert the absorbance value A n Substitute the linear regression equation of the UV spectrophotometer to calculate the concentration value of the sample to be tested, that is, the PFOS residual value; and / or substitute the temperature value T n Substitute the temperature change into the linear regression equation to calculate the concentration value of the sample to be tested, that is, the PFOS residual value.

2. The trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes according to claim 1, characterized in that: The preparation method of the Co@CDs nanozyme comprises the following steps: S1: Chitosan, citric acid, cobalt chloride hexahydrate, and ethylenediamine were added to deionized water containing glacial acetic acid. After sonication for 25-35 minutes, the mixture was transferred to a reactor with a polytetrafluoroethylene-lined reaction chamber. The reaction mixture was kept at 175-185°C for 5-8 hours and then cooled naturally to room temperature. S2: The reactants were centrifuged at 10,000-14,000 rpm for 8-15 minutes, and the supernatant was filtered through a 0.22 µm filter membrane to remove impurities. The resulting product was then placed in a dialysis bag and dialyzed for 24 hours, with the deionized water replaced every 5-7 hours. After drying, the Co@CDs nanozyme was obtained.

3. The trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes according to claim 2, characterized in that: The mass ratio of chitosan, citric acid and cobalt chloride hexahydrate is: 0.04-0.06: 0.04-0.06: 0.2-0.

3.

4. The trimodal detection method for perfluorooctane sulfonate based on cobalt-doped carbon dot nanozymes according to claim 3, characterized in that: The concentration of Co@CDs nanozyme solution is 0.2 mg / mL; the concentration of TMB solution is 2.0 mmol / L; the concentration of H2O2 solution is 2.0 mmol / L. The volume ratio between Co@CDs nanozyme solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 is: 8-12: 9-11:

10.

5. A kit for detecting perfluorooctane sulfonic acid residues, characterized in that: The kit includes Co@CDs nanozyme, H2O2 and 3,3',5,5'-tetramethylbenzidine solution, and the detection method adopts the perfluorooctane sulfonate trimodal detection method based on cobalt-doped carbon dot nanozyme according to any one of claims 1-4.

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