Cyclodextrin polymer self-assembled fluorescent array for portable detection of perfluorinated compounds

Abstract

The invention discloses a perfluorinated compound detection method based on a cyclodextrin polymer sensing array and a portable device, and relates to the field of environmental pollutant detection. The method comprises the following steps: synthesizing a cyclodextrin polymer, self-assembling the cyclodextrin polymer and a fluorescent dye, screening out four probes to construct a sensing array, and measuring the response conditions of the sensing array and six perfluorinated compounds to construct a fingerprint spectrum, so as to distinguish the categories of the perfluorinated compounds in a sample to be detected. A standard curve is also established to quantitatively measure the concentration of the perfluorinated compound, and the detection effect on a mixed sample and an actual water sample is good; and combining with a micro-fluidic chip to construct the portable perfluorine screening device. The detection method disclosed by the invention is rapid, sensitive, low in cost, portable and suitable for detecting the perfluorinated compounds in various environmental samples.

Description

Technical Field

[0001] This invention relates to the field of environmental pollutant detection, specifically to a portable detection technology for perfluorinated compounds based on a cyclodextrin polymer self-assembled fluorescence array. Background Technology

[0002] Perfluorinated compounds (PFCs) are a typical new class of pollutants, diverse in type and structurally similar, such as perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS). PFCs possess excellent stability, high surface activity, and unique hydrophobic and oleophobic properties, leading to their widespread use in industrial production (e.g., surfactants, refrigerants, paints, fire extinguishing foams) and daily life (e.g., cosmetics). However, their large-scale use has resulted in their widespread distribution in the environment, making them a global environmental pollutant. PFCs are persistent and bioaccumulative, potentially causing various health problems, including liver, immune, reproductive, neurological, genotoxic, and endocrine disruption. One of the main routes of human exposure to PFCs is through contaminated drinking water; therefore, the detection of PFCs in water bodies is crucial.

[0003] Currently, while large-scale instrument-based detection methods such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) offer high sensitivity and resolution, they suffer from limitations such as complex sample processing, long detection cycles, and high costs, restricting their application in large-scale screening and rapid on-site detection. Detection methods based on fluorescence sensors have attracted attention due to their advantages of ease of operation, rapid response, and low cost; however, traditional fluorescent dyes suffer from poor water solubility and susceptibility to photobleaching. Summary of the Invention

[0004] The purpose of this invention is to provide a fluorescent sensing array for perfluorinated compounds based on cyclodextrin polymers and a portable detection device thereof, so as to achieve rapid, sensitive, low-cost and portable detection of perfluorinated compounds in water.

[0005] 1. Synthesis of Cyclodextrin Polymer: A cyclodextrin polymer with a specific cavity structure was prepared by chemical synthesis, enabling it to form stable assemblies with fluorescent dyes. 11.35 g (10 mmol) of cyclodextrin monomers (CDs) were accurately weighed and slowly added to 15 mL of 15 wt% NaOH solution. The mixture was stirred at 35 °C for 2 hours to ensure complete dissolution of the CDs monomers. 2 mL of toluene was added to the solution, and the mixture was stirred at a constant temperature for 2 hours. 4.0 mL of epichlorohydrin, a crosslinking agent, was slowly added dropwise. After the addition was complete, the mixture was stirred at 35 °C for another 3 hours. After the reaction was complete, the reaction solution was transferred to a container containing 200 mL of isopropanol for pre-precipitation, and the supernatant was discarded. The precipitate was dissolved in water, and the pH of the solution was adjusted to neutral with dilute hydrochloric acid. The solution was then transferred to an 8000 D dialysis bag and dialyzed for 3 days. After dialysis, the β-cyclodextrin polymer was freeze-dried and stored at -20 °C for later use.

[0006] 2. Assembly and Screening of Fluorescent Probes: The synthesized cyclodextrin polymer was assembled with various fluorescent dyes, and four fluorescent probes showing significant responses to perfluorinated compounds were screened experimentally. Methanol solutions of various fluorescent dyes at a final concentration of 10 μmol / L were incubated with different concentrations of β-CDP (0, 100, 200, 300, 400, and 500 μmol / L, calculated based on the β-CD monomer concentration) in a constant-temperature metal shaking bath for 30 min (25 °C, 150 rpm) to form β-CDP / dye complexes. The fluorescence intensity was measured using a fluorometer, and four fluorescent dyes showing significant changes in fluorescence intensity in the presence of β-CDP were screened.

[0007] 3. Construction of the sensor array: A sensor array was constructed using four selected fluorescent probes. Fingerprint patterns were constructed by measuring the responses of the probes to six perfluorinated compounds. The four selected fluorescent dyes were mixed with β-CDP, and then each of the six perfluorinated compounds (100 μg / L) was added to construct a 4 × 6 fluorescent probe array. The fluorescence intensity of the sensor array after interaction with the perfluorinated compounds was measured using a fluorometer, with six parallel experiments performed for each perfluorinated compound. The obtained data were input into SPSS 17 for linear discriminant analysis (LDA). The analytical results were used to determine the sensor array's ability to distinguish between different perfluorinated compounds, and fingerprint patterns were constructed. The types of perfluorinated compounds in the test samples were then distinguished based on the fingerprint patterns.

[0008] 4. Establishment of Standard Curve: A standard curve was established by comparing the response of perfluorinated compound standards with known concentrations, enabling quantitative measurement of perfluorinated compound concentrations. The fluorescence intensity of different concentrations of perfluorinated compounds (0, 10, 20, 30, 40, 50, 100, 200 μg / L) after incubation with four fluorescent probes was measured.

[0009] 5. Detection of Perfluorinated Mixtures: This study validated the sensor's excellent ability to distinguish between different perfluorinated compound mixtures. Maintaining a total perfluorinated compound concentration of 10 μmol, the developed fluorescence sensor array was used to distinguish between various mixtures of perfluorinated compounds. Using different ratios of PFOA and PFOS (10:0, 8:2, 6:4, 4:6, 2:8, 0:10), a sensor array was constructed to measure fluorescence intensity. Six parallel experiments were performed for each perfluorinated mixture. The obtained data were input into SPSS 17 for linear discriminant analysis (LDA). The analytical results were used to determine the sensor array's ability to distinguish between different perfluorinated mixtures.

[0010] 6. Detection of Real Water Samples: This section verifies the effectiveness of the sensor array in detecting perfluorinated compounds (PFOCs) in real water samples. Real water samples were collected, divided into six equal portions, and diluted to the concentration range of the standard curve. A suitable amount of the diluted water sample was incubated with the constructed sensor array, and the fluorescence intensity was measured. The concentration of pollutants in the real water sample was calculated by substituting the values ​​into the corresponding standard curve. The accuracy of PFOC detection in real water samples was evaluated using liquid chromatography-mass spectrometry (LC-MS / MS).

[0011] 7. Development of a Portable Detection Device: By combining sensor arrays with microfluidic chip technology, a portable perfluorinated compound screening device is constructed to achieve rapid on-site detection. The portable detection device of this invention includes a centrifugal chip disk, a handheld centrifuge device, and a smartphone-based handheld spectrometer. The centrifugal chip disk contains four reaction chambers, each pre-stored with four screening probes; the centrifugal chip disk includes one sample inlet chamber for adding standards or test samples; the centrifugal chip disk includes one waste liquid chamber for storing waste liquid; and the centrifugal chip includes one connector for fixing to the centrifuge device. The handheld centrifuge device is a modified centrifugal fan, and a connector is fabricated using 3D printing to fix the centrifugal chip disk. The smartphone-based spectrometer, purchased from Hongke Company, can collect light signals and generate spectra. Applying the above-mentioned perfluorinated compound detection method based on a cyclodextrin polymer sensor array to this device enables rapid on-site detection of perfluorinated compounds.

[0012] Beneficial effects

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] (1) The cyclodextrin polymer and fluorescent dye used in the detection method of the present invention are readily available. The dye and cyclodextrin polymer are self-assembled and do not require other chemical reagents and methods for synthesis.

[0015] (2) Fluorescent probes based on cyclodextrin polymers have high sensitivity to perfluorinated compounds and can quickly detect low concentrations of perfluorinated compounds.

[0016] (3) The present invention uses a sensor array to detect multiple pollutants and can detect and distinguish pollutants with similar structures, thus expanding the detection range of pollutants.

[0017] (4) The detection device combined with the microfluidic chip is easy to carry, suitable for rapid on-site detection, and can be widely used in the detection of perfluorinated compounds in various environmental samples such as water and soil. Attached Figure Description

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0019] Figure 1 Flowchart of cyclodextrin polymer synthesis.

[0020] Figure 2 Characterization diagrams of cyclodextrin polymers (Figure A: FT-IR, Figure B: TEM)

[0021] Figure 3 Schematic diagram of fluorescent probe assembly and screening.

[0022] Figure 4 Optimization diagram of incubation time for four dyes.

[0023] Figure 5 : Cyclodextrin detection perfluorinated compound sensor array.

[0024] Figure 6 A diagram distinguishing different perfluorinated compounds at the same concentration (10 μg).

[0025] Figure 7 Establishment of standard curves and quantitative measurement of perfluorinated compounds (Figure A: LDA plots of PFOA at different concentrations; Figure B: PFOA standard curve).

[0026] Figure 8 Diagram showing the differentiation of perfluorinated mixtures in different proportions.

[0027] Figure 9 : Structural diagram of a portable perfluorinated compound screening device.

[0028] Legend: PC1 and PC2 are the two principal factors calculated by linear discriminant analysis (LDA) in SPSS 17. Detailed Implementation

[0029] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention. All materials and instruments used in the following embodiments are commercially available.

[0030] Example 1: Preparation of Reagents and Materials

[0031] Prepare reagents and materials such as β-cyclodextrin, pyrene, curcumin, 1,8-naphthalenedimide, coumarin, 2-aminofluorene, rhodamine B, 1-naphthylaminobenzene, fluorescein, naphthol green B, bromophenol blue, perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroheptanoic acid (PFHpA), PFHxA (perfluorohexanoic acid), and 8000D dialysis bags.

[0032] Example 2: Synthesis and Characterization of β-Cyclodextrin Polymers

[0033] Synthesis: Accurately weigh 11.35 g (10 mmol) of β-CD monomer and slowly add it to 15 mL of 15 wt% NaOH solution. Place the solution in a constant-temperature stirring apparatus and stir at a stable speed at 35°C for 2 hours to ensure complete dissolution of β-CD. Add 2 mL of toluene to the above solution and maintain constant temperature stirring for 2 hours to ensure that the toluene is completely embedded in the β-CD cavity. Slowly add 4.0 mL of crosslinking agent epichlorohydrin, controlling the dropping rate to avoid excessively rapid local reactions. After the addition is complete, continue stirring at 35°C for 3 hours to promote the polymerization reaction. After the reaction is complete, transfer the reaction solution to a container containing 200 mL of isopropanol for pre-precipitation. A precipitate will form at this time; discard the supernatant. The precipitate was dissolved in water, and the pH of the solution was carefully adjusted to neutral using dilute hydrochloric acid. The solution was then transferred to an 8000 D dialysis bag and dialyzed for 3 days with magnetic stirring to remove unreacted impurities and small molecules. After dialysis, the solution was freeze-dried to obtain the β-cyclodextrin polymer, which was then stored at –20°C for later use.

[0034] Infrared characterization: Appropriate amounts of pure KBr solid and a mixture of KBr and analytes (cyclodextrin monomers, cyclodextrin polymers, cyclodextrin polymer-coated dyes, and dyes) were ground uniformly and then compressed into tablets using a tablet press. The compressed tablets were placed in an infrared spectrometer at 400–4000 cm⁻¹. - 1The KBr spectrum was scanned within a certain range, and professional software was used to perform background subtraction, atmosphere compensation, and smoothing operations to obtain a clear infrared spectrum. The structure and functional group changes of the polymer were determined based on the characteristic absorption peaks.

[0035] SEM Characterization: A solution of self-assembled nanoparticles encapsulated with fluorescent dye and β-cyclodextrin polymer was prepared. 2 μL of the diluted solution (β-cyclodextrin polymer concentration of 400 μmol and fluorescent dye concentration of 10 μmol) was transferred onto a mica or copper sheet. The sample was placed in a dry, clean environment overnight to allow it to dry completely. The dried sample was then placed in the sample chamber of a scanning electron microscope. After vacuuming, an appropriate magnification and accelerating voltage were selected to observe and photograph the sample surface, obtaining morphological information of the nanoparticles.

[0036] Example 3: Optimization of Experimental Conditions

[0037] β-CDP concentration optimization (taking β-CDP / pyrene fluorescent probes as an example): Four screened fluorescent dyes (pyrene, 1,8-naphthalenedimide, fluorescein, and bromophenol blue) were assembled with 500 μmol / L cyclodextrin to form four fluorescent probes (probes A, B, C, and D). The fluorescence changes of the four fluorescent probes after incubation with 0, 10 μg / L, and 100 μg / L perfluorinated compounds (PFOA, PFOS, PFDA, PFNA, PFHpA, and PFHxA) for 30 min were measured, and the fluorescence intensity data were recorded to analyze the interaction mechanism and response pattern between the probes and the perfluorinated compounds.

[0038] Incubation time optimization: A fluorescent probe was assembled using the optimized cyclodextrin and 10 μmol / L pyrene. The fluorescence changes of the probe after incubation with 0, 10 μg / L and 100 μg / L PFOA for different times (e.g., 5 min, 10 min, 15 min, 20 min, 30 min, 60 min, etc.) were measured to determine the incubation time that allows the probe to fully interact with the perfluorinated compound and ensures a stable fluorescence signal.

[0039] Example 4: Construction of the sensor array and analysis of perfluorinated compounds

[0040] Sensor array construction: Four screened fluorescent dyes (pyrene, 1,8-naphthalenedimide, fluorescein, and bromophenol blue) were mixed with β-CDP, and then six 100 μg / L perfluorinated compounds (PFOA, PFOS, PFDA, PFNA, PFHpA, and PFHxA) were added to construct a 4×6 fluorescent probe array.

[0041] Qualitative analysis: The fluorescence intensity of the sensor array after interaction with the perfluorinated compounds was measured using a fluorescence spectrometer. Six parallel experiments were performed for each perfluorinated compound. The obtained data were input into SPSS 17 analysis software for linear discriminant analysis (LDA). Based on the analysis results, the ability of the sensor array to distinguish different perfluorinated compounds was determined, and fingerprint spectra were constructed. The types of perfluorinated compounds in the test samples were distinguished based on the fingerprint spectra.

[0042] Quantitative analysis: The fluorescence intensity of different concentrations of perfluorinated compounds (0, 10, 20, 30, 40, 50, 100, 200 μg / L) after incubation with four fluorescent probes was measured. Linear discriminant analysis (LDA) was performed on the data using SPSS 17 software to obtain analytical factors. A standard curve was established using these factors to correlate the concentration of perfluorinated compounds with the changes in fluorescence intensity. The linear regression equation was given by (where is the change in fluorescence intensity, is the concentration of the perfluorinated compound, and and are the fitted coefficients). A correlation coefficient reaching a certain value indicates that the standard curve has a good linear relationship.

[0043] Example 5: Detection of perfluorinated mixed samples

[0044] While maintaining a total perfluorinated compound concentration of 10 μmol, PFOA and PFOS were mixed at ratios of 10:0, 8:2, 6:4, 4:6, 2:8, and 0:10. A sensing array was constructed to measure fluorescence intensity, with six parallel experiments performed for each perfluorinated mixture. The obtained data were input into SPSS 17 for linear discriminant analysis (LDA). The absence of overlapping matrices in the LDA plots indicates that the different proportions of perfluorinated mixtures can be well distinguished.

[0045] Example 6: Analysis of perfluorinated compounds in actual water samples

[0046] Actual water samples (such as surface water, groundwater, and tap water) were collected and divided into six equal portions, then diluted to the concentration range of the standard curve. An appropriate amount of the diluted water sample was incubated with the constructed sensor array, and the fluorescence intensity was measured according to the above detection method. The concentration of pollutants in the actual water sample was calculated by substituting the values ​​into the corresponding standard curve. Simultaneously, the accuracy of the detection of perfluorinated compounds in the actual water sample was evaluated using liquid chromatography-mass spectrometry (LC-MS / MS). The consistency between the results of this invention and the LC-MS / MS results was compared to verify the accuracy and reliability of this invention.

[0047] Example 7: Application of a portable perfluorinated compound screening device

[0048] The perfluorinated compound detection method based on a cyclodextrin polymer sensor array constructed in Examples 1-6 was applied to a portable detection device. The water sample to be tested was added to the sample inlet chamber, and a handheld centrifuge was used to propel the sample into four reaction chambers under centrifugal force, ensuring thorough contact and reaction with pre-stored probes in each chamber. Excess solution flowed into the waste chamber. After the reaction, changes in fluorescence signals in the reaction chambers were measured using a smartphone-based handheld spectrometer. The detection of perfluorinated compounds in the water sample was achieved by combining fingerprint spectroscopy and a standard curve.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for detecting perfluorinated compounds based on a β-cyclodextrin polymer (β-CDP) sensor array, characterized in that, Includes the following steps: Step 1: Synthesis of β-CDP: β-CDP is prepared by chemical synthesis methods; Step 2: Self-assembly and screening of fluorescent probes: The synthesized β-CDP was assembled with various fluorescent dyes, and four fluorescent probes that showed significant responses to perfluorinated compounds were screened out. Step 3: Construction of the sensor array: The sensor array is constructed using the four selected fluorescent probes, and its response to six perfluorinated compounds is measured to construct a fingerprint spectrum. The type of perfluorinated compound in the sample is identified based on the fingerprint spectrum. Step 4: Establishment of standard curve: A standard curve is established by comparing the response of perfluorinated compound standards with known concentrations, thereby enabling quantitative measurement of perfluorinated compound concentrations; Step 5: Detection of mixed and actual water samples: Verify the effectiveness of the sensor array in detecting perfluorinated compounds in mixed and actual water samples; Step Six: Development of Portable Detection Devices: By combining sensor arrays with microfluidic chip technology, a portable perfluorinated compound screening device will be constructed to achieve rapid on-site detection.

2. The method for detecting perfluorinated compounds based on a β-CDP sensor array according to claim 1, characterized in that, The synthesis steps of β-CDP are as follows: 11.35 g (10 mmol) of β-cyclodextrin monomer (β-CD) is accurately weighed and slowly added to 15 mL of 15wt% NaOH solution. The mixture is stirred at 35 °C for 2 hours to ensure complete dissolution of β-CD. 2 mL of toluene is added to the above solution, and the mixture is stirred at a constant temperature for 2 hours. 4.0 mL of crosslinking agent epichlorohydrin is slowly added dropwise. After the addition is complete, the mixture is stirred at 35 °C for another 3 hours. After the reaction is complete, the reaction solution is transferred to a container containing 200 mL of isopropanol for pre-precipitation, and the supernatant is discarded. The precipitate is dissolved in water, and the pH of the solution is adjusted to neutral with dilute hydrochloric acid. The solution is then transferred to an 8000 D dialysis bag and dialyzed for 3 days. After dialysis, the solution is freeze-dried to obtain β-CDP, which is then stored at –20 °C for later use.

3. The method for detecting perfluorinated compounds based on a β-CDP sensor array according to claim 1, characterized in that, The assembly and screening of the fluorescent probes are as follows: methanol solutions of various fluorescent dyes with a final concentration of 10 μmol / L are incubated with different concentrations of β-CDP (0, 100, 200, 300, 400, 500 μmol / L, the concentration of which is calculated as the monomer concentration of β-CDP) in a constant temperature metal shaking bath for 30 min (25 °C, 150 rpm) to form β-CDP / dye complexes. Then, the fluorescence intensity is measured by a fluorescence spectrometer to screen out four fluorescent dyes that show significant changes in fluorescence intensity in the presence of β-CDP.

4. The method for detecting perfluorinated compounds based on a cyclodextrin polymer sensor array according to claim 1, characterized in that, The construction of the sensor array and the analysis steps for perfluorinated compounds include: mixing four screened fluorescent dyes with β-CDP, then adding six 100 μg / L perfluorinated compounds to construct a 4 × 6 fluorescent probe array; measuring the fluorescence intensity of the sensor array after interaction with the perfluorinated compounds using a fluorescence spectrometer, performing six parallel experiments for each perfluorinated compound, inputting the obtained data into SPSS17 analysis software for linear discriminant analysis (LDA), determining the sensor array's ability to distinguish different perfluorinated compounds based on the analysis results, and constructing a fingerprint spectrum.

5. The method for detecting perfluorinated compounds based on a cyclodextrin polymer sensor array according to claim 1, characterized in that, The standard curve was established by measuring the fluorescence intensity of different concentrations of perfluorinated compounds (0, 10, 20, 30, 40, 50, 100, 200 μg / L) after incubation with four fluorescent probes. The data were analyzed by linear discriminant analysis (LDA) using SPSS 17 software to obtain the analytical factors. A standard curve was established between the concentration of perfluorinated compounds and the changes in the analytical factors. If the correlation coefficient reached a certain value, it indicated that the standard curve had a good linear relationship.

6. The method for detecting perfluorinated compounds based on a cyclodextrin polymer sensor array according to claim 1, characterized in that, The detection steps for the mixed sample and the actual water sample are as follows: collect the mixed water sample or the actual water sample, divide it into 6 equal parts, dilute it to the concentration range of the standard curve, take an appropriate amount of the diluted water sample and incubate it with the constructed sensor array, measure the fluorescence intensity, substitute it into the corresponding standard curve to calculate the concentration of pollutants in the actual water sample, and at the same time use liquid chromatography-mass spectrometry (LC-MS / MS) to evaluate the accuracy of the detection of perfluorinated compounds in the actual water sample.

7. A portable detection device for perfluorinated compounds based on a cyclodextrin polymer sensor array, characterized in that, The system includes a centrifugal chip disk, a handheld centrifuge device, and a smartphone-based handheld spectrometer. The centrifugal chip disk contains four reaction chambers, each pre-stored with four screening probes; it also includes one sample inlet chamber for adding standards or test samples; one waste liquid chamber for storing waste liquid; and a connector for attaching to the centrifuge device. The handheld centrifuge device is a modified centrifuge fan, and a 3D-printed connector is used to secure the centrifugal chip disk. The smartphone-based spectrometer, purchased from Hongke Company, can collect light signals and generate spectra. The method for detecting perfluorinated compounds based on a cyclodextrin polymer sensor array according to any one of claims 1-6 is applied to this device to achieve rapid on-site detection of perfluorinated compounds.

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