WO2023123136A1

WO2023123136A1 - Fluorescent conjugated polymer quantum dot probe, preparation method therefor and application thereof

Info

Publication number: WO2023123136A1
Application number: PCT/CN2021/142719
Authority: WO
Inventors: 韩亚萌; 郭丽莉; 王蓓丽; 李书鹏; 高艳丽; 瞿婷; 张孟昭; 魏宁; 许铁柱
Original assignee: 北京建工环境修复股份有限公司
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2023-07-06

Abstract

A fluorescent conjugated polymer quantum dot probe, a preparation method therefor and an application thereof. The preparation method for the fluorescent conjugated polymer quantum dot probe comprises: step 1, reacting fluorescent conjugated polymer quantum dots with a silanization reagent to obtain silanized fluorescent conjugated polymer quantum dots; and step 2, modifying a perfluoroalkane chain on the surface of the silanized fluorescent conjugated polymer quantum dots obtained in step 1 to obtain the fluorescent conjugated polymer quantum dot probe. The fluorescent conjugated polymer quantum dot probe is applied to detection of a perfluorinated compound, and overcomes the defect of high detection limit in existing perfluorinated compound detection methods.

Description

A fluorescent conjugated polymer quantum dot probe and its preparation method and application

technical field

The invention belongs to the field of environmental pollutant detection, and in particular relates to a fluorescent conjugated polymer quantum dot probe and a preparation method and application thereof.

Background technique

Perfluorinated compounds have thermal stability, chemical stability and good surface activity, and have been widely used in various production and daily necessities such as food packaging bags, non-stick pans, textiles, waterproofing agents, electroplating agents, and foam fire extinguishing agents. However, with the in-depth research on perfluorinated compounds, it is found that they have serious biological toxicity effects and can cause serious harm to the ecological environment and human health. Perfluorinated compounds have become a new type of persistent organic pollutants, which have a huge harmful effect on the safety of the ecological environment. Due to the high polarity of perfluorinated compounds, water is one of their main transmission media. my country is a big country in the production and use of perfluorinated compounds. In recent years, through the investigation of the pollution status of perfluorinated compounds in the water environment, it has been found that the pollution distribution range of perfluorinated compounds in water bodies is very wide, in oceans, rivers, lakes, surface water, groundwater Perfluorinated compound residues can be detected in both tap water and tap water, posing a serious threat to drinking water safety. Due to the low concentrations of PFCs in groundwater and tap water, sensitive analytical techniques must be used to detect PFC contamination levels in water samples.

At present, the detection of perfluorinated compounds mainly uses high-performance liquid chromatography-mass spectrometry and high-performance liquid chromatography-tandem mass spectrometry. Although these two methods have high sensitivity and good accuracy, they require tedious sample pretreatment. , using a large amount of organic solvents, the detection cycle is longer. Therefore, it is necessary to establish a simpler, faster, greener and more sensitive analytical technique. In recent years, optical sensing technology has been widely used as a simple and rapid analysis method. Chinese patent document CN103558198B discloses a rapid fluorescence detection method for perfluorooctane sulfonic acid (PFOS) in environmental water samples, using PFOS The fluorescence of the metal complex 4-(methylphenyl)-2,2':6',2"-terpyridine-Zn(II) can be quenched by electrostatic quenching, and the change of the fluorescence spectrum intensity can be measured , to achieve rapid fluorescence analysis of PFOS in environmental water samples, the detection limit of this method is high. Although this spectral detection method is relatively simple and fast, the detection limit is high, and it cannot be applied to low-concentration perfluorinated in groundwater or tap water. Sensitive detection of compounds.

Contents of the invention

Therefore, the technical problem to be solved by the present invention is to overcome the defect of high detection limit in the existing detection methods of perfluorinated compounds, so as to provide a fluorescent conjugated polymer quantum dot probe and its preparation method and application.

For this reason, the present invention provides the following technical solutions.

In a first aspect, the present invention provides a method for preparing a fluorescent conjugated polymer quantum dot probe, comprising:

Step 1, reacting fluorescent conjugated polymer quantum dots with a silylating agent to prepare silanized fluorescent conjugated polymer quantum dots;

Step 2, modifying the surface of the silanized fluorescent conjugated polymer quantum dots prepared in step 1 with perfluoroalkane chains to prepare fluorescent conjugated polymer quantum dot probes;

Further, the preparation method of fluorescent conjugated polymer quantum dots includes: mixing PFBT, PSMA and tetrahydrofuran to prepare a mixed solution; injecting the mixed solution into water, reacting, and removing tetrahydrofuran to obtain the product;

The PFBT is poly[(9,9-dioctylfluorenyl-2,7-disubstituent)-alternate-co-(1,4-benzo-2,1′-3-thiadiazole)] and PSMA is poly(styrene-maleic anhydride).

Further, the preparation method of fluorescent conjugated polymer quantum dots also satisfies at least one of the following (1)-(6):

(1) The volume ratio of the mixed solution to water is (1220-1280):5000, the concentration of PFBT in the mixed solution is 0.016-0.125 mg/mL, and the concentration of PSMA is 0.156-0.328 mg/mL;

(2) The reaction temperature is 80-90°C, and the time is 0.5-2h;

(3) The preparation process of the mixed solution comprises dissolving PFBT and PSMA in tetrahydrofuran respectively to obtain a PFBT stock solution with a mass concentration of 1 to 2 mg/mL and a PSMA stock solution with a mass concentration of 1 to 2 mg/mL; Pipette the PFBT stock solution and the PSMA stock solution into tetrahydrofuran respectively to obtain a mixed solution;

(4) Under ultrasonic conditions, inject the mixed solution into water, and continue ultrasonication for 30-150s after the injection is completed;

(5) adopting the mode that feeds nitrogen into the reaction system to remove tetrahydrofuran;

(6) After removing THF, after cooling to room temperature, filter the obtained product solution through a filter membrane; preferably, the micropore of the filter membrane is 0.22 μm.

Further, step 1 includes: adding a silylating agent and an activator to the fluorescent conjugated polymer quantum dot solution, reacting, centrifuging, and collecting the lower solid.

Further, step 1 also satisfies at least one of the following (1)-(5):

(1) The reaction time is 2 to 4 hours;

(2) the silylating agent is aminopropyltriethoxysilane or aminopropyltrimethoxysilane;

(3) The activator is EDC/NHS solution or EDC solution, preferably, the concentrations of EDC and/or NHS in the activator are both 0.3-0.8 mg/mL;

(4) The volume ratio of the activator to the silylating agent is 100:(20～60);

(5) The volume ratio of the silylating agent to the fluorescent conjugated polymer quantum dot solution is (20-60): 4000, wherein the concentration of the fluorescent conjugated polymer quantum dot solution is 55 μg/mL～140 μg/mL .

Further, step 2 includes: dispersing the silanized fluorescent conjugated polymer quantum dots prepared in step 1 into an alcohol-water solution, adding perfluorosilane and a catalyst, reacting, centrifuging, and collecting the lower solid.

Further, the step 2 satisfies at least one of the following (1)-(5):

(1) The concentration of silanized fluorescent conjugated polymer quantum dots in the dispersion is 13 to 30 μg/mL; the volume ratio of the perfluorosilane to the silanized fluorescent conjugated polymer quantum dot solution is (10～40): 5000;

(2) The alcohol-water solution is a mixed solution of methanol and/or ethanol and water, preferably, the volume ratio of alcohol to water is (2-5):1;

(3) Reaction time 4-6h;

(4) The perfluorosilane is perfluorooctyltriethoxysilane and/or perfluorodecyltriethoxysilane;

(5) The catalyst is ammonia water. Preferably, the mass concentration of the ammonia water is 25%-28%, and the volume ratio of perfluorosilane to ammonia water is (10-40):(5-20).

In the second aspect, the present invention provides a fluorescent conjugated polymer quantum dot probe.

In a third aspect, the present invention provides an application of a fluorescent conjugated polymer quantum dot probe in the detection of perfluorinated compounds. Preferably, the perfluorinated compound is selected from perfluorooctanesulfonic acid, perfluorohexanoic acid, At least one of perfluorononanoic acid and perfluorooctane carboxylic acid.

In the fourth aspect, the present invention provides a method for detecting perfluorinated compounds, using the above-mentioned fluorescent conjugated polymer quantum dot probe as the detection probe,

By detecting the number of fluorescently conjugated polymer quantum dot probes using a single-particle fluorescence counting sensing method, the concentration of perfluorinated compounds in the sample is obtained, or,

The fluorescence intensity was measured by a fluorescence spectrophotometer to obtain the concentration of perfluorinated compounds in the sample.

The method of detecting the number of fluorescent conjugated polymer quantum dot probes by the single-particle fluorescence counting sensing method and obtaining the concentration of perfluorinated compounds in the sample can be a standard curve method. Specifically, the standard curve method includes:

Preparation of the standard curve: disperse the fluorescent conjugated polymer quantum dot probe into the solvent to obtain the fluorescent conjugated polymer quantum dot probe solution, mix pure water with the fluorescent conjugated polymer quantum dot probe solution to obtain the blank control Sample solution; prepare N standard solutions containing different concentrations of perfluorinated compounds and mix them with fluorescent conjugated polymer quantum dot probe solutions to prepare standard sample solutions, N≥3; preferably, the solvent is water, ethanol or methanol , more preferably water;

The total internal reflection fluorescence microscopy imaging method was used to detect the blank control sample solution and each standard sample solution respectively, record the number n ₀ of fluorescent conjugated polymer quantum dot probes in the blank control sample solution, and record the fluorescent conjugated polymeric probes of each standard sample solution The number of quantum dot probes n _i , i=1～N;

Make a standard curve of the concentration of perfluorinated compounds and the amount of change in the number of fluorescent conjugated polymer quantum dot probes n _i -n ₀ ;

Determination: After mixing the sample to be tested with the fluorescent conjugated polymer quantum dot probe solution, use the total internal reflection fluorescence microscopy imaging system to detect, record the number of fluorescent conjugated polymer quantum dot probes, and obtain the fluorescent conjugated polymer According to the standard curve, the concentration of perfluorinated compounds in the sample to be tested can be obtained.

The method for measuring the fluorescence intensity by using a fluorescence spectrophotometer to obtain the concentration of the perfluorinated compound in the sample includes: measuring the fluorescence intensity by using a fluorescence spectrophotometer, and establishing the relationship between the fluorescence intensity and the concentration.

Further, when using the single-particle fluorescence counting sensing method to detect the number of fluorescent conjugated polymer quantum dot probes, it is necessary to aminate the slide, and the amination of the slide includes: preparing an amination reagent, and aminating the slide Soak in the reagent for 1-2h to aminate the surface of the glass slide. The glass slide is taken out from the solution, rinsed with ethanol for 2-3 times, washed with ultrapure water for 2-3 times, and then blown dry with a nitrogen flow to prepare an aminated slide.

The preparation of the amination reagent includes: adding 50 μL of aminopropyltriethoxysilane to 2-10 mL of ethanol to prepare an amination reagent solution. Exemplarily, the size of the glass slide is 20mm×20mm.

In the process of total internal reflection fluorescence microscopy imaging, the liquid is added to the aminated glass slide for detection; the excitation light wavelength is 465-475nm, and the emission wavelength is 535-545nm. Preferably, the excitation light wavelength is 470nm, and the emission wavelength is 540nm, under which the fluorescence emission intensity is the maximum.

The technical solution of the present invention has the following advantages:

1. The preparation method of the fluorescent conjugated polymer quantum dot probe provided by the present invention comprises: step 1, reacting the fluorescent conjugated polymer quantum dot with a silylating reagent to obtain a silanized fluorescent conjugated polymer quantum dot ; Step 2, modifying the surface of the silanized fluorescent conjugated polymer quantum dots prepared in step 1 with perfluoroalkane chains to prepare fluorescent conjugated polymer quantum dot probes. The fluorescent conjugated polymer quantum dot probe prepared by the method of the invention has good single-particle fluorescence stability and uniform dispersion, and is beneficial to statistically counting a single quantum dot probe in an image when performing single-particle imaging. The preparation method of the invention is simple and easy to operate, can specifically recognize perfluorinated compounds, and can be used to construct a single-particle fluorescence counting sensing method.

Fluorescent conjugated polymer quantum dots have good water solubility and monodispersity, and are an excellent single-particle quantum dot fluorescent probe carrier, which is conducive to modifying the surface to obtain functionalized single-particle quantum dot fluorescent probes. The silylating agent is an aminosilane compound, and the carboxyl group on the fluorescent conjugated polymer quantum dot can undergo an amide reaction with the amino group on the silane to obtain a silanized fluorescent conjugated polymer quantum dot. Since silane is easily hydrolyzed in water to form silanol, the surface of silanized fluorescent conjugated polymer quantum dots contains a large amount of silanol, and the surface of silanized fluorescent conjugated polymer quantum dots is modified with perfluoroalkane through silane polycondensation reaction. chain, to obtain fluorescent conjugated polymer quantum dots functionalized with perfluoroalkane chains, that is, fluorescent conjugated polymer quantum dot probes. During the detection process, the perfluoroalkane chain can undergo specific fluorine-fluorine interaction with the perfluorinated compound, so that the fluorescence of the fluorescent conjugated polymer quantum dot probe is significantly enhanced.

2. The preparation method of the fluorescent conjugated polymer quantum dot probe provided by the present invention, the preparation method of the fluorescent conjugated polymer quantum dot comprises: mixing PFBT, PSMA and tetrahydrofuran to prepare a mixed solution; injecting the mixed solution into water, Reaction, removal of tetrahydrofuran, that is.

Poly[(9,9-dioctylfluorenyl-2,7-disubstituent)-alternate-co-(1,4-benzo-2,1′-3-thiadiazole)] (PFBT) Fluorescent conjugated polymer quantum dots were prepared by co-precipitation polymerization with poly(styrene-maleic anhydride) (PSMA). Since the structure of PFBT contains abundant π electrons, the fluorescent polymer particles have higher fluorescence intensity. and fluorescence stability. The hydrophobic polystyrene units of PSMA are fixed inside the fluorescent polymer particles, and the maleic anhydride units are hydrolyzed in aqueous solution to generate carboxyl groups on the surface of the fluorescent particles, so the surface of the prepared fluorescent conjugated polymer quantum dots contains a large amount of The carboxyl group makes it have good water solubility and monodispersity.

3. The preparation method of fluorescent conjugated polymer quantum dot probe provided by the present invention, step 1 satisfies the volume ratio of silylating reagent and described fluorescent conjugated polymer quantum dot solution to be (20～60):4000, wherein fluorescent The concentration of the conjugated polymer quantum dot solution is 55 μg/mL˜140 μg/mL. In step 1, it is necessary to control the amount of silylating agent added, so as to avoid the amount of silanol on the surface of the silanized fluorescent conjugated polymer quantum dots prepared, or the amount of silanized fluorescent conjugated polymer quantum dots in the reaction process Agglomeration easily occurs and good monodispersity cannot be maintained.

4. The preparation method of fluorescent conjugated polymer quantum dot probe provided by the present invention, in step 2, in alcohol-water solution, the volume ratio of alcohol and water is (2～5):1; Perfluorosilane and described silanization The volume ratio of the fluorescent conjugated polymer quantum dot solution is (10-40):5000, wherein the concentration of the silanized fluorescent conjugated polymer quantum dot solution is 13-30 μg/mL.

Perfluorosilane is hydrophobic and has low water solubility. Alcohol-water is used as a solvent. When the volume of alcohol is 2 to 5 times that of water, it can not only dissolve perfluorosilane, but also hydrolyze silane into silanol and further generate silane. polycondensation reaction.

It is necessary to control the amount of perfluorosilane added in the reaction process, so as to avoid the perfluoroalkane chains on the surface of the prepared fluorescent conjugated polymer quantum dot probe less, or the fluorescent conjugated polymer quantum dot probe in the reaction process Needles are prone to agglomeration and cannot maintain good monodispersity.

5. The method for detecting perfluorinated compounds provided by the present invention uses single-particle fluorescent sensing technology for the detection of perfluorinated compounds for the first time, and prepares fluorescent conjugated polymer quantum dot probes that can recognize and respond to perfluorinated compounds as Detection probe. The solution of the present invention is not limited to perfluorooctane sulfonic acid in specific applications, and can also be used for perfluorocaproic acid, perfluorononanoic acid, perfluorooctane carboxylic acid and the like.

Based on the total internal reflection fluorescence microscopy imaging system, the fluorescent conjugated polymer quantum dot probe solution is dropped onto the surface of the slide, and the slide is placed on the total internal reflection fluorescence microscopy imaging platform. Through the microscopy imaging system, The number of fluorescent-conjugated polymer quantum dot probes on the slide surface can be detected at the single-particle level. Due to the low fluorescence intensity of fluorescent conjugated polymer quantum dot probes, the number of quantum dot probes detected on the glass slide is less, when perfluorinated compounds are added, the fluorescence conjugated polymer quantum dot probes can be made The fluorescence intensity is enhanced, thereby increasing the number of detected quantum dot probes. In this method, as the concentration of perfluorinated compounds increases, the number of quantum dot probes gradually increases, so the highly sensitive detection of perfluorinated compounds at the single particle level can be achieved by statistically analyzing the change in the number of quantum dot probes . The single-particle fluorescence counting sensing method provided by the invention can be used for the pollution analysis of low-concentration perfluorinated compounds in groundwater or tap water.

Based on the total internal reflection fluorescence microscopy imaging system, the change in the number of fluorescent conjugated polymer quantum dot probes produced in the presence of perfluorinated compounds can be detected at the microscopic level, which greatly reduces the detection limit and improves the detection of perfluorinated compounds. Detection sensitivity, so that it can be used for the pollution detection of low concentration perfluorinated compounds in groundwater or tap water samples.

The single-particle fluorescence counting sensing method provided by the present invention is to perform single-particle fluorescence imaging detection on the quantum dots in the 100nm solution layer on the surface of the glass slide. The required sample amount to be tested is very small, avoiding the use of a large amount of organic solvents, and is environmentally friendly. Advantages, and the detection background is low, and the matrix interference is small.

6. The perfluorinated compound detection method provided by the present invention is to aminate the glass slide. When the total internal reflection fluorescence microscopy imaging system is used for single particle fluorescence counting sensing, the aminated glass slide is first placed on the stage, and then the fluorescent conjugated polymer quantum dot probe solution is dropped onto the aminated glass slide On the surface, since the surface of the fluorescent conjugated polymer quantum dot probe contains fluorine atoms, hydrogen bonding can occur with the amino group, so that the fluorescent conjugated polymer quantum dot probe can be fixed on the glass slide, and the total internal reflection fluorescence microscopy system can The number of fluorescent conjugated polymer quantum dot probes on the glass surface is imaged by single particle, and the number of quantum dot probes in the image can be statistically analyzed by ImageJ software. If the slide is not aminated, the fluorescent conjugated polymer quantum dot probe cannot be stably fixed on the surface of the slide, and single particle fluorescence imaging analysis cannot be performed.

7. The fluorescent conjugated polymer quantum dot probe prepared by the present invention has good single-particle fluorescence stability and uniform dispersion, and is suitable for measuring the concentration of perfluorinated compounds by total internal reflection fluorescence microscopy imaging technology. In imaging, each signal output comes from a single probe particle, and each probe particle is associated with the target. By statistically counting the number of probe particles related to the target concentration at the single particle level, a sensitive single particle can be established. Particle Fluorescence Counting Sensing Method. The method is simple and sensitive, has low background interference, requires a very small amount of sample to be tested, avoids the use of a large amount of organic solvents, and has the advantages of environmental protection.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

Fig. 1 is a schematic flow chart of the preparation method of fluorescent conjugated polymer quantum dot probe in Example 1 of the present invention;

2 is a schematic diagram of a total internal reflection fluorescence microscopy imaging system in Example 2 of the present invention;

Fig. 3 is the standard curve obtained in Example 2 of the present invention.

Reference signs:

1-slide; 2-stage; 3-objective lens; 4-spotlight; 5-dichroic mirror; 6-emission filter; 7-excitation light source; 8-EMCCD; 9-display.

Detailed ways

The following examples are provided in order to further understand the present invention better, are not limited to the best implementation mode, and do not limit the content and protection scope of the present invention, anyone under the inspiration of the present invention or use the present invention Any product identical or similar to the present invention obtained by combining features of other prior art falls within the protection scope of the present invention.

If no specific experimental steps or conditions are indicated in the examples, it can be carried out according to the operation or conditions of the conventional experimental steps described in the literature in this field. The reagents or instruments used, whose manufacturers are not indicated, are all commercially available conventional reagent products.

Example 1

This embodiment provides a method for preparing a fluorescent conjugated polymer quantum dot probe, as shown in Figure 1, including:

Step 1. Poly[(9,9-dioctylfluorenyl-2,7-disubstituent)-alternate-co-(1,4-benzo-2,1′-3-thiadiazole)] (PFBT) and poly(styrene-maleic anhydride) (PSMA) were dissolved in THF, respectively, to prepare 1 mg/mL PFBT stock solution and PSMA stock solution.

Pipette 50 μL of PFBT stock solution and 200 μL of PSMA stock solution into 1 mL of tetrahydrofuran and mix to obtain a uniform mixed solution. Under ultrasonication (40kHz), the mixture was quickly injected into 5mL ultrapure water, and after ultrasonication for 90 seconds, the reaction solution was transferred to a water bath at 85°C, and stirred for 1 hour with magnetic stirring, and THF was slowly passed into nitrogen flow to remove THF . After cooling to room temperature, the obtained product solution was filtered through a 0.22 μm filter membrane, and finally a 4 mL solution of carboxyl-modified fluorescent conjugated polymer quantum dots with a concentration of 62.5 μg/mL was prepared.

Step 2. Add 40 μL of aminopropyltriethoxysilane and 100 μL of 0.5 mg/mL EDC/NHS solution to 4 mL of fluorescent conjugated polymer quantum dot solution. After stirring for 4 hours, the solution is centrifuged to remove the upper liquid, and the lower layer is obtained 75 μg of silanized fluorescent conjugated polymer quantum dots.

EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) are activators that can promote the reaction of amino and carboxyl groups, first prepare 1mg/mL EDC solution and 1mg/mL NHS solution, then take 50μL each and mix to obtain 100μL 0.5mg/mL EDC/NHS solution.

Step 3. Disperse 75 μg of silanized fluorescent conjugated polymer quantum dots prepared in step 2 into 5 mL of ethanol-water solution with a volume ratio of 4:1, add 20 μL of perfluorooctyltriethoxysilane and 10 μL of mass percent Ammonia water (commercially available industrial ammonia water) with a concentration of 25% to 28% was used. After reacting for 6 hours, the solution was centrifuged to remove the liquid in the upper layer, and the solid in the lower layer was obtained, that is, 90 μg of fluorescent conjugated polymer quantum dot probes.

Example 2

This embodiment provides a method for detecting perfluorinated compounds, including:

(1) Take 90 μg of the fluorescent conjugated polymer quantum dot probe prepared in Example 1 and disperse it in 5 mL of ultrapure water to prepare a 18 μg/mL fluorescent conjugated polymer quantum dot probe solution.

(2) Amination of slides, including:

Add 50 μL of aminopropyltriethoxysilane to 5 mL of ethanol to prepare an amination reagent solution. Soak a 20mm×20mm slide in the amination reagent solution, let it stand for 2 hours to aminate the surface of the slide, then take the slide out of the solution, wash it with ethanol for 3 times, wash it with ultrapure water for 3 times, and then The slides were blown dry with a stream of nitrogen to prepare aminated slides.

(3) Obtain the standard curve of the concentration-the change in the number of single-particle quantum dot probes:

A, the single-particle quantum dot probe solution in (1) is fixed on the aminated glass slide 1, and the aminated glass slide 1 is detected on the stage of the total internal reflection fluorescence microscopy imaging system, and recorded The number n ₀ of single-particle quantum dot probes.

Specifically, the total internal reflection fluorescence microscopy imaging system, as shown in Figure 2, includes a spotlight 4, an objective lens 3, an object stage 2, an excitation light source 7, an emission filter 6, a dichroic mirror 5, an EMCCD 8 and a display 9 . Place the aminated glass slide on stage 2, mix 10 μL fluorescent conjugated polymer quantum dot probe solution with 5 μL pure water to obtain a blank control sample solution, and add the blank control sample solution dropwise to the aminated glass slide. Slice 1 surface and place a circular coverslip over the droplet. Adjust the distance between the objective lens 3 and the stage 2, the excitation light wavelength is 470nm, and the emission wavelength is 540nm. Quantum dot probes perform single-particle fluorescence imaging, and output single-particle fluorescence pictures on the monitor. The number of fluorescent conjugated polymer quantum dot probes in the picture can be statistically analyzed by ImageJ software, and the fluorescent conjugated polymer The number of quantum dot probes is denoted as n ₀ , where n ₀ =52.

B. Prepare six standard solutions of perfluorooctane sulfonic acid with concentrations of 0.3pg/L, 1.5pg/L, 4.5pg/L, 9pg/L, 18pg/L, and 30pg/L, respectively.

Take 10 μL fluorescent conjugated polymer quantum dot probe solution and 5 μL perfluorooctane sulfonic acid with concentrations of 0.3pg/L, 1.5pg/L, 4.5pg/L, 9pg/L, 18pg/L and 30pg/L respectively Mix the standard solutions so that the final concentrations of PFOS in the standard sample solutions are 0.1pg/L, 0.5pg/L, 1.5pg/L, 3pg/L, 6pg/L, and 10pg/L, respectively. Then 6 parts of the mixed solution were dropped onto the aminated glass slide 1 on the stage respectively, and a circular cover glass was covered on the droplet. Based on the total internal reflection fluorescence microscopy imaging system, the distance between the objective lens and the stage is adjusted, and under the illumination of the excitation light source, the fluorescent conjugated polymer quantum dot probe within 100nm in the thin layer droplet on the glass surface is detected by EMCCD Perform single-particle fluorescence imaging, and output the single-particle fluorescence picture on the monitor. The number of fluorescent conjugated polymer quantum dot probes in the picture can be statistically analyzed by ImageJ software, and the number of quantum dot probes in the 6 sample solutions can be recorded. , are respectively n ₁ =95, n ₂ =112, n ₃ =141, n ₄ =232, n ₅ =335, n ₆ =503. The amount of change in the number of fluorescent conjugated polymer quantum dot probes is Δn ₁ = 43, Δn ₂ = 60, Δn ₃ = 89, Δn ₄ = 180, Δn ₅ = 283, Δn ₆ = 451, the production concentration-single particle As shown in Figure 3, the standard curve for the change in the number of quantum dot probes has a linear range of 0.1pg/L-10pg/L for the detection of perfluorooctane sulfonate.

Measure the _n of 5 parts of blank control samples to be 52, 51, 50, 53, 52 respectively, thus calculate the standard deviation of the blank sample to be 1.1, according to the formula: detection limit=3 times the standard deviation of the blank sample/standard curve Slope, the calculated detection limit of the method=3×1.1/41.283=0.08pg/L.

(4) Mix 10 μL fluorescent conjugated polymer quantum dot probe solution with 5 μL groundwater sample to be tested, drop the mixed solution onto the surface of an aminated glass slide, and record the fluorescence in the sample droplet based on total internal reflection fluorescence microscopy imaging The number of conjugated polymer quantum dot probes is 414, and the amount of change in the number of quantum dots is brought into the standard curve of step (3) by 362, so that the concentration of perfluorooctane sulfonic acid in the groundwater sample to be tested is 7.8 pg/L.

Example 3

This embodiment provides a detection method for perfluorinated compounds, which is the same as that in Embodiment 2, except that the sample to be tested in this embodiment is tap water.

Mix 10 μL fluorescent conjugated polymer quantum dot probe solution with 5 μL tap water sample to be tested, drop the mixed solution onto the surface of an aminated glass slide, and record the fluorescent conjugated polymerization in the sample droplet based on total internal reflection fluorescence microscopy imaging. The number of quantum dot probes is 176, and the amount of change in the number of quantum dots is 124 into the standard curve, so that the concentration of perfluorooctane sulfonic acid in the tap water sample to be tested is 2.1pg/L.

Example 4

This embodiment provides a method for preparing a fluorescent conjugated polymer quantum dot probe, including:

Step 1. Poly[(9,9-dioctylfluorenyl-2,7-disubstituent)-alternate-co-(1,4-benzo-2,1′-3-thiadiazole)] (PFBT) and poly(styrene-maleic anhydride) (PSMA) were respectively dissolved in THF to prepare 1 mg/mL stock solutions of PFBT and PSMA.

Pipette 40 μL of PFBT stock solution and 200 μL of PSMA stock solution into 1 mL of tetrahydrofuran and mix to obtain a uniform mixed solution. Under ultrasonication (40kHz), the mixture was quickly injected into 5mL ultrapure water, and after ultrasonication for 30 seconds, the reaction solution was transferred to a water bath at 80°C, and reacted with magnetic stirring for 0.5 hours, and slowly introduced nitrogen flow to remove Tetrahydrofuran. After cooling to room temperature, the obtained product solution was filtered through a 0.22 μm filter membrane, and finally 4 mL of carboxyl-modified fluorescent conjugated polymer quantum dot solution was prepared.

Step 2. Add 20 μL of aminopropyltriethoxysilane and 100 μL of 0.3 mg/mL EDC/NHS solution to the fluorescent conjugated polymer quantum dot solution obtained in step 1. After stirring for 2 hours, centrifuge the solution to remove the upper liquid , the lower layer obtains silanized fluorescent conjugated polymer quantum dots.

EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) are activators that can promote the reaction of amino and carboxyl groups, firstly prepare 0.6mg/ mL EDC solution and 0.6mg/mL NHS solution, then take 50μL each and mix to obtain 100μL 0.3mg/mL EDC/NHS solution.

Step 3. Disperse all the silanized fluorescent conjugated polymer quantum dots prepared in step 2 into 5 mL of ethanol-water solution with a volume ratio of 2:1, add 10 μL of perfluorooctyltriethoxysilane and 10 μL of mass Ammonia water (commercially available industrial ammonia water) with a percentage concentration of 25% to 28% was reacted for 4 hours, and the solution was centrifuged to remove the upper layer liquid, and the lower layer obtained fluorescent conjugated polymer quantum dot probes.

The detection method of perfluorinated compounds is the same as that of Example 2, and the detection limit of this example is 0.23pg/L.

Example 5

Step 1. Poly[(9,9-dioctylfluorenyl-2,7-disubstituent)-alternate-co-(1,4-benzo-2,1′-3-thiadiazole)] (PFBT) and poly(styrene-maleic anhydride) (PSMA) were respectively dissolved in THF to prepare 2 mg/mL stock solutions of PFBT and PSMA.

Pipette 60 μL of PFBT stock solution and 200 μL of PSMA stock solution into 1 mL of tetrahydrofuran and mix to obtain a uniform mixed solution. Under an ultrasonic water bath, the mixture was rapidly injected into 5 mL of ultrapure water. After ultrasonication for 130 seconds, the reaction solution was transferred to a 90° C. water bath, and stirred for 2 hours with magnetic stirring, and THF was slowly passed through nitrogen flow to remove THF. After cooling to room temperature, the obtained product solution was filtered through a 0.22 μm filter membrane, and finally 4 mL of carboxyl-modified fluorescent conjugated polymer quantum dot solution was prepared.

Step 2. Add 60 μL of aminopropyltriethoxysilane and 100 μL of 0.8 mg/mL EDC/NHS solution to the fluorescent conjugated polymer quantum dot solution prepared in step 1. After stirring for 3 hours, centrifuge the solution to remove the upper liquid , the lower layer obtains silanized fluorescent conjugated polymer quantum dots.

EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and NHS (N-hydroxysuccinimide) are activators that can promote the reaction of amino and carboxyl groups, firstly prepare 1.6mg/ mL EDC solution and 1.6mg/mL NHS solution, then take 50μL each and mix to obtain 100μL 0.8mg/mL EDC/NHS solution.

Step 3. Disperse all the silanized fluorescent conjugated polymer quantum dots prepared in step 2 into 5 mL of ethanol-water solution with a volume ratio of 5:1, add 40 μL of perfluorooctyltriethoxysilane and 10 μL of mass Ammonia water (commercially available industrial ammonia water) with a concentration of 25% to 28%, after reacting for 5 hours, centrifuge the solution to remove the upper layer liquid, and obtain the fluorescent conjugated polymer quantum dot probe in the lower layer, and then disperse it into ultrapure water , to obtain the fluorescent conjugated polymer quantum dot probe solution.

The detection method of perfluorinated compounds is the same as in Example 2, and the detection limit of this example is 0.15pg/L.

Example 6

This embodiment provides a method for detecting perfluorinated compounds. The preparation method of fluorescent conjugated polymer quantum dot probes is basically the same as in Example 1, and the method for detecting perfluorinated compounds is the same as in Example 2. The difference is that this In the preparation process of the fluorescent conjugated polymer quantum dot probe in the embodiment, in step 2, the amount of aminopropyltriethoxysilane added was 80 μL.

The detection limit of this embodiment is 0.75pg/L.

Example 7

This embodiment provides a method for detecting perfluorinated compounds. The preparation method of fluorescent conjugated polymer quantum dot probes is basically the same as in Example 1, and the method for detecting perfluorinated compounds is the same as in Example 2. The difference is that this In the preparation process of the fluorescent conjugated polymer quantum dot probe in the example, in step 2, the amount of aminopropyltriethoxysilane added was 10 μL.

The detection limit of this embodiment is 0.83pg/L.

Example 8

This embodiment provides a method for detecting perfluorinated compounds. The preparation method of fluorescent conjugated polymer quantum dot probes is basically the same as in Example 1, and the method for detecting perfluorinated compounds is the same as in Example 2. The difference is that this In the preparation process of the fluorescent conjugated polymer quantum dot probe in the embodiment, in step 3, the amount of perfluorooctyltriethoxysilane added was 60 μL.

The detection limit of this embodiment is 0.97pg/L.

Example 9

This embodiment provides a method for detecting perfluorinated compounds. The preparation method of fluorescent conjugated polymer quantum dot probes is basically the same as in Example 1, and the method for detecting perfluorinated compounds is the same as in Example 2. The difference is that this In the preparation process of the fluorescent conjugated polymer quantum dot probe in the embodiment, in step 3, the amount of perfluorooctyltriethoxysilane added was 5 μL.

The detection limit of this embodiment is 0.71pg/L.

Comparative example 1

This comparative example provides a method for detecting perfluorinated compounds. The silanized fluorescent conjugated polymer quantum dot quantum dots prepared in Example 1 are used as probes to detect perfluorinated compounds. The detection method is the same as in Example 2. The measured number of silanized fluorescent conjugated polymer quantum dots does not change significantly, that is, the silanized fluorescent conjugated polymer quantum dots cannot be used for the detection of perfluorinated compounds.

Comparative example 2

The implementation of the patent document CN103558198B is used to detect perfluorooctane sulfonic acid, and the detected detection limit is 20 μg/L, which is obviously higher than the detection limit of the detection method provided by the present invention.

Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, other changes or changes in different forms can be made on the basis of the above description. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims

A method for preparing a fluorescent conjugated polymer quantum dot probe, characterized in that it comprises:

Step 1, reacting fluorescent conjugated polymer quantum dots with a silylating agent to prepare silanized fluorescent conjugated polymer quantum dots;

Step 2, modifying the surface of the silanized fluorescent conjugated polymer quantum dots prepared in step 1 with perfluoroalkane chains to prepare fluorescent conjugated polymer quantum dot probes.
The preparation method of fluorescent conjugated polymer quantum dot probe according to claim 1, is characterized in that, the preparation method of fluorescent conjugated polymer quantum dot comprises: mixing PFBT, PSMA and tetrahydrofuran to obtain a mixed solution; The mixed solution is poured into water, reacted, and tetrahydrofuran is removed to obtain the product.
The method for preparing fluorescently conjugated polymer quantum dot probes according to claim 2, wherein the method for preparing fluorescently conjugated polymer quantum dots also satisfies at least one of the following (1)-(5):

(1) The volume ratio of the mixed solution to water is (1220-1280):5000, the concentration of PFBT in the mixed solution is 0.016-0.125 mg/mL, and the concentration of PSMA is 0.156-0.328 mg/mL;

(2) The reaction temperature is 80-90°C, and the time is 0.5-2h;

(3) Under ultrasonic conditions, inject the mixed solution into water, and continue ultrasonication for 30-150s after the injection is completed;

(4) adopting the mode that feeds nitrogen into the reaction system to remove tetrahydrofuran;

(5) After removing THF, after cooling to room temperature, filter the obtained product solution through a filter membrane; preferably, the micropore of the filter membrane is 0.22 μm.
The preparation method of fluorescent conjugated polymer quantum dot probe according to claim 1, is characterized in that, step 1 comprises: in fluorescent conjugated polymer quantum dot solution, add silylating agent and activator, react, centrifuge, The lower solid was collected.
The method for preparing fluorescently conjugated polymer quantum dot probes according to claim 4, wherein step 1 also satisfies at least one of the following (1)-(5):

(1) The reaction time is 2 to 4 hours;

(2) the silylating agent is aminopropyltriethoxysilane or aminopropyltrimethoxysilane;

(3) The activator is EDC/NHS solution or EDC solution, preferably, the concentration of EDC and/or NHS in the activator is 0.3-0.8 mg/mL;

(4) The volume ratio of the activator to the silylating agent is 100:(20～60);

(5) The volume ratio of the silylating agent to the fluorescent conjugated polymer quantum dot solution is (20-60): 4000, wherein the concentration of the fluorescent conjugated polymer quantum dot solution is 55 μg/mL～140 μg/mL .
The method for preparing fluorescent conjugated polymer quantum dot probes according to claim 1, wherein step 2 comprises: dispersing the silanized fluorescent conjugated polymer quantum dots prepared in step 1 into an alcohol-water solution , to obtain a dispersion, add perfluorosilane and a catalyst, react, centrifuge, and collect the lower layer of solids.
The method for preparing fluorescent conjugated polymer quantum dot probes according to claim 6, wherein said step 2 satisfies at least one of the following (1)-(5):

(1) The concentration of silanized fluorescent conjugated polymer quantum dots in the dispersion is 13 to 30 μg/mL; the volume ratio of the perfluorosilane to the silanized fluorescent conjugated polymer quantum dot solution is (10～40): 5000;

(2) The alcohol-water solution is a mixed solution of methanol and/or ethanol and water, preferably, the volume ratio of alcohol to water is (2-5):1;

(3) Reaction time 4-6h;

(4) The perfluorosilane is perfluorooctyltriethoxysilane and/or perfluorodecyltriethoxysilane;

(5) The catalyst is ammonia water. Preferably, the mass concentration of the ammonia water is 25%-28%, and the volume ratio of perfluorosilane to ammonia water is (10-40):(5-20).
A fluorescent conjugated polymer quantum dot probe prepared by the method according to any one of claims 1-7.
The application of the fluorescent conjugated polymer quantum dot probe described in claim 8 in the detection of perfluorinated compounds, preferably, the perfluorinated compound is selected from perfluorooctane sulfonic acid, perfluorocaproic acid, perfluorononanoic acid , at least one of perfluorooctane carboxylic acid.
A method for detecting perfluorinated compounds, characterized in that the fluorescent conjugated polymer quantum dot probe according to claim 8 is used as a detection probe,

By detecting the number of fluorescently conjugated polymer quantum dot probes using a single-particle fluorescence counting sensing method, the concentration of perfluorinated compounds in the sample is obtained, or,

The fluorescence intensity was measured by a fluorescence spectrophotometer to obtain the concentration of perfluorinated compounds in the sample.