CN107907522B - Perfluorinated compound molecularly imprinted fluorescent probe and use method and application thereof - Google Patents

Abstract

The invention provides a perfluorinated compound molecularly imprinted fluorescent probe and application thereof. The fluorescent probe is prepared by the following steps: swelling chitosan with a weak acid solution, adding perfluorinated compound template molecules, uniformly mixing, adding epoxy chloropropane and amino carbon quantum dots, carrying out a crosslinking reaction for 4-12 h, and eluting the template molecules with an eluent for 4-12 h to obtain a perfluorinated compound molecularly imprinted fluorescent probe; wherein the mass ratio of the chitosan to the perfluorinated compound template molecule is 300-800: 1-5; the mass ratio of the chitosan to the amino carbon quantum dots to the epichlorohydrin is 300-800: 0.05-0.2: 0.12-0.35. The fluorescent probe integrates the enrichment and the detection of a sample, the detection is more convenient, namely the fluorescent probe captures the perfluorinated compounds and then directly detects the fluorescence intensity of the perfluorinated compounds to obtain the content of the perfluorinated compounds, so that the detection of the perfluorinated compounds is quicker and simpler; and the detection sensitivity and the specific selectivity are higher, and the interference resistance is stronger.

Description

A kind of perfluorinated compound molecularly imprinted fluorescent probe and its using method and application

technical field

The invention belongs to the field of analytical chemistry, and more particularly, relates to a perfluorinated compound molecularly imprinted fluorescent probe and a method and application thereof.

Background technique

Perfluorinated compounds (PFCs) are a class of pollutants that are ubiquitous in the environment. At the same time, PFCs are difficult to degrade, resulting in their persistent existence in the environment and enrichment by biological organisms through the transmission of biological chains. Many countries and regions around the world have It has been reported that PFCs have been detected in environmental and various biological samples, including human blood, serum and other matrices. Such compounds have received increasing attention due to the bioaccumulation of PFCs and their potential biotoxicity. Among them, perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) are the two most frequently detected and discussed perfluorinated compounds, and these two compounds are the final transformation products of various PFCs in the environment. At present, chromatography-mass spectrometry is mainly used for the analysis and detection of perfluorinated compounds. This method requires tedious sample pretreatment methods such as solid-phase extraction, and consumes a large amount of reagents and is time-consuming. Therefore, it is of great significance to establish a fast, sensitive and highly selective method for the analysis and detection of PFCs in biological samples such as blood and urine.

Carbon quantum dots are nanomaterials with high fluorescence properties after semiconductor quantum dots. Semiconductor quantum dots, such as cadmium selenide and cadmium telluride, contain heavy metal ions, which limit their use. The synthesis method of carbon quantum dots is simple, the water solubility is good, and the toxicity is small, and its application is more and more extensive. Molecular imprinting technology refers to the preparation of molecularly imprinted polymers with molecular recognition properties (specific selectivity) for a specific target molecule (template molecule, imprinted molecule). Molecular recognition means that the host molecule with the spatial structure, size and chemical functional group complementary to the guest molecule can recognize and bind the guest molecule. It has the characteristics of high affinity and selectivity, strong anti-interference and good stability, simple preparation and long service life. Therefore, molecular imprinting technology is widely used in the field of analytical chemistry such as chromatographic separation, solid-phase extraction, and solid-phase microextraction.

In recent years, there are also research works that introduce fluorescent material quantum dots into molecular imprinting technology to prepare molecularly imprinted fluorescent materials with specific recognition. However, molecularly imprinted fluorescent probes using perfluorinated compounds as template molecules have not yet been reported. Therefore, there is an urgent need to provide a molecularly imprinted fluorescent probe for perfluorinated compounds, which makes the detection of perfluorinated compounds more sensitive and accurate.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a perfluorinated compound molecularly imprinted fluorescent probe. The fluorescent probe of the present invention is obtained by combining the perfluorinated compound molecularly imprinted polymer prepared from chitosan and perfluorinated compound with carbon quantum dots. The fluorescent probe integrates the enrichment and detection of the sample, and the detection is more convenient; and the detection limit of the perfluorinated compound is lower, the specific selectivity is higher, and the anti-interference property is stronger, so that the perfluorinated compound is detected. detection is more accurate and sensitive.

Another object of the present invention is to provide a method for using the perfluorinated compound molecularly imprinted fluorescent probe.

Another object of the present invention is to provide the application of the perfluorinated compound molecularly imprinted fluorescent probe in the detection of perfluorinated compounds.

Above-mentioned purpose of the present invention is achieved through the following scheme:

A perfluorinated compound molecularly imprinted fluorescent probe is prepared by the following steps: swelling chitosan with a weak acid solution, adding perfluorinated compound template molecules to mix evenly, and then adding epichlorohydrin and amino carbon quantum dots for cross-linking reaction 4-12h, and finally the template molecule is eluted with the eluent, and the elution time is 4-12h, that is, the perfluorinated compound molecularly imprinted fluorescent probe is obtained;

The mass ratio of chitosan to perfluoro compound template molecules is 160-300:1; the mass ratio of chitosan, amino carbon quantum dots and epichlorohydrin is 4000-8000:1:1770-2365.

The fluorescent probe of the present invention not only has molecular recognition ability, but also a signal detector, which integrates traditional sample enrichment and detection, that is, after the fluorescent probe captures the perfluorinated compound, the fluorescent probe can directly detect its fluorescence intensity to obtain the The content of fluorine compounds makes the analysis and detection of perfluorinated compounds more convenient and faster.

If the mass ratio of chitosan and perfluorinated compound template molecules is too low, a large number of template molecules may exist in the internal pores of the chitosan molecule, and it is difficult for the template molecules to elute completely, which will ultimately affect the accuracy of the detection results; If the dosage is too large, the fluorescence of the quantum dots will be too strong, which will cover the reaction signal of the target product, resulting in inaccurate fluorescence detection results. If the cross-linking agent epichlorohydrin is used too little, the molecular imprinting will be unsuccessful and the target molecules cannot be captured well; if the cross-linking agent is used too much or the reaction time is too long, the chitosan will be completely modified into a rigid structure , the molecular fluorescence detection injection cannot be performed.

In the present invention, chitosan is used as the functional monomer, and by controlling the amount of the cross-linking agent and the cross-linking reaction time, the chitosan undergoes a cross-linking reaction, and the modification of the chitosan is controlled in a moderate range, so that the prepared The fluorescent probe microspheres are transparent and have excellent swelling properties under slightly acidic conditions, so that the fluorescent detection process will not be affected, and errors that may be caused by the color of the fluorescent probe itself are reduced.

Ordinary fluorescent probes are prone to fluorescence quenching in the presence of interfering substances, so that accurate detection results cannot be obtained. However, there are many non-target molecules in biological samples, which have great interference with fluorescent probes. When ordinary fluorescent probes are used in biological samples, the possibility of fluorescence quenching is extremely large, which limits their use in biological samples. Application in biological sample detection. In the present invention, the chitosan molecule is used as the functional monomer, which is specifically combined with the template molecule, and the carbon quantum dots are located inside the chitosan molecule. The phenomenon of stable fluorescence enhancement occurs, and the phenomenon of fluorescence quenching is not easy to occur. Therefore, the fluorescent probe of the present invention has good anti-interference performance, and is not easily affected by the presence of other substances to cause fluorescence quenching when detecting biological samples.

In the process of eluting the template molecules in the eluent, if the elution time is too short, the template molecules are not completely eluted, resulting in inaccurate detection results during the detection process; however, if the elution time is too long, carbon quantum is easily lost The carbon quantum dots on the dot fluorescent probes lead to insignificant changes in fluorescence intensity and reduced detection sensitivity.

Preferably, the mass ratio of the chitosan to the perfluoro compound template molecule is 200-280:1-2.

Preferably, the mass ratio of chitosan, amino carbon quantum dots and epichlorohydrin is 4000-8000:1:1770-2365.

Preferably, the crosslinking reaction time is 12h, and the elution time is 8h. The crosslinking reaction time was calculated from the addition of epichlorohydrin and aminocarbon quantum dots.

Preferably, the perfluoro compound template molecule is PFOS or PFOA.

Preferably, the weak acid solution used for chitosan swelling is acetic acid solution. The volume fraction of the acetic acid solution used is 1-3%.

Preferably, the eluent is an alkaline solution of an organic solvent; more preferably, the eluent is a mixed solution of acetone and sodium hydroxide with a volume ratio of 1:1-3.

Preferably, the carbon quantum dots are prepared by the following method: after mixing citric acid and ethylenediamine evenly, the solution is transferred to a hydrothermal reactor for reaction to obtain an amino carbon quantum dot solution; Then the carbon quantum dot solid is obtained.

Preferably, the mass ratio of citric acid to ethylenediamine is 3-4:0.9-2.7.

Preferably, the hydrothermal reaction temperature is 160-200°C; and the reaction time is 4-6h.

Preferably, the retention volume of the dialysis bag is 300-600D.

The use method of the fluorescent probe provided by the present invention is as follows: adjusting the pH of the sample to be tested to 3-4 with a weak acid solution, adding the fluorescent probe to react for 20-90 min at 30°C to 50°C, filtering and separating the fluorescent probe needle and perform fluorescence detection.

The sample is adjusted to weak acid, on the one hand, because the perfluorinated compound is more stable under weak acid conditions and has better solvent properties; on the other hand, because the fluorescent probe of the present invention has better swelling performance under weak acid conditions , which can be uniformly dispersed in the sample under weak acid conditions, and can quickly and completely capture the perfluorinated compounds in the sample, making the detection result more accurate.

Preferably, the pH of the sample adjusted by the weak acid is 3-4; more preferably, the pH of the sample adjusted by the weak acid is 4.

Preferably, the reaction temperature is 40°C; the reaction time is 30min.

The invention also protects the application of the fluorescent probe in detecting the content of perfluorinated compounds in biological samples.

Preferably, the fluorescent probe is used in the detection of PFOS or PFOA in biological samples.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The fluorescent probe of the present invention not only has molecular recognition ability, but also a signal detector, which integrates traditional sample enrichment and detection, that is, after the fluorescent probe captures the perfluorinated compound, the fluorescent probe can directly detect its fluorescence intensity to obtain the The content of fluorine compounds makes the detection of perfluorinated compounds faster and easier.

At the same time, the fluorescent probe of the present invention has higher detection sensitivity and specific selectivity for perfluorinated compounds, and stronger anti-interference property, thereby making the detection result more accurate.

Description of drawings

1 is a transmission electron microscope image of the carbon quantum dots used in Example 1.

FIG. 2 is the standard curve of the fluorescent probes prepared in Examples 1-3 for detecting perfluorinated compounds.

specific embodiment

The content of the present invention is further described below in conjunction with specific embodiments, but should not be construed as a limitation of the present invention. Without deviating from the spirit and essence of the present invention, simple modifications or substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention; unless otherwise specified, the technical means used in the embodiments are those of ordinary skill in the art well-known conventional means.

The preparation of the perfluorinated compound molecularly imprinted carbon quantum dot fluorescent probe of the present invention, wherein PFOS is used as a template molecule for preparation.

Example 1

(1) Weigh 3.0 g of citric acid, add 33 mL of deionized water, and stir magnetically for 5 min to dissolve all the solids. Add 1.0 mL of ethylenediamine (in a fume hood) and stir magnetically for 10 min. The solution was transferred to a hydrothermal reactor, screwed into an oven, and reacted at 160 °C for 4 h. The dialysis bag with the retention volume of 300D was dialyzed for 48h, during which the water was changed continuously. The dialysate was freeze-dried in a glass petri dish to obtain a brownish-yellow viscous carbon quantum dot solid.

(2) Add 1% acetic acid to 0.3 g of chitosan for swelling; add 1 mg of template molecule PFOS. Stir at room temperature for 3 hours; add 1 mL of an aqueous solution containing 50 μg of carbon quantum dots, stir evenly with 100 μL of epichlorohydrin, and then conduct cross-linking at a constant temperature of 40 °C for 4 hours. Afterwards, 20 g of chitosan molecularly imprinted fluorescent material was taken with a wet weight, and 30 mL of a mixed solution of acetone and 0.5 M sodium hydroxide with a volume ratio of 1:1 was used, and the mixture was stirred and eluted at room temperature for 4 h.

In step (2), the mass ratio of chitosan and perfluorinated compound template molecules is 300:1; the mass ratio of chitosan, amino carbon quantum dots and epichlorohydrin is 6000:1:2362.

Example 2

(1) The preparation process of carbon quantum dots is the same as that in Example 1, except that the citric acid used is 3.5 g, the ethylenediamine is 2.0 mL, the reaction temperature is 170 °C, and the reaction time is 5 h. The retention volume of the dialysis bag is 400D.

(2) Add 2% acetic acid to 0.5 g of chitosan for swelling; add 3 mg of template molecule PFOS. Stir at room temperature for 3 h; add 1 mL of an aqueous solution containing 100 μg of carbon quantum dots, stir evenly with 200 μL of epichlorohydrin, and then conduct cross-linking at a constant temperature of 40 °C for 8 h. Afterwards, 20 g of the chitosan molecularly imprinted fluorescent material was taken with a wet weight, and 45 mL of a mixed solution of acetone and 0.5 M sodium hydroxide in a volume ratio of 1:2 was used to elute at room temperature for 8 h under stirring conditions.

In step (2), the mass ratio of chitosan and perfluorinated compound template molecules is 166.67:1; the mass ratio of chitosan, amino carbon quantum dots and epichlorohydrin is 5000:1:2362.

Example 3

(1) The preparation process of carbon quantum dots is the same as that in Example 1, except that the citric acid used is 4.0 g, the ethylenediamine is 1.5 mL, the reaction temperature is 180 °C, and the reaction time is 6 h. The hold-up volume of the dialysis bag is 500D.

(2) Add 3% acetic acid to 0.8 g of chitosan for swelling, add 5 mg of template molecule PFOS, and stir at room temperature for 3 h; add 1 mL of an aqueous solution containing 200 μg of carbon quantum dots, stir evenly with 300 μL of epichlorohydrin, and then keep at a constant temperature of 40 °C Cross-linking was carried out for 12h. Afterwards, 20 g of the chitosan molecularly imprinted fluorescent material was taken with a wet weight, and 60 mL of a mixed solution of acetone and 0.5 M sodium hydroxide in a volume ratio of 1:3 was used to elute at room temperature for 12 h under stirring conditions.

In step (2), the mass ratio of chitosan and perfluorinated compound template molecules is 160:1; the mass ratio of chitosan, amino carbon quantum dots and epichlorohydrin is 4000:1:1771.

Example 4

The types, amounts and methods of all raw materials are the same as in Example 1, except that in step (2), the mass ratio of chitosan and perfluoro compound template molecules is 240:1; chitosan, amino carbon quantum dots and epoxy The mass ratio of chloropropane is 4000:1:1800.

Example 5

The types, amounts and methods of all raw materials are the same as those in Example 1, except that in step (2), the mass ratio of chitosan to perfluoro compound template molecules is 260:1; chitosan, amino carbon quantum dots and epoxy The mass ratio of chloropropane is 5000:1:2000.

Example 6

The types, amounts and methods of all raw materials are the same as in Example 1, except that in step (2), the mass ratio of chitosan to perfluoro compound template molecule is 280:1; chitosan, amino carbon quantum dots and epoxy The mass ratio of chloropropane is 6000:1:2200.

Comparative Example 1

The type, amount and preparation process of the raw materials used in this comparative example are the same as those in Example 1, except that agarose of equal quality is used instead of chitosan.

Comparative Example 2

The kind of raw material, consumption and preparation process adopted in this comparative example are the same as in Example 1, except that glutaraldehyde of equal quality is adopted to replace epichlorohydrin.

Comparative Example 3

The raw material types, dosage and preparation process adopted in this comparative example are the same as those in Example 1, except that the mass ratio of chitosan, carbon quantum dots and epichlorohydrin is 4000-8000:1:3000.

Comparative Example 4

The raw material type, amount and preparation process adopted in this comparative example are the same as those in Example 1, except that the mass ratio of chitosan and template molecule PFOS is 100:1.

Comparative Example 5

The kind, amount and preparation process of the raw materials used in this comparative example are the same as those in Example 1, except that the cross-linking reaction time is 16h.

Comparative Example 6

The kind, amount and preparation process of the raw materials used in this comparative example are the same as those in Example 1, except that the elution time is 2h.

Performance test: Test the sensitivity, specific selectivity and anti-interference of fluorescent probes.

(1) Test the sensitivity of fluorescent probes

A standard curve of the fluorescence response of the fluorescent probes prepared in Examples 1 to 6 to PFOS was prepared.

PFOS was prepared into a standard solution with a concentration of 1-1000 pg/L with a volume fraction of 2% HAc. At 40 °C, 10 mL of the standard solution was mixed with 1 g of the fluorescent probe for 30 min, and the fluorescence intensity of the fluorescent probe was detected after capturing the perfluorinated compound, and the change of the fluorescence intensity with the concentration of PFOS was observed. According to the Stern-Volmer equation F ₀ /F=1+Ksv[C], with the concentration [C] as the abscissa and the relative fluorescence intensity F ₀ /F as the ordinate, the fluorescence response curve was drawn according to the detected results.

The resulting standard curve is shown in Figure 2. Through calculation, the detection limit of the fluorescent probe and the detection method of the present invention is 5.1 pg/L. It can be seen that the detection limit of the fluorescent probe of the present invention is relatively low and has good sensitivity. .

(2) Test the specific selectivity of fluorescent probes

The fluorescent probes prepared in Examples 1 to 6 and Comparative Examples 1 to 6 were used to detect PFOS samples with known concentrations. Except for PFOS, no other substances were added to the samples. The detection results were recorded as the first detection. After the detection, perfluorooctanoic acid (PFOA), sodium dodecylbenzenesulfonate (SDBS), sodium dodecyl sulfate, sodium dodecylsulfonate and perfluoro-1-butanesulfonic acid were added to the samples, and then The same method is used for detection, and this detection is recorded as the second detection. The specific selection performance of the fluorescent probe was verified by comparing the two detection results with known concentrations and the difference between the two detection results. The detection results are shown in Table 1.

Table 1 Fluorescent probes prepared in Examples 1-6 and Comparative Examples 1-6 to detect PFOS results (pg/L)

serial number Molecular concentration of perfluorinated compounds in the sample/pg/L The first detection concentration/pg/L Second detection concentration/pg/L The third detection concentration/pg/L Fourth detection concentration/pg/L The fifth detection concentration/pg/L Example 1 twenty two twenty one twenty two twenty two twenty one twenty two Example 2 twenty two twenty two 20 twenty one twenty one twenty one Example 3 twenty two twenty two twenty two twenty one twenty one twenty one Example 4 twenty two twenty two twenty two 20 twenty one twenty two Example 5 twenty two 20 20 twenty one 20 20 Example 6 twenty two twenty one 20 20 20 twenty one Comparative Example 1 twenty two 0 0 0 0 0 Comparative Example 2 twenty two 10 5 7 9 5 Comparative Example 3 twenty two 0 0 0 0 0 Comparative Example 4 twenty two 16 14 12 10 11 Comparative Example 5 twenty two 0 0 0 0 0 Comparative Example 6 twenty two 11 8 9 7 7

It can be seen from Table 1 that the results of the second detection of the fluorescent probe prepared by the present invention are very close to the actual content of PFOS in the sample, and the maximum difference is only 2pg/L, and the results of the five detections have almost no difference. The difference is only 2 pg/L, indicating that the fluorescent probe prepared by the present invention has good specific selectivity for PFOS, and can specifically identify PFOS when a variety of compounds with similar structures exist at the same time, which improves the accuracy of detection results. sex. However, the fluorescent probes prepared in Comparative Examples 1, 3 and 5 could not detect the presence of PFOS in the samples, while the results detected in Comparative Examples 2, 4 and 6 were quite different from the actual content of PFOS in the samples, and the results of multiple detections The difference between them is also large, the detection results are extremely inaccurate, and the repeatability is not good.

(3) Test the anti-interference of fluorescent probes

Taking human blood as a sample, adding a certain amount of PFOS to the sample, and using the fluorescent probes prepared in Examples 1-6 and Comparative Examples 1-7 to detect the samples. During the detection process, the fluorescence changes of the fluorescent probes were observed, and the anti-interference performance of the fluorescent probes was identified according to whether the fluorescence was quenched or not.

Table 2 Fluorescent probes prepared in Examples 1-6 and Comparative Examples 1-6

serial number first test second test The third test Example 1 No fluorescence quenching or weakening No fluorescence quenching or weakening No fluorescence quenching or weakening Example 2 No fluorescence quenching or weakening No fluorescence quenching or weakening No fluorescence quenching or weakening Example 3 No fluorescence quenching or weakening No fluorescence quenching or weakening No fluorescence quenching or weakening Example 4 No fluorescence quenching or weakening No fluorescence quenching or weakening No fluorescence quenching or weakening Example 5 No fluorescence quenching or weakening No fluorescence quenching or weakening No fluorescence quenching or weakening Example 6 No fluorescence quenching or weakening No fluorescence quenching or weakening No fluorescence quenching or weakening Comparative Example 1 no fluorescence no fluorescence no fluorescence Comparative Example 2 Fluorescence increases first and then decreases Fluorescence increases first and then decreases Fluorescence increases first and then decreases Comparative Example 3 no fluorescence no fluorescence no fluorescence Comparative Example 4 Fluorescence is enhanced first and then quenched Fluorescence is enhanced first and then quenched Fluorescence is enhanced first and then quenched Comparative Example 5 no fluorescence no fluorescence no fluorescence Comparative Example 6 Fluorescence increases and then decreases Fluorescence is first enhanced and then quenched Fluorescence is first enhanced and then quenched

It can be seen from Table 2 that the fluorescent probe prepared by the present invention has no fluorescence quenching or weakening phenomenon in the three detection processes, which proves that the fluorescent probe prepared by the present invention has good anti-interference ability and is not prone to fluorescence weakening. or quenching phenomenon, to ensure the accuracy of the detection results. However, comparative examples 2, 4 and 6 all showed a certain degree of fluorescence weakening or quenching during the detection process, which eventually led to inaccurate detection results.

Claims (10)

1. A perfluorinated compound molecularly imprinted fluorescent probe is characterized by being prepared by the following steps: swelling chitosan with a weak acid solution, adding perfluorinated compound template molecules, uniformly mixing, adding epoxy chloropropane and amino carbon quantum dots, carrying out a crosslinking reaction for 4-12 h, and eluting the template molecules with an eluent for 4-12 h to obtain a perfluorinated compound molecularly imprinted fluorescent probe;

wherein the mass ratio of the chitosan to the perfluorinated compound template molecule is 160-300: 1; the mass ratio of the chitosan to the amino carbon quantum dots to the epichlorohydrin is 4000-8000: 1: 1770-2365.

2. The fluorescent probe of claim 1, wherein the mass ratio of chitosan to perfluorinated compound template molecule is 200-280: 1.

3. The fluorescent probe according to claim 1, wherein the mass ratio of the chitosan to the amino carbon quantum dots to the epichlorohydrin is as follows: 4000-6000: 1: 1770-2365.

4. The fluorescent probe of claim 1, wherein the crosslinking reaction time is 12 hours; the elution time was 8 h.

5. The fluorescent probe of claim 1, wherein the perfluorinated compound template molecule is PFOS or PFOA.

6. The fluorescence probe of claim 1, wherein the weak acid solution used for swelling the chitosan is an acetic acid solution.

7. The fluorescent probe of claim 1, wherein the elution solution is an alkaline solution of an organic solvent.

8. The method for using the fluorescent probe according to any one of claims 1 to 7, which comprises the following steps: and adjusting the pH value of a sample to be detected to 3-4 by using a weak acid solution, adding the fluorescent probe to react for 20-90 min at the temperature of 30-50 ℃, filtering and separating the fluorescent probe, and performing fluorescence detection.

9. The method of using the fluorescent probe according to claim 7, wherein the pH of the sample is 4 after the sample is adjusted with a weak acid; the reaction temperature is 40 ℃; the reaction time was 30 min.

10. Use of the fluorescent probe according to any one of claims 1 to 7 for detecting the content of perfluorinated compounds in a biological sample.

Images