Method for ultrasensitively detecting biodegradable microplastics and application

Abstract

The invention relates to a method for ultrasensitively detecting biodegradable micro-plastics and application, and particularly discloses a method for ultrasensitively detecting biodegradable micro-plastics, which can specifically identify and quantitatively analyze micro-plastics in various samples. By using a specific compound, the method realizes top-speed detection, has excellent selectivity, can eliminate interference of common metal ions and substances, keeps ultrahigh sensitivity, and can accurately detect biodegradable micro-plastics even at a low concentration level. In addition, the synthesis process of the compound used in the method is simple and convenient, the reagent cost is low, and the method has wide commercialization and industrialization application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microplastic detection technology, and in particular to a method and application for ultra-sensitive detection of biodegradable microplastics. The fluorescence method can achieve rapid, ultra-sensitive and specific detection of biodegradable microplastics. Background Art

[0002] Plastic, as a multifunctional polymer material derived from petroleum, has penetrated into all aspects of social economy with its light weight, flexibility, durability and low cost, greatly enriching and facilitating human life. However, its difficult to degrade naturally, coupled with improper management and disposal, has led to the accumulation of a large amount of plastic waste, which is then converted into microplastics (less than 5mm in size), seriously polluting the earth's water, soil and atmosphere, posing an unprecedented challenge to the ecological balance.

[0003] Microplastics, an emerging environmental pollutant, can be divided into two categories: primary and secondary. The former originates directly from the production process and is commonly found in personal care products and industrial products; the latter is a byproduct of large pieces of plastic that undergo weathering, photolysis and biodegradation under natural conditions. The ubiquity of microplastics not only disrupts the normal operation of the marine ecosystem, but also poses a potential threat to human health through its accumulation in the food chain, especially when microplastics and the harmful substances they adsorb enter the human body.

[0004] Although the scientific research community has begun to pay attention to the impact of microplastics on the environment and organisms, existing detection technologies still have many shortcomings. Traditional microplastic detection methods rely on filtration, microscopy and chemical analysis, which are time-consuming and labor-intensive and difficult to achieve on-site real-time monitoring. Fluorescent probe technology, due to its rapid response and high sensitivity, has opened up a new way for real-time detection of microplastics. However, the current fluorescent probe technology still needs to be broken through in terms of specificity, anti-interference ability, sensitivity and ease of preparation, which hinders its widespread application in the field of microplastic detection.

[0005] In view of this, it is urgent to develop a microplastic detection method based on fluorescent probe technology, which can achieve specific identification of microplastics, has high sensitivity, strong stability and anti-interference ability, and can adapt to complex environmental samples. In addition, the ideal microplastic detection technology should also be easy to operate, suitable for real-time monitoring under field and laboratory conditions, and provide a powerful tool for the assessment and control of microplastic pollution. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a method for ultrasensitive detection of biodegradable microplastics. The method for detecting biodegradable microplastics of the present invention can achieve rapid, ultrasensitive and specific detection of biodegradable microplastics.

[0007] Specifically, the present invention provides a method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample, the method comprising the following steps:

[0008] a) contacting the compound of formula (I) with a sample to form a product with a fluorescence change;

[0009] Wherein, the structural formula of the compound of formula (I) is as follows:

[0010]

[0011] b) measuring the fluorescence property of the product of said fluorescence change.

[0012] In some specific embodiments of the present invention, the method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample is characterized by further comprising the following steps:

[0013] A series of biodegradable microplastic solutions of standard concentrations were prepared, the compound of formula (I) was used to react with biodegradable microplastic solutions of different standard concentrations, the fluorescence emission spectra of the solutions after the reaction were measured, and a standard working curve was drawn.

[0014] In some specific embodiments of the present invention, the time for the compound of formula (I) to contact the sample is 1-3 minutes.

[0015] In some specific embodiments of the present invention, the specific step of determining the fluorescence property of the fluorescent substance includes: using a fluorescence spectrometer to test the fluorescence intensity of the fluorescent substance.

[0016] In some embodiments of the invention, the sample is an environmental sample, a chemical sample, a food sample or a biological sample.

[0017] In some specific embodiments of the present invention, the sample is a biological sample including water, blood, microorganisms or animal cells.

[0018] In some specific embodiments of the present invention, the environmental sample is an aqueous environmental sample.

[0019] In some specific embodiments of the present invention, the specific detection steps of the method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample are as follows:

[0020] S1. Prepare a series of biodegradable microplastic solutions of standard concentrations, add them to the solutions of the compound of formula (I) of known concentrations, mix them evenly, and then use a fluorescence spectrometer to test the fluorescence emission spectrum of the solution after the reaction after standing for 1-3 minutes, and construct a linear relationship between the fluorescence intensity of the fluorescence spectrum at 600nm and the corresponding biodegradable microplastic concentration, so as to obtain a standard working curve for detecting the concentration of biodegradable microplastics;

[0021] S2. Add the sample to a solution of the compound of formula (Ι) of known concentration, mix evenly, and then let it stand for reaction for 1-3 minutes, and then use a fluorescence spectrometer to test the fluorescence emission spectrum of the solution after the reaction. According to the fluorescence intensity at 600nm and the standard working curve, the content of biodegradable microplastics in the test sample is obtained.

[0022] In some specific embodiments of the present invention, the reaction systems for the static reactions in steps S1 and S2 are both PBS buffer solution systems with a pH of 7.4.

[0023] In some specific embodiments of the present invention, the reaction systems of the static reactions in steps S1 and S2 are both 10 mM PBS buffer solution systems with a pH of 7.4.

[0024] The present invention also provides the use of a compound of formula (I) in detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample. The structural formula of the compound of formula (I) is as follows:

[0025]

[0026] The present invention also provides the use of a compound of formula (I) in the preparation of a reagent for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample. The structural formula of the compound of formula (I) is as follows:

[0027]

[0028] The present invention also provides a kit or composition for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample, characterized in that it comprises a compound of formula (I), wherein the compound of formula (I) has the following structural formula:

[0029]

[0030] In some specific embodiments of the present invention, the kit or composition for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample further comprises a solvent, an acid, a base, a buffer solution or a combination thereof.

[0031] The present invention also provides a method for preparing the compound of formula (I), comprising the following steps:

[0032] The compound of formula (I) is prepared by reacting 4-[4-(dimethylamino)phenylvinyl]pyridine with 4-bromomethylphenylacetate.

[0033]

[0034] In some specific embodiments of the present invention, the preparation method of the compound of formula (I) is as follows: 4-[4-(dimethylamino)phenylvinyl]pyridine is dissolved in toluene, 4-bromomethylphenylacetate is added, and the mixture is heated in an oil bath and refluxed overnight. After the reaction is completed, the mixture is cooled and allowed to stand until the substance precipitates, and then the solvent is removed by suction to obtain a red solid powder. The filter cake is washed three times with cold ether and dried under vacuum at 60°C for 6 hours to obtain a red solid powder; the mixture is further separated by a chromatographic column, and a mixed system of dichloromethane and methanol is used as an eluent to obtain a pure compound of formula (I).

[0035] In some specific embodiments of the present invention, the 4-[4-(dimethylamino)phenylvinyl]pyridine and 4-bromomethylphenylacetate are heated to reflux at 110°C.

[0036] In some specific embodiments of the present invention, the molar ratio of 4-[4-(dimethylamino)phenylvinyl]pyridine to 4-bromomethylphenylacetate is 1:1.

[0037] In some specific embodiments of the present invention, the reaction time of heating reflux reaction of 4-[4-(dimethylamino)phenylvinyl]pyridine and 4-bromomethylphenylacetate is 12 hours.

[0038] Compared with the prior art, the present invention has the following significant advantages and outstanding effects:

[0039] (1) Ultra-high sensitivity

[0040] The method for detecting biodegradable microplastics provided by the present invention uses a compound of formula (I) that reacts extremely sensitively with biodegradable microplastics and can accurately detect biodegradable microplastics at low concentration levels, with a detection limit of 0.477 μg / mL.

[0041] (2) Extremely fast detection

[0042] The method for detecting biodegradable microplastics provided by the present invention, wherein the compound of formula (I) used can react rapidly with biodegradable microplastics, and this property greatly promotes the rapid and even real-time detection of biodegradable microplastics.

[0043] (3) Excellent selectivity

[0044] The method for detecting biodegradable microplastics provided by the present invention, the formula (I) compound used therein has a selective specific reaction ability to microplastics, and the reaction generates a product with fluorescence change. Compared with many common other metal ions and substances, such as calcium ion, iron ion, ferrous ion, sodium ion, potassium ion, copper ion, zinc ion, carbonate ion, nitrite ion, nitrate ion, acetate ion, bromide ion, sulfate ion, bicarbonate ion, chloride ion, sulfide ion, hypochlorous acid, iodide ion, hydrogen peroxide, sodium ascorbate, glutathione, cysteine, beta-galactosidase, ribonuclease, trypsin, lysozyme, etc., the formula (I) compound can effectively eliminate the interference of most substances, demonstrating an extremely excellent selectivity.

[0045] (4) Low testing cost

[0046] The method for detecting biodegradable microplastics provided by the present invention has a simple synthesis process of the compound of formula (I) used, and the reagent cost used in the detection is low. This advantage makes the detection method very potential in the promotion and application of commercialization and industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that may be involved. It should be emphasized that the drawings described below are only some embodiments of the present invention, and for ordinary technicians working in this field, other embodiments can be obtained based on these drawings without creative work.

[0048] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound of formula (I) synthesized in Example 1;

[0049] Figure 2 is the carbon-1NMR spectrum of the compound of formula (I) synthesized in Example 1;

[0050] Figure 3 is a high-resolution mass spectrum of the compound of formula (I) synthesized in Example 1;

[0051] Figure 4 is the fluorescence spectrum of the compound of formula (I) before and after adding biodegradable microplastics;

[0052] Figure 5 is a response time spectrum of the compound of formula (I) before and after adding biodegradable microplastics;

[0053] Figure 6 It is the effect of different analytes (glutathione and cysteine ​​are 1 mM, and the others are 100 μM) on the fluorescence intensity of the compound of formula (I). The bar graph represents the fluorescence intensity of the compound of formula (I) at 600 nm in the presence of different analytes.

[0054] Figure 7 is the fluorescence spectrum of the compound of formula (I) after adding different concentrations of biodegradable microplastics;

[0055] Figure 8 is a linear relationship diagram of the fluorescence intensity of the compound of formula (I) at 600 nm and the concentration of biodegradable microplastics; DETAILED DESCRIPTION

[0056] In conjunction with the drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be clear that the described embodiments only represent a part of the implementation methods of the present invention and do not limit the protection scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without performing creative work belong to the protection scope of the present invention.

[0057] Example 1: Synthesis of the compound of formula (I)

[0058] The synthetic route is as follows:

[0059]

[0060] The specific steps are as follows:

[0061] 4-[4-(Dimethylamino)phenylvinyl]pyridine (224g, 1mmol) was dissolved in 20mL toluene, 4-bromomethylphenylacetate (229g, 1mmol) was added, and the mixture was heated in an oil bath at 110°C and refluxed for 12 hours. After the reaction was completed, the mixture was cooled and allowed to stand until the substance was precipitated, and then the solvent was removed by suction to obtain a red solid powder. The filter cake was washed three times with cold ether and dried under vacuum at 60°C for 6h to obtain a red solid powder. The mixture was further separated by a chromatographic column, and a mixed system of dichloromethane and methanol was used as the eluent to obtain 330mg of the pure compound of formula (I), with a yield of 72.8%.

[0062] The hydrogen spectrum, carbon spectrum and high-resolution mass spectrum of the compound of formula (I) synthesized by this method are shown in the attached Figure 1 , Attachment Figure 2 , Attachment Figure 3 shown.

[0063] Example 2: Changes in fluorescence spectra of the compound of test formula (I) before and after the reaction with biodegradable microplastics

[0064] A test system of the compound of formula (I) with a concentration of 5 μM was prepared, and the volume of each system was 10 mL. After shaking, the fluorescence spectrum was measured using a fluorescence spectrometer. Next, 5 mg of biodegradable microplastics (PBAT) was added to each test system, and after shaking, the fluorescence spectrum was measured again using a fluorescence spectrometer. All experiments were carried out in PBS buffer solution (10 mM PBS, pH = 7.4), and the measurement temperature of the fluorescence spectrum was 25 ° C. The compound of formula (I) used was prepared in Example 1. The test results are shown in the attached Figure 4 shown.

[0065] Depend on Figure 4 It can be clearly seen that after adding biodegradable microplastics, the emission peak of the fluorescence spectrum of the compound of formula (I) at 600nm is significantly enhanced.

[0066] Example 3: Test of the detection time required for the method for detecting biodegradable microplastics provided by the present invention

[0067] First, a 10 mL test system with a concentration of 5 μM of the compound of formula (I) was prepared. Then, 5 mg of biodegradable microplastics (PBAT) was added to the test system, and the change in fluorescence intensity was immediately tested using a fluorescence spectrometer after shaking. All experiments were carried out in PBS buffer solution (10 mM PBS, pH = 7.4), the measurement temperature of the fluorescence spectrum was 25 ° C, and the compound of formula (I) used was prepared in Example 1. The test results are shown in the attached Figure 5 shown.

[0068] from Figure 5 It can be clearly observed that the fluorescence intensity of the compound of formula (I) itself remains stable; when biodegradable microplastics are added, the fluorescence intensity of the compound of formula (I) increases over time. In less than 1 minute, the fluorescence intensity reaches the maximum value, which indicates that the compound of formula (I) has a rapid response to biodegradable microplastics and provides an effective analytical method for the rapid detection of biodegradable microplastics.

[0069] Example 4: Testing the selectivity of the method for detecting biodegradable microplastics provided by the present invention

[0070] A plurality of compounds of formula (I) with a concentration of 5 μM were prepared in a 10 mL colorimetric tube, and then different analytes (the analytes were blank, calcium ion, iron ion, ferrous ion, sodium ion, potassium ion, copper ion, zinc ion, carbonate ion, nitrite ion, nitrate ion, acetate ion, bromide ion, sulfate ion, bicarbonate ion, chloride ion, sulfur ion, hypochlorous acid, iodide ion, hydrogen peroxide, sodium ascorbate, glutathione, cysteine, β-galactosidase, ribonuclease, trypsin, lysozyme and biodegradable microplastics (PBAT, 1 μm, 500 μg / mL); the concentrations of cysteine ​​and glutathione were 1 mM, and the concentrations of other analytes were 100 μM) were added to the test system, shaken evenly, left to stand for 3 minutes, and then the change in fluorescence intensity was tested using a fluorescence spectrometer. The above determination was carried out in a PBS buffer solution (10 mM PBS, pH = 7.4) system, the fluorescence spectrum was measured at a temperature of 25°C, and the compound of formula (I) used was prepared in Example 1. The test results are shown in the attached Figure 6 shown.

[0071] from Figure 6 The results show that only when biodegradable microplastics are added, the fluorescence intensity of the compound of formula (I) will change significantly, while the influence of other analytes is almost negligible. This proves that the method for detecting biodegradable microplastics provided by the present invention has high selectivity for microplastics and can be used as a specific identification and analysis tool for biodegradable microplastics in the environment and water.

[0072] Example 5: Drawing a standard working curve for detecting biodegradable microplastics

[0073] Multiple compounds of formula (I) with a concentration of 5 μM were prepared in 5 mL cuvettes. Then, different concentrations (0-500 μg / mL) of biodegradable microplastics (PBAT) were added to each sample, shaken and allowed to stand for 3 minutes, and the change in fluorescence intensity was tested using a fluorescence spectrometer. All experiments were conducted in PBS buffer solution (10 mM PBS, pH = 7.4), the fluorescence spectrum was measured at 25 ° C, and the compounds of formula (I) used were prepared in Example 1. The test results are shown in the attached Figure 7 The standard working curve is shown in the attached Figure 8 .

[0074] from Figure 7 It is clearly seen that with the increase of the concentration of biodegradable microplastics, the emission peak of the compound of formula (I) at 600nm gradually increases. Figure 8It can be seen that after the compound of formula (Ι) (5 μM) reacts with biodegradable microplastics (0-500 μg / mL), its fluorescence intensity shows a good linear relationship with the concentration of biodegradable microplastics, indicating that the method for detecting biodegradable microplastics provided by the present invention can be used for quantitative analysis of biodegradable microplastics.

[0075] Example 6: Sensitivity of the method for detecting biodegradable microplastics provided by the present invention

[0076] The method for detecting biodegradable microplastics provided by the present invention calculates the detection limit of biodegradable microplastics as follows:

[0077] Detection limit = 3σ / k

[0078] σ is the standard deviation of the blank experiment, k is the fluorescence intensity F measured by the fluorescence concentration titration experiment 600 The slope of the straight line obtained by linear fitting of the biodegradable microplastic concentration (see Appendix Figure 8 ).

[0079] It is calculated that the detection limit of the method for detecting biodegradable microplastics provided by the present invention is 0.477 μg / mL.

[0080] Example 7: Analysis and detection of biodegradable microplastic content

[0081] Real water samples from Jixia Lake of Shandong University of Technology were collected and tested, and no microplastics were found. Then, two groups of test systems were configured for each water sample, and 5 μM of the compound of formula (I) was added to each test system. The compound of formula (I) used was prepared in Example 1, and then 100 μg / mL and 200 μg / mL of biodegradable microplastics (PBAT) were added to each water sample and each test system, respectively. The mixture was shaken evenly and allowed to stand for 3 minutes. The fluorescence spectrum was then tested, and the content of biodegradable microplastics in each test system was calculated based on the fluorescence intensity at 600 nm and the standard working curve drawn in Example 5.

[0082] The above steps were repeated three times.

[0083] The test results are shown in the table below. The test results show that the recovery rate of water samples is 99.72%-102.54%, which further confirms that the method provided by the present invention for detecting the presence of biodegradable microplastics in samples or determining the content of biodegradable microplastics in samples can effectively detect biodegradable microplastics in real water samples.

[0084]

[0085] Although the present invention is described using the above embodiments, it should be understood that the present invention may be further modified and varied without departing from the spirit of the present invention, and these modifications and variations are all within the scope of protection of the present invention.

Claims

1. A method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample, characterized in that: The method comprises the following steps: a) contacting the compound of formula (I) with a sample to form a product with a fluorescence change; Wherein, the structural formula of the compound of formula (I) is as follows: b) measuring the fluorescence property of the product of said fluorescence change.

2. The method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample according to claim 1, characterized in that: The following steps are also included: A series of biodegradable microplastic solutions of standard concentrations were prepared, the compound of formula (I) was used to react with biodegradable microplastic solutions of different standard concentrations, the fluorescence emission spectra of the solutions after the reaction were measured, and a standard working curve was drawn.

3. The method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample according to claim 1, characterized in that: The time for the compound of formula (I) to contact with the sample is 1-3 minutes.

4. The method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample according to claim 1, characterized in that: The specific step of determining the fluorescence property of the fluorescent substance includes: using a fluorescence spectrometer to test the fluorescence intensity of the fluorescent substance.

5. The method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample according to claim 1, characterized in that: The sample is an environmental sample, a chemical sample, a food sample or a biological sample.

6. The method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample according to any one of claims 1 to 5, characterized in that: The specific detection steps are as follows: S1. Prepare a series of biodegradable microplastic solutions of standard concentrations, add them to the solutions of the compound of formula (I) of known concentrations, mix them evenly, and then test the fluorescence emission spectrum of the solution after the reaction using a fluorescence spectrometer after standing for 1-3 minutes, and construct a linear relationship between the fluorescence intensity of the fluorescence spectrum at 600nm and the corresponding biodegradable microplastic concentration, so as to obtain a standard working curve for detecting the concentration of biodegradable microplastics; S2. Add the sample to a solution of the compound of formula (Ι) of known concentration, mix evenly, and then let it stand for reaction for 1-3 minutes, and then use a fluorescence spectrometer to test the fluorescence emission spectrum of the solution after the reaction. According to the fluorescence intensity at 600nm and the standard working curve, the content of biodegradable microplastics in the test sample is obtained.

7. The method for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample according to claim 6, characterized in that: The reaction systems for the static reactions in steps S1 and S2 are both PBS buffer solution systems with a pH of 7.

4.

8. Use of a compound of formula (I) in detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample, characterized in that: The structural formula of the compound of formula (I) is as follows:

9. Use of a compound of formula (I) in the preparation of a reagent for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample, characterized in that: The structural formula of the compound of formula (I) is as follows:

10. A kit or composition for detecting the presence of biodegradable microplastics in a sample or determining the content of biodegradable microplastics in a sample, characterized in that: Contains a compound of formula (I), wherein the compound of formula (I) has the following structural formula:

Images