Flavonoid fluorescent dye, supramolecular fluorescent probe, preparation method of supramolecular fluorescent probe and pyriproxyfen detection method

Abstract

The invention discloses a flavonoid fluorescent dye, a supramolecular fluorescent probe, a preparation method of the supramolecular fluorescent probe and a pyriproxyfen detection method. The probe is constructed by spontaneously assembling the albumin derivative and the flavonoid fluorescent dye in a buffer solution. The detection mechanism is shown as follows: the flavonoid dye presents characteristic red fluorescence in a free state, and obvious fluorescence blue shift is triggered after the flavonoid dye is combined with albumin. When pyriproxyfen is introduced into the system, hydrophobic molecules enter an albumin hydrophobic cavity in a competitive binding manner, so that dye molecules have a replacement effect. In the process, along with the bidirectional response of a fluorescence signal, namely, the yellow fluorescence intensity and the pyriproxyfen concentration are in negative correlation, and the reconstructed dye aggregation state triggers the enhancement of a green fluorescence channel signal, so that the quantitative conversion of the fluorescence chroma of the solution from yellow to green is finally realized. Based on the ratio-type fluorescence signal, the probe system has an obvious colorimetric effect when being used for detecting pyriproxyfen in environment, food and the like, and the color change can be directly observed by naked eyes.

Description

Technical Field

[0001] This invention relates to the field of pyriproxyfen detection technology, and in particular to a flavonoid fluorescent dye, a supramolecular fluorescent probe for pyriproxyfen detection, and their preparation and detection methods. Background Technology

[0002] Pyriproxyfen is a benzyl ether insect growth regulator developed by Sumitomo Chemical Co., Ltd. of Japan in the 1980s. It works by mimicking insect juvenile hormones to specifically inhibit larval molting and adult emergence, exhibiting high efficiency, low mammalian toxicity, and environmental compatibility. It is widely used for the control of agricultural pests (such as whiteflies and aphids) and mosquito and fly larvae. Its mechanism of action includes interfering with chitin synthesis, blocking egg development, and disrupting hormonal balance, with particularly significant effects on final-instar larvae and eggs. It is often used in integrated pest management (IPM) to reduce the risk of resistance to traditional insecticides. However, pyriproxyfen's high chemical stability (soil half-life 30-90 days) leads to its easy accumulation in crops and water bodies, posing a potential endocrine disruption risk to crustaceans and aquatic organisms. The EU and Japan have set strict limits for its residues (0.01-0.5 mg / kg for fruits and vegetables, and 0.02 mg / kg for tea). Existing detection technologies rely on chromatography (GC-MS / LC-MS), which requires complex pretreatment and cannot be performed on-site, while immunoassay is susceptible to cross-interference from similar substances such as phenoxycarb. Summary of the Invention

[0003] To address this bottleneck, this invention develops a ratiometric fluorescence sensing technology based on an albumin-flavonoid supramolecular fluorescent probe: by using a pyriproxypyridine competitive substitution probe to trigger the conversion of yellow-green dual-channel fluorescence signals, high-sensitivity detection is achieved, and quantitative detection in the 0-10μM range is completed by combining it with RGB analysis on a smartphone, providing a portable solution for rapid on-site detection of pesticide residues.

[0004] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0005] In a first aspect, the present invention provides a flavonoid fluorescent dye, wherein the structural formula of the flavonoid fluorescent dye is as follows:

[0006]

[0007] Due to the limitations of short-wavelength emission of flavonol dyes, a flavonoid fluorescent dye with the structure described above was designed and synthesized based on the flavonoid skeleton characteristics. Compared with common flavonoid dyes, the flavonoid fluorescent dye of this invention protects the hydroxyl groups on the chromotropic ketone ring of flavonoids, shielding the intramolecular proton transfer process in the excited state, thereby enhancing its fluorescence emission performance. The supramolecular fluorescent probe obtained by self-assembly using this flavonoid fluorescent dye and albumin derivatives exhibits superior fluorescence stability and anti-interference performance.

[0008] A second aspect of the present invention provides a method for preparing the flavonoid fluorescent dye of the present invention, comprising the steps of:

[0009] 2-(4-(dimethylamino)styryl)-3-hydroxy-4H-chromen-4-one (HSF) was dissolved in N,N-dimethylformamide (DMF) with azircycloheptan-1-carboxyl chloride. Cesium carbonate (Cs₂CO₃) was added, and the mixture was stirred under an inert atmosphere for 12-24 hours (e.g., 12 hours, 18 hours, 20 hours, 24 hours, etc.) to obtain a mixture.

[0010] The mixture was purified to obtain the flavonoid fluorescent dye.

[0011] Specifically, HSF and azircycloheptan-1-formyl chloride were dissolved in N,N-dimethylformamide, cesium carbonate was added, and the mixture was stirred for 12 hours at room temperature under nitrogen protection. After the reaction was completed, the mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with sodium bicarbonate solution and concentrated under reduced pressure. The crude product was purified by recrystallization from a dichloromethane / n-hexane mixed solvent to obtain an orange solid, which is the flavonoid fluorescent dye.

[0012] A third aspect of the present invention provides a supramolecular fluorescent probe for the detection of pyriproxyfen, wherein the supramolecular fluorescent probe is self-assembled from an albumin derivative and a flavonoid fluorescent dye in a buffer solution; the structural formula of the flavonoid fluorescent dye is as follows:

[0013]

[0014] In this invention, the supramolecular fluorescent probe is obtained by self-assembly of an albumin-based derivative host and a flavonoid fluorescent dye guest in a buffer solution after mixing them in a specific ratio. Specifically, the flavonoid fluorescent dye and the albumin-based derivative form a complex (i.e., the supramolecular fluorescent probe) in the buffer solution through hydrophobic interactions and π-π stacking. The buffer solution can be PBS buffer, but is not limited to it, with a concentration of 0.1-1 mM (preferably 0.1 mM) and pH = 7.4. The supramolecular fluorescent probe is used at a concentration of 10 μM-1 mM, such as 1 mM. Its detection mechanism is as follows: the flavonoid fluorescent dye exhibits characteristic red fluorescence in its free state. Upon binding with the albumin-based derivative, it triggers a significant blue shift in fluorescence, producing yellow fluorescence. When pyriproxyfen is introduced into the system, this hydrophobic molecule enters the hydrophobic cavity of the albumin-based derivative through competitive binding, causing the flavonoid fluorescent dye molecules to be displaced and expelled from the cavity. This process is accompanied by a bidirectional response of fluorescence signals—the intensity of yellow fluorescence is negatively correlated with the concentration of pyriproxyfen, while the reconstructed flavonoid fluorescent dye aggregates induce an enhancement of the green fluorescence channel signal, ultimately achieving a quantitative conversion of the solution fluorescence color from yellow to green, forming a significant yellow-green colorimetric response. Based on this ratiometric fluorescence signal, the supramolecular fluorescent probe of this invention exhibits significant colorimetric effects when used to detect pyriproxyfen in the environment, food, etc., and the color change can be directly observed with the naked eye. This method provides a highly sensitive and visualized novel sensing platform for pesticide residue monitoring, and has advantages such as low cost and excellent anti-interference performance.

[0015] The following technical solutions are preferred embodiments of the present invention, but should not be considered as limitations on the scope of protection of the present invention. By implementing the following optimized configuration scheme, the stability of the detection system can be ensured, and the fluorescence response characteristics of the probe can be optimized, thereby achieving the technical objectives and advantages of the present invention more efficiently.

[0016] Specifically, in the supramolecular fluorescent probe for pyriproxyfen detection, human serum albumin (HSA) is preferentially selected as the host recognition unit. This protein has a unique three-dimensional cavity structure and abundant binding sites, and its hydrophobic surface region exhibits good spatial adaptation with the pyriproxyfen molecule, providing a structural basis for constructing a highly selective host-guest recognition system.

[0017] Furthermore, the optimal molar ratio of albumin derivatives to flavonoid fluorescent dyes in the supramolecular fluorescent probe for pyriproxyfen detection is 1:1. This stoichiometric relationship was determined through preliminary fluorescence quenching effect analysis. At this ratio, effective loading of dye molecules on the protein carrier is ensured while avoiding fluorescence self-quenching caused by excessive dye, thus maintaining the stable fluorescence emission characteristics of the probe system. It should be noted that this ratio can be dynamically adjusted by ±10% according to parameters such as pH and ionic strength of the specific detection environment.

[0018] A fourth aspect of the present invention provides a method for preparing a supramolecular fluorescent probe for the detection of pyriproxyfen, comprising the steps of: self-assembling an albumin derivative and a flavonoid fluorescent dye in a buffer solution to obtain the supramolecular fluorescent probe.

[0019] A fifth aspect of the present invention provides a method for detecting pyriproxyfen content, comprising:

[0020] Take the sample to be tested;

[0021] Add a buffer solution (such as PBS buffer) and a supramolecular fluorescent probe to the sample to be tested to obtain a mixture; the supramolecular fluorescent probe is the supramolecular fluorescent probe described above.

[0022] The fluorescence intensity of the mixture was detected, and the content of pyriproxyfen in the sample was calculated based on the standard curve of pyriproxyfen concentration change.

[0023] In the aforementioned pyriproxyfen detection method, the detection targets include two types of matrices: tap water and tea leaves. For tap water samples, pretreatment must ensure that the system is free of suspended particulate matter and pigment interference; while tea leaves samples require treatment using a combination of polyacrylamide solid-phase extraction column and activated carbon adsorption column to effectively remove insoluble substances and pigment components from the plant matrix.

[0024] For tea sample testing, the specific steps include: transferring 0.5 mL of the tea extract to be tested into a fluorescent cuvette, adding 1.5 mL of PBS buffer (pH = 7.4 ± 0.2) and 20 μL of supramolecular fluorescent probe stock solution (concentration 1 mM, HSA:flavonoid fluorescent dye = 1:1 molar ratio), followed by quantitative injection of pyriproxyfen standard solution. After vortexing for 10 seconds to complete molecular recognition, the fluorescence signal changes are recorded using a fluorescence spectrophotometer or a smartphone-assisted optical detection platform. A quantitative curve is constructed by determining the gradient concentration of the standard, and the pyriproxyfen content in the actual sample is then analyzed.

[0025] This detection system features rapid response (molecular recognition completed within 10 seconds), a signal acquisition time window of 10 seconds to 6 minutes, and a detection limit of 1.03 μM. Method specificity experiments show that common coexisting substances in tap water and tea matrix (including Na+) are detected. + K + Ca 2+ Plasma, amino acids such as glutamic acid and alanine, and small molecule alcohols such as ethanol and glycerol did not produce significant signal interference (relative error <5%) within the physiological concentration range, confirming that the method has good resistance to matrix interference.

[0026] Compared to traditional detection methods, the supramolecular fluorescent probe constructed in this invention exhibits unique dynamic optical response characteristics. Based on the host-guest competition mechanism, the fluorescence emission peak shifts from blue to yellow after the flavonoid fluorescent dye binds to albumin derivatives (such as HSA). When pyriproxyfen molecules enter the HSA cavity through hydrophobic interactions and replace the flavonoid fluorescent dye, a significant yellow-green visual contrast is formed, allowing the detection of trace amounts of target substances to be directly interpreted with the naked eye.

[0027] This probe system is achieved through a molecular self-assembly strategy, resulting in a simple preparation process and readily available raw materials. In terms of detection performance, it is not only suitable for complex matrices (such as tea extracts containing pigments and polyphenols), but can also identify target analytes within 10 seconds. Combined with portable optical equipment, it enables rapid on-site detection (detection limit 1.03 μM). Specificity experiments confirm that the probe system tolerates concentrations of common interfering substances (such as metal ions, amino acids, and alcohols) up to 50 times their environmental background levels, ensuring the reliability of actual sample detection. This technology combines high sensitivity, strong anti-interference capabilities, and visual detection advantages, providing a novel solution for on-site screening of pesticide residues. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the structure of the supramolecular fluorescent probe provided in an embodiment of the present invention;

[0029] Figure 2 This is the fluorescence spectrum of the titration of pyriproxypyridine and supramolecular fluorescent probe in PBS buffer in Example 1 of the present invention;

[0030] Figure 3 This is the concentration-dependent curve of pyriproxyfen in Example 1 of the present invention;

[0031] Figure 4 The fluorescence spectrum of pyriproxypyridine and supramolecular fluorescent probe in treated black tea in Example 2 of this invention is shown.

[0032] Figure 5 The figures show the on-site detection results of pyriproxyfen with mobile phone assistance in Examples 2 and 3 of this invention;

[0033] Figure 6 The fluorescence spectrum of pyriproxyfen and supramolecular fluorescent probe in treated black tea is shown in Example 3 of this invention. Detailed Implementation

[0034] This invention relates to a supramolecular fluorescent probe system and a matching detection method based on the subject-guest recognition principle, specifically for the rapid quantitative analysis of pyriproxyfen residues in the environment and agricultural products. To clarify the technical objective, implementation scheme, and beneficial effects of this invention, the technical solution is explained in detail below with reference to specific embodiments. It should be specifically noted that the embodiments described below are only for illustrating the technical principles and do not constitute a limitation on the scope of protection. Those skilled in the art can reasonably extend the scope within the framework of the claims. The technical features described in the specification can be combined and applied according to actual needs to form various equivalent implementation schemes. In the practice of the method steps, those skilled in the art can reasonably adjust the operation process or optimize the sequence based on common sense. It should be emphasized that the sequence of steps shown in the specification and its accompanying drawings is only a visual expression of the technical solution. Except for the process steps that are specifically indicated as mandatory, the remaining operation sequences are not mandatory. Furthermore, ordinal numbers in the specification (such as "first," "second") are only used for identification and do not involve technical weight or implementation order. The reagents and equipment used in the experiments are all commercially purchased standardized products; specific model information can be found in general technical manuals in this field.

[0035] The following specific preparation examples will further explain and illustrate the supramolecular fluorescent probe and detection method for pyriproxyfen provided by the present invention.

[0036] Example 1

[0037] The structure of flavonoid fluorescent dyes is as follows:

[0038]

[0039] The main synthetic steps included: dissolving HSF (0.307 g) and azepane-1-carbonyl chloride (0.477 g) in 25 mL of N,N-dimethylformamide (DMF), adding cesium carbonate (Cs₂CO₃, 1 g), and stirring the mixture at room temperature under nitrogen protection for 12 hours. After the reaction was complete, the resulting mixture was poured into ice water and extracted with ethyl acetate. The organic phase was washed with sodium bicarbonate (NaHCO₃) solution and concentrated under reduced pressure. The crude product was purified by recrystallization from a dichloromethane / n-hexane mixed solvent to give 0.099 g of an orange solid, with a yield of 23%. 1H NMR(500MHz, CDCl3):8.21(d,J=8.0Hz,1H),7.65(t,1H),7.58–7.51(m,2H),7.48(d,J=8.8Hz,2H),7.36(d,J=7.8Hz,1H),6.05 (ddt,J=15.9,10.4,5.3Hz,1H),5.91(ddd,J=16.9,10.7,5.4Hz,1H),5.37–5.25(m,4H),4.15(s,2H),4.04(s,2H),3.05(s,6H). 13 C NMR(125MHz, CDCl3):172.04,156.04,155.14,153.51,137.93,133.71,133.29,132.99,132.33,129.54,12 5.98,124.51,124.15,117.77,117.64,116.65,112.01,109.57,49.87,49.33,40.23.HRMS:m / z:calcdforC 26 H 26 N2O4+[M+H]+:431.1965; found:431.1969.

[0040] The preparation of the supramolecular fluorescent probe specifically includes the following steps: 66.5 mg of human serum albumin (HSA) was dissolved in 2 mL of PBS buffer (pH = 7.4, concentration 0.5 mM) to obtain a concentration of 0.5 × 10⁻⁶. -3 A stock solution of HSA at mol / L. 4.32 mg of flavonoid fluorescent dye was dissolved in 10 mL of DMSO to obtain a concentration of 1 × 10⁻⁶. -3 A stock solution of flavonoid fluorescent dye at a concentration of mol / L was prepared. 40 μL of HSA stock solution and 20 μL of flavonoid fluorescent dye stock solution were transferred to 2 mL of PBS buffer, shaken for 1 minute, and then allowed to stand for 1 minute to obtain a supramolecular fluorescent probe with a concentration of 10 μM. Its structural schematic diagram is shown below. Figure 1 As shown.

[0041] Dissolving 4.32 mg of pyriproxyfen in 10 mL of DMSO yields a concentration of 1 × 10⁻⁶. -3 A 0–200 μL volume of pyriproxyfen stock solution was transferred into the prepared supramolecular fluorescent probe solution. The excitation wavelength for fluorescence spectroscopy was set to 440 nm, and after shaking for 30 seconds, the solution was placed in a fluorescence spectrometer for testing. Figure 2 As shown, without the addition of pyriproxyfen, the emission peak of the supramolecular fluorescent probe is located at 575 nm. As the concentration of pyriproxyfen increases, the emission peak shifts to blue and appears at 535 nm. Figure 3This is a standard curve of fluorescence changes caused by changes in pyriproxyfen concentration, which is obtained by using the change in the ratio of fluorescence peaks to determine the concentration of pyriproxyfen.

[0042] Example 2

[0043] The specific steps for the black tea test include: using a syringe to extract the pre-treated black tea sample and transfer it into a filter containing a polyacrylamide filter column and a silica gel filter column; using a piston to pressurize and push the black tea sample into a sample vial and storing it in a freezer; adding 0.5 mL of the treated black tea extract and 1.5 mL of PBS buffer to a cuvette, adding 40 μL of HSA stock solution and 20 μL of chalcone fluorescent dye stock solution, shaking for 30 seconds, then adding pyriproxyfen standard solution, shaking again for 30 seconds, and then testing in a fluorescence spectrometer to obtain the results. Figure 4 , Figure 4 This demonstrates that as the concentration of pyriproxyfen increases, the fluorescence color of the probe changes from brown to green. After the test, cuvettes containing gradient concentrations of pyriproxyfen were placed in a dark chamber and photographed under 365nm UV light to obtain fluorescence response maps (e.g., Figure 5 (As shown).

[0044] Example 3

[0045] The specific steps for the dark tea test include: using a syringe to extract the pre-treated dark tea sample and transfer it into a filter containing a polyacrylamide filter column and a silica gel filter column; using a piston to pressurize and push the dark tea sample into a sample vial and storing it in a freezer; adding 0.5 mL of the treated dark tea extract and 1.5 mL of PBS buffer to a cuvette, adding 40 μL of HSA stock solution and 20 μL of chalcone fluorescent dye stock solution, shaking for 30 seconds, then adding pyriproxyfen standard solution, shaking again for 30 seconds, and then testing in a fluorescence spectrometer to obtain the results. Figure 6 , Figure 6 This demonstrates that as the concentration of pyriproxyfen increases, the fluorescence color of the probe changes from brown to green. After the test, cuvettes containing gradient concentrations of pyriproxyfen were placed in a dark chamber and photographed under 365nm UV light to obtain fluorescence response maps (e.g., Figure 5 (As shown).

[0046] First, due to the limitations of short-wavelength emission of flavonol dyes, a flavonoid fluorescent dye with the structure shown above was designed and synthesized based on the characteristics of the flavonoid skeleton. Compared with common flavonoid dyes, the flavonoid fluorescent dye of this invention protects the hydroxyl groups on the chromotropic ketone ring of flavonoids, shielding the intramolecular proton transfer process in its excited state, thereby enhancing its fluorescence emission performance. Second, this invention constructs a supramolecular fluorescent probe system based on host-guest competition, which is built through the directional self-assembly of albumin derivatives and flavonoid fluorescent dyes in a buffer solution. The colorimetric mechanism of this probe originates from the fluorescence shift effect induced by molecular recognition: based on the host-guest molecular recognition mechanism, the flavonoid fluorescent dye exhibits red fluorescence emission in its free state. When it forms a complex with human serum albumin through hydrophobic interactions and π-π stacking, the fluorescence emission peak shifts from blue to the yellow band. When pyriproxyfen molecules competitively enter the hydrophobic cavity of the HSA due to their hydrophobicity and spatial adaptability, the flavonoid fluorescent dye dissociates from the host-guest complex, and the fluorescence shifts from blue to green, forming a significant yellow-green colorimetric response that can be directly identified with the naked eye. Experimental verification shows that this probe system maintains stable fluorescence response characteristics in complex environmental matrices (such as tea extracts containing polyphenols and pigments), and the detection of pyriproxyfen can be completed within 10 seconds. It also exhibits excellent anti-interference ability (tolerance concentration of common coexisting substances ≥50 times the background value). Its low raw material cost and simple preparation process provide a reliable solution for rapid on-site screening of pesticide residues.

[0047] It should be specifically stated that the embodiments of the present invention are not limited to the examples listed in the specification. Any optimization of probe structure based on the subject-guest competition color development principle (such as selecting different protein carriers or fluorescent dyes), improvement of detection process (such as using microfluidic chip integrated detection), or innovation of signal acquisition method (such as developing a dedicated portable optical sensor), any adaptive improvement or equivalent substitution that does not depart from the essence of the technical solution of the present invention, shall fall within the scope of protection defined by the claims.

Claims

1. A flavonoid fluorescent dye, characterized in that, The structural formula of the flavonoid fluorescent dye is as follows:

2. A method for preparing the flavonoid fluorescent dye according to claim 1, characterized in that, Including the following steps: 2-(4-(dimethylamino)styryl)-3-hydroxy-4H-chromen-4-one and azircycloheptane-1-carboxyl chloride were dissolved in N,N-dimethylformamide, cesium carbonate was added, and the mixture was stirred for 12-24 hours under an inert atmosphere to obtain a mixture. The mixture was purified to obtain the flavonoid fluorescent dye.

3. The method for preparing flavonoid fluorescent dye according to claim 2, characterized in that, The step of purifying the mixture specifically includes: The mixture was poured into ice water, extracted with ethyl acetate, and the organic phase was collected. The organic phase was washed with sodium bicarbonate solution and then concentrated under reduced pressure to obtain a crude product. The crude product was purified by recrystallization using a mixed solvent of dichloromethane and n-hexane to obtain the flavonoid fluorescent dye.

4. A supramolecular fluorescent probe for the detection of pyriproxyfen, characterized in that, The supramolecular fluorescent probe is self-assembled from albumin derivatives and flavonoid fluorescent dyes in a buffer solution; the structural formula of the flavonoid fluorescent dye is as follows:

5. The supramolecular fluorescent probe for the detection of pyriproxyfen according to claim 4, characterized in that, The albumin derivatives are selected from human serum albumin.

6. The supramolecular fluorescent probe for the detection of pyriproxyfen according to claim 4, characterized in that, The molar ratio of albumin derivatives and flavonoid fluorescent dyes in the supramolecular fluorescent probe is 1:

1.

7. A method for preparing a supramolecular fluorescent probe for the detection of pyriproxyfen according to any one of claims 4-6, characterized in that, The steps include: self-assembling albumin derivatives and flavonoid fluorescent dyes in a buffer solution to obtain the supramolecular fluorescent probe.

8. A method for detecting pyriproxyfen content, characterized in that, Including the following steps: Take the sample to be tested; A buffer solution and a supramolecular fluorescent probe are added to the sample to be tested to obtain a mixture; the supramolecular fluorescent probe is the supramolecular fluorescent probe according to any one of claims 4-6. The fluorescence intensity of the mixture was detected, and the content of pyriproxyfen in the sample was calculated based on the standard curve of pyriproxyfen concentration change.

9. The method for detecting pyriproxyfen content according to claim 8, characterized in that, The plotting of the standard curve for the change in pyriproxyfen concentration includes: The buffer solution and supramolecular fluorescent probe were mixed to obtain a mixture; Different concentrations of pyriproxyfen standard solutions were added dropwise to the mixture, and the changes in fluorescence were recorded using a fluorescence spectrophotometer or the photographic function of a terminal device. A standard curve of pyriproxyfen concentration change was plotted based on the recorded results.

10. The method for detecting pyriproxyfen content according to claim 8, characterized in that, The sample to be tested is tap water or tea leaves; when the sample to be tested is tap water, it does not contain suspended particulate matter or pigments; when the sample to be tested is tea leaves, it does not contain insoluble matter or pigments. The concentration of the supramolecular fluorescent probe in the mixture is 1 mmol / L.

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