Off-On type fluorescent probe compound as well as preparation method and application thereof in specific detection of mercury ions

Abstract

The invention discloses an Off-On type fluorescent probe compound, the chemical name of the Off-On type fluorescent probe compound is thiocarbonic acid O-(2-butyl-1, 3-dioxo-2, 3-dihydro-1H-benzo [de] isoquinoline-6-yl) O-(4-fluorophenyl) ester (short for 2B6NI-4FP-TC), a preparation method of the Off-On type fluorescent probe compound and application of the Off-On type fluorescent probe compound to detection of mercury ions in complex matrix samples such as ashing soil and tea leaves. During detection, Hg < 2 + > is combined with sulfur atoms of thiocarbonate and catalyzes ester bond breakage, 4-OH of a fluorescent parent nucleus is exposed, and a system fluorescent signal is remarkably amplified. The probe has extremely high selectivity and sensitivity, the detection limit is as low as 1.5 * 10 <-8 > M, the stability in a wide pH range is good, qualitative and quantitative detection of Hg < 2 + > can be realized through visual colorimetry and fluorescence spectrum, and the probe is particularly suitable for rapid and high-throughput detection of trace Hg < 2 + > in complex matrix samples such as traditional Chinese medicines and tea leaves subjected to ashing treatment.

Description

Technical Field

[0001] This invention relates to the fields of analytical chemistry and environmental monitoring technology, specifically to an Off-On type fluorescent probe compound, its preparation method, and its application in the specific detection of mercury ions. Background Technology

[0002] Mercury is a typical highly toxic heavy metal element in the environment, existing in various forms, including elemental mercury, inorganic mercury, and organic mercury (such as methylmercury). Among these, Hg... 2+ Due to its highest chemical activity, as well as its core characteristics of being non-biodegradable, having strong environmental migration capabilities, and being capable of bioaccumulation and amplification, it poses the most direct and significant threat to ecosystems and human health.

[0003] To control the risk of mercury pollution, precise and efficient Hg testing is needed. 2+ Detection technology is crucial. Currently, Hg... 2+ Routine detection still relies primarily on traditional large-scale instrumental analysis techniques, typically including inductively coupled plasma atomic emission spectrometry (ICP-OES), graphite furnace atomic absorption spectrometry (GFAAS), and atomic fluorescence spectrometry (AFS). The core advantages of these techniques lie in their low detection limits, high quantitative accuracy, and strong resistance to matrix interference, making them essential tools for precise quantification in laboratories. However, they suffer from inherent limitations: firstly, the cost of equipment purchase and maintenance is high, requiring a specialized laboratory environment; secondly, the sample pretreatment process is cumbersome, involving multiple steps such as digestion, separation, and enrichment, making the operation complex and time-consuming; and thirdly, the detection process depends on specialized technical personnel, resulting in a high operational threshold and failing to meet the practical needs of rapid on-site detection and large-scale sample screening, significantly limiting their widespread application in fields such as environmental monitoring and food and drug safety supervision.

[0004] Compared to traditional detection technologies, fluorescent molecular probe detection technology has become the preferred choice for Hg testing due to its advantages such as ease of operation, no need for complex pretreatment, compact equipment size, and controllable cost. 2+ A research hotspot in the field of Hg detection. However, currently reported methods for Hg detection... 2+ The fluorescent probes used for detection still suffer from three major defects, which severely limit their practical application effectiveness: First, their selectivity is poor, making accurate detection in complex matrices difficult. Most existing fluorescent probes rely on Hg. 2+ Recognition is achieved through coordination with the recognition site, while Ag... + Cu 2+ Other heavy metal ions have a strong affinity for coordinating groups such as thiol groups and readily combine with Hg. 2+ Competition for binding sites significantly interferes with the probe's fluorescence response, making it difficult to achieve Hg² binding in complex matrices containing multiple coexisting ions (such as soil, food, and traditional Chinese medicine). +Accurate identification and quantification.

[0005] Second, they have weak resistance to environmental interference and insufficient stability of detection signals. The fluorescence response mechanism of traditional fluorescent probes is sensitive to environmental factors. Fluorescence fluctuations, changes in ionic strength, etc., can affect the configuration or electronic distribution of probe molecules, leading to fluctuations in fluorescence intensity. This, in turn, results in larger errors when analyzing actual samples, making it difficult to guarantee the reliability and reproducibility of detection results.

[0006] Third, insufficient water solubility and biocompatibility. The core structure of some probes (such as aromatic fused ring structures) is highly hydrophobic, making it difficult to disperse uniformly in aqueous systems and effectively penetrate biological membranes. This not only limits their application in trace detection in aqueous systems such as environmental water samples and biological samples, but also fails to meet the needs of emerging detection scenarios such as in vivo biological imaging.

[0007] Therefore, it is necessary to develop a Hg precipitant with high selectivity, strong anti-interference ability, good water solubility, and suitability for detection in complex matrices. 2+ Fluorescent probes have become a pressing technical challenge in this field, and are of great significance for promoting the development of environmental monitoring and food and drug safety testing technologies. Summary of the Invention

[0008] The present invention aims to provide an Off-On fluorescent probe compound, its preparation method, and its application in the specific detection of mercury ions. This Off-On fluorescent probe has extremely high selectivity and sensitivity, and can be used for visual and colorimetric detection of mercury ions, overcoming the shortcomings of traditional fluorescent probes such as poor selectivity and weak anti-interference.

[0009] To achieve the above objectives, this application provides the following technical solution: The first aspect of this invention provides an Off-On type fluorescent probe compound having the following structural formula: .

[0010] The second aspect of the present invention provides a method for preparing an Off-On type fluorescent probe compound, wherein the fluorescent probe compound is obtained by a condensation reaction of 2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione with thiochloroformate-O-(4-fluorophenyl) ester.

[0011] Specifically, 2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione was dissolved in a solvent, a catalyst was added, and then thiochloroformate-O-(4-fluorophenyl) ester was added dropwise. After reacting at room temperature, the fluorescent probe compound was obtained by solid-liquid separation, washing, and drying.

[0012] Furthermore, the solvent is dichloromethane, the catalyst is potassium carbonate, and the reaction conditions are heating under reflux for 12–18 hours.

[0013] The third aspect of the present invention provides the application of the Off-On type fluorescent probe compound as described in the first aspect in mercury ion sensing and detection.

[0014] Furthermore, the sensing detection includes selective sensing detection and competitive sensing detection, and the detection method is fluorescence detection, specifically qualitative detection and quantitative detection under fluorescence spectroscopy.

[0015] The optimized sensing targets include water environment samples and ashing-treated traditional Chinese medicine samples and tea samples.

[0016] The fourth aspect of the present invention provides Hg of the Off-On type fluorescent probe compound as described in the first aspect. 2+ The detection method includes the following steps: (1) Dissolve the fluorescent probe compound in a mixture of a polar organic solvent and a buffer solution to prepare a fluorescent probe solution; (2) Add the sample to be tested to the fluorescent probe solution obtained in step (1) and mix well to obtain a mixed solution; (3) Qualitatively determine whether the sample contains Hg by observing the color change of the mixed solution. 2+ The concentration of Hg in the sample was quantitatively determined by detecting the fluorescence signal intensity of the mixed solution and combining it with a fluorescence quantitative standard curve. 2+ The content of.

[0017] More preferably, the concentration of the fluorescent molecular probe in the fluorescent molecular probe solution in step (1) is 5 to 10 μM.

[0018] In some preferred embodiments, the polar organic solvent is dimethyl sulfoxide, and the buffer solution is Hepes buffer.

[0019] Furthermore, the pH of the fluorescent molecular probe solution is 7.4.

[0020] Even better, the volume ratio of DMSO to HEPES buffer is 1:99, and the concentration of HEPES buffer is 0.01M.

[0021] Preferably, the fluorescence intensity of the mixed solution is measured at an excitation wavelength of 450 nm and an emission wavelength of 550 nm.

[0022] Working principle and beneficial effects of the present invention: (1) The Off-On fluorescent molecular probe provided by the present invention can be used for the specific identification of mercury ions in water environment and traditional Chinese medicine. It can demonstrate different functions through ultraviolet and fluorescence spectra and can be used for quantitative and sensing detection of mercury ions. It exhibits good selectivity, strong anti-interference performance against other metal ions, and low detection limit.

[0023] (2) The Off-On fluorescent molecular probe of the present invention can provide real-time qualitative and quantitative information through fluorescence spectroscopy detection, and can undergo color change, making it a highly specific indicator suitable for visual colorimetric detection.

[0024] (3) The raw materials for the synthesis of the Off-On fluorescent molecular probe of the present invention are economical and readily available, the synthesis method is simple to operate, and the post-reaction processing is relatively simple.

[0025] (4) The Off-On fluorescent molecular probe has a weak fluorescence intensity in the absence of mercury ions, but a significant fluorescence enhancement after the addition of mercury ions. It has high selectivity and anti-interference ability, thus achieving the "Off-On" function. Attached Figure Description

[0026] Figure 1 This is a high-resolution mass spectrum of the fluorescent molecular probe 2B6NI-4FP-TC synthesized in Example 1 of this invention; Figure 2 The nuclear magnetic resonance (NMR) of the fluorescent molecular probe 2B6NI-4FP-TC synthesized in Example 1 of this invention is shown. 1 H NMR spectrum; Figure 3 The nuclear magnetic resonance (NMR) of the fluorescent molecular probe 2B6NI-4FP-TC synthesized in Example 1 of this invention is shown. 13 C NMR spectrum; Figure 4 For 2B6NI-4FP-TC, 2B6NI-4FP-TC+Hg 2+ Fluorescence spectra of 2B6NI-4FP-TC+ and other metal cations; Figure 5 The probe 2B6NI-4FP-TC was used to recognize (10 μM) various common metal ions (all at 50 μM, Hg) in HEPES buffer solution. 2+ A bar chart of the selectivity experiment results (at 10 μM); Figure 6 The reaction of 2B6NI-4FP-TC (1 μM) with different concentrations of Hg in HEPES buffer (pH 7.4) was investigated. 2+ Fluorescence emission spectrum (0-1 μM) (excitation wavelength λex = 450 nm), inset shows fluorescence intensity at 550 nm and Hg.2+ Concentration relationship curve; Figure 7 The probe is used to detect Hg in ashed Chinese medicine and tea samples. 2+ Results of the spiked recovery experiment. Detailed Implementation

[0027] The following detailed description illustrates the specific implementation method: Example 1: This Example 1 illustrates the synthetic route of the target compound 2B6NI-4FP-TC, including: (1) The synthesis route of the intermediate 6-bromo-2-butyl-1H-benzo[de]isoquinoline-1,3(2H)-dione (compound 2) is as follows:

[0028] At room temperature, 6-bromo-1H,3H-benzo[de]isochromene-1,3-dione (compound 1, 5 g, 18.05 mmol) was added to anhydrous methanol. While stirring vigorously, n-Butylamine (1.32 g, 18.05 mmol) was added, and the mixture was refluxed at 80 °C for 10 h. After the reaction was complete, ice water (250 mL) was added, and the precipitated product was filtered. The filter cake was dissolved in ethyl acetate (50 mL), washed with saturated sodium chloride solution (30 mL × 1), and dried over anhydrous sodium sulfate. After thorough drying, the solvent was evaporated, and the crude product was subjected to column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give compound 2.

[0029] (2) The synthetic route of intermediate 6H2BNI (2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione) is as follows:

[0030] Compound 2 (330 mg, 1 mmol), N-hydroxyphthalimide (NHPI, 195.7 mg, 1.2 mmol), and potassium carbonate (208 mg, 1.5 mmol) were added sequentially to DMSO (20 mL), and the mixture was stirred and refluxed at 80 °C for 6 h. After the reaction was complete, ice water (50 mL) was added, the pH was adjusted to 2-3 with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (50 mL × 3), washed with water (30 mL × 1), washed with saturated sodium chloride solution (30 mL × 1), and dried over anhydrous sodium sulfate. The crude product was subjected to column chromatography (petroleum ether / ethyl acetate = 4 / 1) to give compound 6H₂BNI, which was a white powder in 60-70% yield.

[0031] (3) Off-On fluorescent probe compound, chemically named O-(2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-yl)O-(4-fluorophenyl) ester (abbreviation: 2B6NI-4FP-TC), synthetic route is as follows:

[0032] 6H₂BNI (564 mg, 1.99 mmol) was dissolved in DCM (25 mL) and stirred until completely dissolved. Anhydrous potassium carbonate (385 mg, 2.78 mmol) was then added. O-(4-fluorophenyl)chlorothiocarbamate (437 mg, 2.53 mmol) was slowly added dropwise to the reaction system, and the reaction was carried out at room temperature for 12 h. After the reaction was complete, the residue was removed by filtration, and the mixture was washed with water (30 mL × 1), washed with saturated sodium chloride solution (30 mL × 1), and dried over anhydrous sodium sulfate. The crude product was subjected to column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give compound 2B₆NI-4FP-TC, which was a white powder in 50-60% yield.

[0033] The target compound 2B6NI-4FP-TC obtained in Example 1 is a novel compound that has not been previously reported. After purification, it was subjected to high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR). 1 H NMR, 13 Characterized by C NMR. NMR of probe 2B6NI-4FP-TC. 1 H NMR such as Figure 1 As shown, the nuclear magnetic resonance of probe 2B6NI-4FP-TC 13 C NMR such as Figure 2 As shown, the high-resolution mass spectra of probe 2B6NI-4FP-TC are as follows: Figure 3 As shown.

[0034] High-resolution mass spectrometry (HRMS): The target peak in the mass spectrum corresponds to the molecular ion peak of the compound, confirming the correctness of the product structure (as shown in Figure 1). ¹H NMR (400 MHz, DMSO-d6): δ = 8.64 – 8.55 (m, 2H), 8.51 (dd, J=8.5, 1.1, 1H), 8.00 (dd, J=8.5, 7.3, 1H), 7.93 (d, J=8.0, 1H), 7.57 (dd, J=8.6, 7.2, 2H), 7.52 – 7.46 (m, 2H), 7.41 (td, J=7.1, 1.3, 1H), 4.06 (t, J=7.4, 2H), 1.64 (tt, J=8.0, 6.5, 2H), 1.37 (h, J=7.4, 2H), 0.94 (t, J=7.3, 3H) (as shown in Figure 2); Carbon nuclear magnetic resonance spectroscopy (NMR) 13 C NMR, 101 MHz, DMSO-d6): δ = 193.93, 163.68, 163.12, 153.76, 153.49, 131.93, 131.88, 130.50, 129.23, 128.88, 128.23, 127.76, 124.89, 123.21, 122.24, 121.70, 121.28, 30.10, 20.30, 14.22 (as shown in Figure 3).

[0035] The fluorescent molecular probe 2B6NI-4FP-TC synthesized in Example 1 was dissolved in DMSO to prepare a 10 μM DMSO / water (1:99, v / v, 0.01 M Hepes, pH = 7.4) buffer system (hereinafter referred to as: probe 2B6NI-4FP-TC solution) for the following tests.

[0036] Test Example 1 Different metal cations (Sn) were added to the probe 2B6NI-4FP-TC solution. 2+ Cd 2+ Cu 2+ Pb 2+ Zn 2+ Ca 2+ Mn 2+ Cs 2+ Cr 3+ Co 2+ Cu + Al 3+ Ba 2+ K + Ag + and Hg² + Fluorescence spectrum as follows Figure 4 As shown in A. From Figure 4 As can be seen from A, adding Hg² + Afterward, the fluorescence intensity significantly increased, and the addition of other metal ions did not cause a significant change in fluorescence intensity. From Figure 4 As can be seen from B, the addition of Hg² + Subsequently, the fluorescence intensity at 550 nm was significantly enhanced, and the addition of other metal ions did not cause a significant change in fluorescence intensity. These results indicate that Hg² + The probe 2B6NI-4FP-TC is unique compared to other ions and can specifically recognize Hg². + .

[0037] Test Example 2 Hg² was detected using probe 2B6NI-4FP-TC solution. + For different metal cations Sn 2+ Cd 2+ Cu 2+ Pb 2+ Zn 2+ Ca 2+ Mn 2+ Cs 2+ Cr 3+ Co 2+ Cu + Al 3+ Ba 2+ K + and Ag + The competitiveness of the probe was measured in 2B6NI-4FP-TC solution and after adding different metal cations and then adding Hg². + The fluorescence intensity of the subsequent system, such as Figure 5 As shown in the figure. It can be seen that probe 2B6NI-4FP-TC is effective against Hg². + Its recognition has excellent anti-interference ability, and the presence of other metal ions in the system will not affect its recognition effect.

[0038] Test Example 3 Different concentrations of mercury ions were added to solution 2B6NI-4FP-TC, and changes in their fluorescence response were detected. Figure 6 As shown. Figure 6 This shows the changes in the system and Hg² + The relationship between concentrations shows that as the concentration of mercury ions increases, the fluorescence response at a wavelength of 550 nm gradually increases, and the change in fluorescence intensity is significant, proving that probe 2B6NI-4FP-TC can be used for the detection of Hg²⁺. + It enables quantitative detection, and the phenomena are obvious and easily identifiable, with detection limits as low as nanomolar levels.

[0039] Test Example 4 Figure 7 A is the addition of 1×10 to the 2B6NI-4FP-TC probe solution. -5 The color change of the solution was observed under sunlight using mol / L mercury ions. It was found that the color of the 2B6NI-4FP-TC probe solution deepened after the addition of mercury ions. Figure 7 B is the addition of 1×10 to the 2B6NI-4FP-TC probe solution. -5 The color change of the solution was observed under a 365 nm UV lamp with added mol / L mercury ions. It was found that the fluorescence of the 2B6NI-4FP-TC solution with added mercury ions was significantly enhanced.

[0040] The above series of spectroscopic experiments demonstrate that 2B6NI-4FP-TC and Hg² + There are interactions between them, which could potentially lead to the development of a fluorescent molecular sensor capable of simultaneously detecting mercury ions, allowing for both ratio and colorimetric detection of mercury ions with the naked eye.

[0041] Test Example 5 The detection capability of probe 2B6NI-4FP-TC in actual water samples, Codonopsis pilosula, and tea ash extracts was studied using traditional Chinese medicine ash extracts. Codonopsis pilosula and tea leaves were ashed in a muffle furnace at 500℃ for 12 hours until constant weight. The ash was then extracted with dilute nitric acid (1 mM) in an ultrasonic bath for 12 hours. The ash extract was then filtered to remove insoluble impurities, and an appropriate amount of sodium bicarbonate (1 mM) solution was added to adjust the pH to neutral or weakly alkaline. The filtered extract was then mixed with probe 2B6NI-4FP-TC to prepare a test solution, and its fluorescence spectrum was measured. The fluorescence intensity showed almost no change, indicating that Hg²⁺ in the traditional Chinese medicine extract was low. + The content was very low, below the detection limit of 2B6NI-4FP-TC. Subsequently, concentrations of 2.5 × 10⁻⁶ were prepared using tap water, Codonopsis pilosula, and tea ash extracts, respectively. - 5 mol / L, 5×10 -5 mol / L, 10×10 -5 mol / L Hg² + Solution.

[0042] Table 1 shows the Hg levels in tap water, wolfberry extract, and honeysuckle extract determined by the fluorescent molecular probe 2B6NI-4FP-TC. 2+ The experimental results.

[0043] Table 1

[0044] Note: n represents the number of times the test was repeated. As shown in Table 1, the spiked recoveries in real water samples and ash extracts of Codonopsis pilosula and tea were 98.8-103.4%, with relative standard deviations all less than 10%. Therefore, probe 2B6NI-4FP-TC can be used for the quantitative detection of Hg in tap water and ash extracts of traditional Chinese medicine. 2+ content.

[0045] This invention designs and synthesizes an "off-on" fluorescent probe for mercury(II) ions. Probe 2B6NI-4FP-TC exhibits weak fluorescence intensity in the absence of mercury(II) ions, but shows significant fluorescence enhancement upon the addition of mercury(II) ions, demonstrating high selectivity and anti-interference properties. Furthermore, probe 2B6NI-4FP-TC has a low detection limit for mercury(II) ions, with a fluorescence detection limit of 1.5 × 10⁻⁶. -8 M.

[0046] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An Off-On type fluorescent probe compound, characterized in that, It has the following structural formula: 。 2. A method for preparing the fluorescent probe compound as described in claim 1, characterized in that, The fluorescent probe compound was obtained by a condensation reaction of 2-butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione with thiochloroformate-O-(4-fluorophenyl) ester.

3. The preparation method according to claim 2, characterized in that, 2-Butyl-6-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione was dissolved in a solvent, a catalyst was added, and then thiochloroformate-O-(4-fluorophenyl) ester was added dropwise. After reacting at room temperature, the fluorescent probe compound was obtained by solid-liquid separation, washing, and drying.

4. The preparation method according to claim 3, characterized in that, The solvent is dichloromethane, the catalyst is potassium carbonate, and the reaction conditions are heating under reflux for 12–18 hours.

5. The application of the fluorescent probe compound as described in claim 1 in mercury ion sensing detection.

6. The application according to claim 5, characterized in that, The sensing detection includes selective sensing detection and competitive sensing detection, and the detection methods are qualitative detection and quantitative detection under fluorescence spectroscopy.

7. The application according to claim 6, characterized in that, The objects detected by the sensors include water environment samples and ashing-treated traditional Chinese medicine samples and tea samples.

8. An Hg based on the fluorescent probe compound of claim 1 2+ The detection method is characterized by, Includes the following steps: (1) Dissolve the fluorescent probe compound in a mixture of a polar organic solvent and a buffer solution to prepare a fluorescent probe solution; (2) Add the sample to be tested to the fluorescent probe solution obtained in step (1) and mix well to obtain a mixed solution; (3) Qualitatively determine whether the sample contains Hg by observing the color change of the mixed solution. 2+ ; The Hg content in the sample was quantitatively determined by detecting the fluorescence signal intensity of the mixed solution and combining it with a fluorescence quantitative standard curve. 2+ The content of.

9. The detection method according to claim 8, characterized in that, The concentration of the fluorescent probe compound in the fluorescent probe solution is 5-10 μM. Step (3) involves measuring the fluorescence intensity of the mixed solution at an excitation wavelength of 450 nm and an emission wavelength of 550 nm.

10. The detection method according to any one of claims 8 or 9, characterized in that, The polar organic solvent is dimethyl sulfoxide, the buffer solution is Hepes buffer, the volume ratio of dimethyl sulfoxide to Hepes buffer is 1:99, and the concentration of Hepes buffer is 0.01M.

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