Application of near-infrared fluorescent probe of dicyanoisophorone derivative in preparation of bacillus anthracis detection product

Abstract

The invention discloses application of a near-infrared fluorescent probe of a dicyanoisophorone derivative in preparation of a product for detecting bacillus anthracis, and belongs to the technical field of fluorescent probes. The probe has a low detection limit (40 nM), high selectivity and strong anti-interference performance, can accurately identify DPA in a complex sample, is wide in applicable pH range (pH = 3-12) and extremely short in response time (1-2s), and is suitable for detection of various actual samples, including DPA in bacterial endophytic spores; the probe can also realize instant color development and distinguish dormant and germinated spores, and a new method is provided for the study of bacillus anthracis; the probe is simple in synthesis and purification steps, high in yield, low in cost and beneficial to large-scale production; by combining the fluorescence imaging technology and the probe, the existence and concentration of DPA can be visualized, rapid diagnosis and monitoring can be realized, and the probe is particularly suitable for early diagnosis of bacillus anthracis infection and improvement of treatment effect and patient survival rate, has potential clinical application prospects, and provides an efficient and sensitive new tool for bacillus anthracis detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent probes, and particularly relates to application of a near-infrared fluorescent probe of a dicyanoisophorone derivative in preparation of a detection product of Bacillus anthracis. BACKGROUND

[0002] In recent years, organic small-molecule fluorescent probes have been widely used in the fields of biological imaging and environmental monitoring due to their good biocompatibility and high sensitivity. The working principle of small-molecule fluorescent probes is that, according to the characteristics of the target to be detected, the application scene and the biocompatibility requirements, a fluorescent group (fluorescein (FITC), rhodamine (Rhodamine), naphthalimide (Naphthalimide) and the like) is connected to different recognition groups through a linking arm, so as to realize high specificity and high sensitivity detection of the target. Isophorone derivatives have attracted widespread attention from researchers due to their many advantages. A large Stokes shift helps to reduce interference between fluorescence emission and excitation, thereby improving the accuracy of detection. The conjugated diene structure which is easy to modify provides convenience for the introduction of different recognition groups. Excellent quantum yield and molar absorption coefficient ensure that the probe has high fluorescence intensity and sensitivity. Lower biological toxicity makes it suitable for biological related detection. Simple synthesis steps reduce the preparation cost and are conducive to large-scale application. Bacillus anthracis widely exists in nature, and its spores have a unique dormant structure that can resist heat, radiation, chemical attack and ultraviolet radiation and other adverse environmental factors. At the same time, Bacillus anthracis spores have the characteristics of strong concealment, wide spread, high morbidity and mortality, and long-term impact. In addition, the production cost is low, and the detection method is limited. Therefore, it is particularly important to develop timely, rapid and effective detection means to achieve early warning of anthrax.

[0003] Dipicolinic acid (DPA) is the main component of Bacillus anthracis spores, accounting for 5%-15% of the dry weight of bacteria, and can be used as a specific biomarker of Bacillus anthracis. The mortality rate of inhalation anthrax is as high as 25% to 90%, and the proportion of DPA in the total load of Bacillus anthracis can determine that the minimum infectious dose detected by DPA should be 60 μM (lethal dose). Therefore, it is of great significance to develop a probe that can quickly, efficiently and sensitively detect DPA in the field of biological safety.

[0004] Researchers have developed a variety of analytical methods for detecting DPA, including atomic absorption spectroscopy, electrochemistry, spectrophotometry, immunoassays, and fluorescence spectroscopy. Fluorescence spectroscopy is widely used due to its ease of use, practicality, and rapid response. The design and synthesis of fluorescent probes is crucial for accurate detection. However, existing fluorescent probes for DPA detection suffer from numerous limitations, including low sensitivity, poor selectivity, a narrow pH range, and long response times. Furthermore, the lack of fluorescent probes specifically designed for DPA detection hinders the demand for accurate and rapid detection of Bacillus anthracis. Summary of the Invention

[0005] Existing methods for detecting DPA require lanthanide-sensitized luminescence, which presents technical problems such as poor biocompatibility, insufficient dynamic monitoring capabilities, and poor long-term storage stability. The present invention aims to provide a near-infrared fluorescent probe based on a dicyanoisophorone derivative for use in the preparation of anthrax detection products. The near-infrared fluorescent probe based on a dicyanoisophorone derivative does not require lanthanide metals, yet can still rapidly and specifically respond to the anthrax biomarker DPA.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides the use of a near-infrared fluorescent probe of a dicyanoisophorone derivative in the preparation of a product for detecting anthrax Bacillus. The near-infrared fluorescent probe of the dicyanoisophorone derivative has a structural formula as shown in Formula 1: .

[0007] The near-infrared fluorescent probe of the dicyanoisophorone derivative is prepared by the following steps: mixing compound 2 with 4-(diethylamino) salicylaldehyde and anhydrous sodium sulfate under nitrogen atmosphere, refluxing, and purifying to obtain the near-infrared fluorescent probe of the dicyanoisophorone derivative; The structural formula of compound 2 is ; The molar ratio of the compound 2 to 4-(diethylamino) salicylaldehyde and anhydrous sodium sulfate is 1:(1-2):(3-4).

[0008] Furthermore, the molar ratio of the compound 2 to 4-(diethylamino) salicylaldehyde and anhydrous sodium sulfate is 1:(1-2):(3-4).

[0009] Furthermore, the reflux reaction temperature is 70-80° C., and the reflux reaction time is 10-14 h.

[0010] The product for detecting Bacillus anthracis is used to measure, screen and detect the Bacillus anthracis biomarker DPA, and to achieve specific identification through hydrogen bonding and steric hindrance effects between a near-infrared fluorescent probe of a dicyanoisophorone derivative and DPA.

[0011] The product for detecting Bacillus anthracis is a test paper, a test kit, a chip or a reagent for detecting Bacillus anthracis in a biological sample.

[0012] The product is a product that uses the near-infrared luminescence of a dicyanoisophorone derivative as a near-infrared fluorescent probe to perform bacterial imaging.

[0013] Furthermore, the product is a product that uses a near-infrared fluorescent probe of a dicyanoisophorone derivative to distinguish between the dormant state and the germination state of anthrax Bacillus spores.

[0014] The invention provides a kit for detecting anthrax bacillus. The kit comprises a near-infrared fluorescent probe of the dicyanoisophorone derivative.

[0015] The kit also includes reagents for preparing the probe solution.

[0016] The present invention provides a method for detecting DPA, a biomarker of Bacillus anthracis, based on a dicyanoisophorone derivative fluorescent probe, comprising: mixing the near-infrared fluorescent probe solution of the dicyanoisophorone derivative with the sample solution to be tested, incubating, and obtaining a mixture; The fluorescence signal of the mixture is detected to determine whether the sample to be tested contains DPA and its content.

[0017] The incubation temperature is 25° C. and the incubation time is 30 min.

[0018] Furthermore, the volume ratio of the near-infrared fluorescent probe solution of the dicyanoisophorone derivative to the sample solution is 1:8~10.

[0019] Furthermore, the concentration of the near-infrared fluorescent probe solution of the dicyanoisophorone derivative is 10 μM.

[0020] Compared with the prior art, the present invention achieves the following technical effects: The present invention provides a near-infrared fluorescent probe based on a dicyanoisophorone derivative for use in the preparation of anthrax detection products. The probe can effectively detect DPA with a detection limit as low as 40 nM. This extremely low detection limit indicates that the probe can respond to trace amounts of DPA and can accurately detect the presence of DPA in the early stages of anthrax infection when the concentration is low, greatly improving the sensitivity of detection and facilitating early warning and diagnosis of anthrax. The probe exhibits high selectivity and strong anti-interference properties, and can specifically identify DPA. In complex biological or environmental samples, which contain numerous other substances, the probe can accurately identify the target substance, DPA, without being affected by these interfering substances, thereby ensuring the accuracy and reliability of the detection results. The probe has a wide pH adaptability range (pH = 3-12), which enables the probe to be used in environments with different pH values. Whether it is testing environmental samples such as water and soil samples, or samples inside organisms, it can maintain stable detection performance, greatly expanding its application scenarios; the response time is extremely short, only 1-2 seconds, and it can quickly provide test results at biosafety testing sites or environmental emergency monitoring, providing an important basis for timely prevention and control measures; not only can DPA detection be performed on various actual samples (such as tap water and soil), but also on actual bacterial endospores, indicating that the probe has a wide range of applicability and can meet the detection needs of different types of samples, providing an effective tool for monitoring anthrax in different environments; the probe can achieve instant color development function, so it can distinguish between single dormant spores and germinated spores in real-time imaging, which is of great significance for studying the life cycle and infection mechanism of anthrax and evaluating the effectiveness of prevention and control measures, and provides new ideas and methods for the sensing detection of bacterial biomarkers DPA.

[0021] Furthermore, the near-infrared fluorescent probe of the dicyanoisophorone derivative provided by the present invention has relatively simple synthesis steps and separation and purification steps, and can obtain a high yield. The fluorescent probe can be prepared at a relatively low cost and with high efficiency, which is conducive to large-scale production and promotion and application, and provides a guarantee for the stable supply of anthrax detection products.

[0022] The present invention provides a method for detecting DPA, a biomarker for Bacillus anthracis, using a dicyanoisophorone derivative fluorescent probe. The near-infrared fluorescent probe, a dicyanoisophorone derivative, can specifically bind to DPA, generating a fluorescent signal. Combined with fluorescence imaging technology, the presence and concentration of DPA can be visualized. This intuitive and rapid visualization method facilitates rapid diagnosis and monitoring of Bacillus anthracis. The rapid generation of a fluorescent signal facilitates real-time monitoring and rapid diagnosis, and can detect low concentrations of DPA. This method facilitates accurate diagnosis of Bacillus anthracis infection in its early stages, improving treatment efficacy and patient survival, and has potential clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) in DMSO-d6; Figure 2 This is the carbon NMR spectrum of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) in DMSO-d6; Figure 3 Figure 3 shows the effect of the near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) on the detection of DPA under different conditions of the present invention, wherein (a) is the UV spectrum and fluorescence emission; (b) is the fluorescence emission spectrum of different types of solvents; (c) is the comparison of fluorescence emission intensity at different pH values; (d) is the comparison of fluorescence emission intensity at different reaction times; Figure 4 Response graph of the near-infrared fluorescent probe (Compound 3) of the dicyanoisophorone derivative with different solvent compositions to DPA; Figure 5 The fluorescence emission graphs of different analytes added to the solution of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) are shown, wherein (a) is a comparison of fluorescence emission spectra; (b) is a comparison of fluorescence emission intensities; Figure 6 The fluorescence emission spectra of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) reacting with DPA solutions of different concentrations, wherein (a) is a comparison of fluorescence emission spectra; (b) is a comparison of fluorescence emission intensities; Figure 7 The results of the detection of DPA by the near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) of the present invention under different environments, wherein (a) is a soil sample; (b) is a water sample; Figure 8Comparative imaging of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) detecting DPA before and after germination of Bacillus anthracis, wherein (a) shows the staining of spores before and after germination; (b) shows the co-localization staining of the probe with DAPI; Figure 9 The DPA graph of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) detected at different concentrations of anthrax germination, wherein (a) the fluorescence intensity changes with concentration after thermal lysis of the spores; (b) the fluorescence intensity changes with concentration after thermal lysis of the spores before and after germination; Figure 10 This is a DPA imaging diagram of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) detecting the time-dependent changes in the germination of Bacillus anthracis. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0025] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0026] In the present invention, unless otherwise specified, the experimental raw materials used are commercially available products well known to those skilled in the art, and isophorone, malononitrile, p-aminobenzaldehyde and 4-(diethylamino) salicylaldehyde are equal to those purchased from Anaiji Chemical.

[0027] The attenuated anthrax vaccine strain A16R (pXO 1+ , pXO 2- The A16R vaccine was derived from a wild-type Bacillus anthracis strain isolated from the carcass of a mule that died of anthrax in Langfang, Hebei Province in 1953 and exposed to ultraviolet radiation, resulting in the release of the capsule-encoding plasmid pXO 2- Lost (https: / / doi.org / 10.1371 / journal.pone.0057959).

[0028] Example 1 This embodiment provides a method for preparing a near-infrared fluorescent probe of a dicyanoisophorone derivative. The near-infrared fluorescent probe of the dicyanoisophorone derivative has a structure as shown in Formula 1. The specific preparation process is as follows: .

[0029]

[0030] (1) Synthesis of compound 1 Accurately weigh isophorone (36.20 mmol), malononitrile (47.06 mmol), and sodium acetate trihydrate (25.35 mmol). At room temperature, place the reaction flask on a stirring device, add 40 mL of anhydrous ethanol as the solvent, and sequentially add the weighed malononitrile and sodium acetate trihydrate to the reaction flask, stirring to fully dissolve. During the stirring process, slowly add isophorone, raise the temperature to 50°C, install a reflux device, and reflux the reaction system at 50°C for 24 h. During the reaction, the reaction is checked by thin layer chromatography (TLC) to determine whether the reaction is complete. After the reaction is completed, the reaction system is cooled to room temperature and the solvent is removed under reduced pressure to obtain a reaction mixture. An appropriate amount of deionized water is added to the reaction mixture, and the mixture is extracted with dichloromethane. After multiple extractions, the organic phases are combined, dried over anhydrous sodium sulfate, and the solvent is removed under reduced pressure again to obtain a crude product. The crude product is purified by column chromatography and purified by filtration using a mixed solvent of petroleum ether and ethyl acetate (volume ratio of 200: 1) Gradient elution was performed with ethyl acetate as the eluent, and the eluate containing the target product was collected. The solvent in the eluate was removed under reduced pressure to obtain 4.45 g of a white solid, which was the target compound 1, with a yield of 62%.

[0031] (2) Synthesis of compound 2 Compound 1 (12 mmol), p-aminobenzaldehyde (10 mmol), and piperidine (50 mmol) were accurately weighed and set aside. Compound 1 and p-aminobenzaldehyde were placed in a reaction flask, 45 mL of anhydrous ethanol was added as a solvent, and piperidine was slowly added dropwise to the reaction flask using a disposable syringe. The mixture was stirred and refluxed at 85°C for 4 h. The reaction progress was monitored by TLC during the reaction. After the reaction was completed, the reaction system was cooled to room temperature, and the solvent was removed under reduced pressure to obtain a reaction mixture. The mixture was purified by column chromatography and the solvent was removed under reduced pressure to obtain 1.44 g of a red solid, namely compound 2, with a yield of 40%.

[0032] The structural formula of compound 2 is: .

[0033] (3) Synthesis of compound 3 4-(Diethylamino)salicylaldehyde (1.8 mmol), compound 2 (2.2 mmol), and anhydrous sodium sulfate (14.08 mmol) were accurately weighed and set aside. Under a nitrogen atmosphere, compound 2, 4-(diethylamino)salicylaldehyde, and anhydrous sodium sulfate were placed in a Shrek tube. 15 mL of anhydrous ethanol was added as a solvent using a disposable syringe. A reflux apparatus was installed and the reaction system was heated to 75°C to reflux for 12 hours. During the reaction, the progress of the reaction was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature and filtered using a sand core funnel to remove anhydrous sodium sulfate. After removing the solvent under reduced pressure, the target product was eluted with a mixed solvent of petroleum ether and ethyl acetate as the eluent. After removing the solvent under reduced pressure, 0.23 g of a red solid was obtained, which was the target compound 3 with a yield of 68%.

[0034] The structural characterization of target compound 3 is shown in the attached Figures 1 and 2 : H NMR spectrum: 1 H NMR (600 MHz, DMSO- d6 ) δ 13.61 (s, 1H), 8.76 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 16.0 Hz, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.34– 7.29 (m, 2H), 6.89 (s, 1H), 6.34 (dd, J = 8.9, 2.4 Hz, 1H), 6.07 (d, J = 2.5Hz, 1H), 3.40 (q, J = 7.1 Hz, 4H), 2.61 (s, 2H), 2.55 (s, 2H), 1.13 (t, J = 7.0Hz, 6H), 1.02 (s, 6H). 13 C NMR (151 MHz, DMSO- d6) δ 170.71, 162.45, 156.78, 147.70, 137.81,137.21, 135.61, 134.92, 132.23, 131.93, 128.48, 127.67, 127.53, 123.16,122.69, 122.30, 120.34, 118.49, 117.83, 116.31, 114.48, 113.70, 112.28,75.83, 44.31, 42.79, 40.03, 32.16, 27.93, 26.81, 12.93, 0.57. The structural formula of the target compound 3 is confirmed as follows: .

[0035] Example 2 This example is based on Example 1, and provides a method for detecting the anthrax biomarker DPA by using the near-infrared fluorescent probe of the dicyanoisophorone derivative, and the parameters in the detection are optimized. The specific test process is as follows: (1) Effect of near-infrared fluorescent probe solution prepared by different solvents on detection of DPA The near-infrared fluorescent probe (compound 3) of the dicyanoisophorone derivative prepared in Example 1 was prepared into a near-infrared fluorescent probe solution with a concentration of 10 μM by using different solvents (dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, deionized water, methanol, ethanol); 5.0 mL of the near-infrared fluorescent probe solution was set for 3 min, and three parallel samples were set for each solution; after the reaction was completed, the fluorescence intensity of 18 reaction samples was determined, and the specific results are shown in the following table. N, N Figure 3

[0036] From the data, it can be seen that methanol has the best effect on the recognition of compound 3 to DPA, and therefore methanol is selected as the solvent for subsequent experiments. Figure 3

[0037] (2) Effect of near-infrared fluorescent probe solution prepared by different proportions of methanol and deionized water on detection of DPA ​​​2.49 mg of the near-infrared fluorescent probe of the dicyanoisophorone derivative prepared in Example 1 (Compound 3) was weighed and 5.0 mL of a mixed solvent was added (the mixed solvent was prepared by mixing deionized water and methanol in different volume ratios, and the volume ratios of deionized water and methanol were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively) to obtain a near-infrared fluorescent probe solution with a concentration of 10 μM; 50 μL of a 10 μM DPA solution was added to each reaction, and three parallel reactions were set up; after the reaction was complete, the fluorescence intensity of each of the 27 reactants was measured. For specific results, see the attached figure. Figure 4 shown.

[0038] By the attached Figure 4 The data show that when the ratio of deionized water to methanol is 2:8, the effect of compound 3 on recognition of DPA is the best. Therefore, a ratio of deionized water to methanol of 2:8 was selected as the solvent ratio for subsequent experiments.

[0039] (3) Effect of different pH values ​​on specific recognition 2.49 mg of the near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) prepared in Example 1 was weighed and 5.0 mL of a mixed solvent (deionized water and methanol in a ratio of 2:8) was added to obtain a near-infrared fluorescent probe solution with a concentration of 10 μM; the pH of the PBS buffer solution was set to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; 10 μM DPA was added to the 10 μM solutions of Compound 3 at different pH concentrations, respectively, and three parallel reactions were set; after the reaction was complete, the fluorescence intensity of each of the 39 reactants was measured. For specific results, see the attached Figure 3 shown.

[0040] By the attached Figure 3 The data show that the pH value of 5-12 has little effect on the recognition of DPA by compound 3. Considering the subsequent imaging experiments in vivo, the physiological pH of 7.4 was selected as the pH of the subsequent experimental solution.

[0041] (4) Different reaction times The near-infrared fluorescent probe of the dicyanoisophorone derivative prepared in Example 1 (Compound 3) was prepared into a near-infrared fluorescent probe solution with a concentration of 10 μM. The solvent used was a mixed solvent of deionized water and methanol in a volume ratio of 2:8, and the pH value of the buffer was 7.4. 50 μL of a 10 mM DPA solution was added to the near-infrared fluorescent probe solution and reacted for different time periods. After the reaction was completed, three parallel experiments were performed at each time point to measure the fluorescence intensity. The specific results are shown in the attached figure. Figure 3 shown.

[0042] By the attached Figure 3 The data show that compound 3 and DPA react for 60 minutes to reach equilibrium.

[0043] Example 3 This example tests the specificity of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention on the basis of Examples 1 and 2. The specific process is as follows: (1) Preparation of near-infrared fluorescent probe solution The near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) prepared in Example 1 was prepared into a near-infrared fluorescent probe solution with a concentration of 10 μM. The solvent was deionized water and methanol in a volume ratio of 2:8, and the pH value was 7.4. (2) Analyte to be tested There are 14 analytes to be tested, including aromatic carboxylic acids: benzoic acid, isophthalic acid, terephthalic acid, 2-picolinic acid, 3-picolinic acid; amino acids: glycine (Gly), phenylalanine (Phe), glutamic acid (Glu), aspartic acid (Asp); and oxygen-philic biological analytes: citrate, oxalate, ascorbic acid, sodium sulfite (Na2SO3), and disodium hydrogen phosphate (Na2HPO4). The analytes to be tested are prepared into a 5 mM analyte solution.

[0044] (3) Detection Take 42 5 mL portions of 10 μM solution of compound 3, add 50 μL of 5 mM solution of the analyte obtained in step (2) to it. Each analyte is composed of 3 parallel units, and 42 responses are obtained. After the response, the fluorescence of each response is measured. The specific results are shown in the attached Figure 5 shown.

[0045] By the attached Figure 5 The data show that in the presence of DPA alone, the fluorescence intensity and ultraviolet absorption intensity of the detection system are significantly enhanced, indicating that the near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) of the present invention has high specificity for DPA; other interfering analytes do not affect the response of the near-infrared fluorescent probe (Compound 3) to DPA, which shows that the near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) of the present invention has strong anti-interference ability.

[0046] Example 4 This example tests the sensitivity of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention on the basis of Examples 1 to 3. The specific experimental process is as follows: (1) Preparation of near-infrared fluorescent probe solution The near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) prepared in Example 1 was prepared into a near-infrared fluorescent probe solution with a concentration of 10 μM. The solvent used was deionized water and methanol in a volume ratio of 2:8, and the pH value was 7.4.

[0047] (2) Preparation of DPA solution DPA solutions with different concentrations (0 μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10.0 μM, 20.0 μM, 30.0 μM, 40.0 μM, 50.0 μM) were prepared.

[0048] (3) Fluorescence detection To 45 5.0 mL portions of 10 μM near-infrared fluorescent probe solution were added different concentrations (0 μM, 1.0 μM, 2.0 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10.0 μM, 20.0 μM, 30.0 μM, 40.0 μM, 50.0 μM) of DPA solution for reaction. Three parallel units were set for each concentration. After the reaction, 45 reactants were obtained. The fluorescence intensity of each of the 45 reactants was measured. The specific results are shown in the attached figure. Figure 6 shown.

[0049] By the attached Figure 6 The data show that as the concentration of DPA continues to increase, the fluorescence intensity increases accordingly, indicating that the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (compound 3) responds to DPA, and there is a positive correlation between the fluorescence intensity and the DPA concentration; the fluorescence intensity has a good linear relationship with the concentration of DPA. Through linear regression analysis, it was calculated that the detection limit of the near-infrared fluorescent probe for DPA is as low as 40 nM (far lower than the DPA concentration of 60 μM corresponding to anthrax infection), that is, the near-infrared fluorescent probe can quantitatively detect the presence of trace amounts of DPA in the solution, indicating that the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (compound 3) can quantitatively detect the presence of trace amounts of DPA in the solution.

[0050] Example 5 In this example, based on Examples 1 to 4, the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention is used to quantitatively detect DPA in environmental samples.

[0051] (1) Preparation of near-infrared fluorescent probe solution The near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) prepared in Example 1 was prepared into a near-infrared fluorescent probe solution with a concentration of 10 μM. The solvent was methanol and deionized water in a volume ratio of 8:2, and the pH was 7.4.

[0052] (2) Fluorescence detection of exogenous DPA in environmental water samples Water sample collection and treatment: Environmental water samples were collected from different areas and filtered through a 0.02 μm aqueous filter membrane to obtain treated environmental water samples; DPA addition: Different concentrations of DPA were added to the treated environmental water samples to simulate the possible DPA contamination in the reagent water samples.

[0053] Fluorescence detection: Mix the near-infrared fluorescent probe solution with environmental water samples containing different concentrations of DPA, perform fluorescence detection using a fluorescence spectrometer, and record the fluorescence intensity. For specific results, see the attached Figure 7 As shown in Table 1.

[0054] Table 1: Fluorescent probe recovery of DPA in actual water samples

[0055] From Table 1 and Appendix Figure 7 The data show that the recoveries of DPA for tap water and pond water are between 90% and 111.2% and 94.6% and 114.6%, respectively, indicating that compound 3 can accurately detect DPA in water samples; the relative standard deviations (RSDs) are between 1.2% and 3.3% and 1.2% and 5.9%, respectively, indicating that the repeatability and accuracy of the test are high; this indicates that the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (compound 3) can quantitatively detect the presence of DPA in environmental water samples, verifying its application potential in actual environmental water sample detection.

[0056] (3) Fluorescence detection of exogenous DPA in environmental soil samples Soil Sample Collection and Processing: Environmental soil samples were collected from different areas and soaked in deionized water overnight to dissolve the soluble substances in the soil samples. The soaking solution was then filtered through a 0.22 μm aqueous filter membrane to obtain the treated environmental soil sample filtrate.

[0057] DPA addition: DPA at different concentrations was added to the treated filtrate.

[0058] Fluorescence detection: The near-infrared fluorescent probe solution was mixed with the filtrate containing different concentrations of DPA, and fluorescence detection was performed using a fluorescence spectrometer to record the fluorescence intensity. The specific results are shown in Table 2 and Appendix. Figure 7 shown.

[0059] Table 2: Fluorescent probe recovery of DPA in actual soil samples

[0060] From Table 2 and Appendix Figure 7 The data show that under the conditions of pH 5.0 and 7.4, the recovery rates of DPA are between 90% and 113.2% and 88.5% and 121%, respectively, indicating that the near-infrared fluorescent probe of the dicyanoisophorone derivative (compound 3) of the present invention can adapt to the detection of DPA under different pH conditions; under the conditions of pH 5.0 and 7.4, the relative standard deviation (RSD) is between 1.0% and 6.7% and 0.5% and 3.8%, respectively, indicating that the test has high repeatability and accuracy under different pH conditions; it shows that the near-infrared fluorescent probe of the dicyanoisophorone derivative (compound 3) of the present invention can quantitatively detect the presence of DPA in the filtrate of environmental soil samples, verifying its application potential in actual environmental soil sample detection.

[0061] Example 6 In this example, based on Examples 1 to 5, the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention is used for the quantitative detection of endogenous DPA in Bacillus anthracis samples.

[0062] (1) Preparation of near-infrared fluorescent probe solution The near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) prepared in Example 1 was prepared into a near-infrared fluorescent probe solution with a concentration of 10 μM. The solvent was methanol and deionized water in a volume ratio of 8:2, and the pH was 7.4.

[0063] (2) Preparation of the Bacillus anthracis culture to be tested After heat activation (72°C, 1 h) of a reserved anthrax dormant spore suspension, 50 μL of the bacterial solution was added to 450 μL (100 mM, L-alanine) and incubated at 37°C for half an hour. Subsequently, a near-infrared fluorescent probe solution of a dicyanoisophorone derivative (compound 3, 10 μM) and 4', 6-diamidino-2-phenylindole (DAPI, 2.5 μM) were used for incubation at 37°C for half an hour. Then, 10 μL of the bacterial solution was fixed on a glass slide at 4°C, covered with a coverslip, and imaged using an ultra-high-resolution live cell imaging system.

[0064] A near-infrared fluorescent probe (compound 3) is excluded from the spore core during dormancy but penetrates the core after germination in response to changes in environmental DPA (Supplementary Figure 8). To better assess changes in localization signals between dormant and germinating spores, B. anthracis spores were co-stained with the near-infrared fluorescent probe (compound 3) and DAPI. Notably, co-staining with the DNA intercalator DAPI revealed chromatin-like ring-shaped crystalline cores, with which compound 3 highly overlapped, consistent with previous findings. In summary, super-resolution confocal microscopy results demonstrate that the near-infrared fluorescent probe (compound 3) is a valuable tool for high-resolution bacterial spore studies, compared to other markers.

[0065] Example 7 This example, based on Examples 1 to 6, uses the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention to quantitatively detect endogenous DPA in Bacillus anthracis samples.

[0066] (1) Preparation of near-infrared fluorescent probe solution The near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) prepared in Example 1 was prepared into a near-infrared fluorescent probe solution with a concentration of 10 μM. The solvent was methanol and deionized water in a volume ratio of 8:2, and the pH was 7.4.

[0067] (2) Preparation of the Bacillus anthracis culture to be tested Inoculation and initial culture: Bacillus anthracis AR16 was inoculated on a solid LB agar plate at 37°C overnight. The next day, a single colony was inoculated into liquid LB agar medium and incubated in a 37°C constant temperature incubator until OD 600 The initial culture was obtained at a concentration of 0.3-0.5.

[0068] Expansion culture: The primary culture (25 mL) was re-inoculated into liquid LB medium (225 mL, pH = 7.4) using an Erlenmyer gas-permeable flask with a gas-permeable membrane for adequate ventilation, and then cultured with Bacillus anthracis AR16 in a shaker (130 rpm, 37°C) for 92 h.

[0069] (3) Detection of endogenous DPA after germination of Bacillus anthracis Spore suspension grouping: The spore suspension was divided into two groups. The first group was left untreated and stored at 4°C until ready for use. The second group was treated with the spore germination agent L-alanine (100 μM) to promote spore germination. Gradient dilution: The above two groups of spore suspensions were diluted in a gradient manner to make the final concentration of the test 10 5 spores / mL; Fluorescence intensity determination: The gradient diluted spore suspension was added to the near-infrared fluorescent probe (compound 3) solution to measure the fluorescence intensity. The specific results are shown in the attached Figure 9 shown.

[0070] By the attached Figure 9 The data show that the curve of fluorescence intensity changing with the concentration of the spore suspension shows a linear trend between the fluorescence intensity of the spore suspension after germination and the spore concentration, indicating that the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) can quantitatively detect endogenous DPA in the spore suspension after germination. Based on the fluorescence intensity measurement curve, the detection limit of the near-infrared fluorescent probe of the dicyanoisophorone derivative of the present invention (Compound 3) for Bacillus anthracis was calculated to be 2.71 × 10 4 spores / mL, indicating that it has high sensitivity and can detect lower concentrations of anthrax Bacillus; under different spore suspension volumes, the error between the observed DPA concentration and the theoretical DPA concentration is within a reasonable range, indicating that it can detect endogenous DPA in bacterial spores and the test results are accurate and reliable.

[0071] Table 3. Quantitative properties of bacteria (Bacillus anthracis) in spore studies using DHEY (10 μM, 1:1, λex = 500 nm) in buffered medium. Stock suspension concentration: 1.0 × 10 8 spores / mL, 0 ~ 5 × 10 -19 Moore's anthracis DPA.

[0072] Table 3: Quantitative analysis of Bacillus anthracis spores in buffered medium using DHEY (10 μM, 1:1 volume ratio, excitation wavelength λex = 500 nm)

[0073] Example 8 In this example, based on Examples 1 to 7, a near-infrared fluorescent probe (Compound 3) of a dicyanoisophorone derivative of the present invention is used for real-time imaging detection of Bacillus anthracis spore germination.

[0074] (1) Preparation of near-infrared fluorescent probe solution The near-infrared fluorescent probe of the dicyanoisophorone derivative (Compound 3) prepared in Example 1 was prepared into a near-infrared fluorescent probe solution with a concentration of 10 μM. The solvent was deionized water and methanol in a volume ratio of 2:8, and the pH value was 7.4.

[0075] (2) Preparation of test bacterial solution Inoculation and initial culture: B. anthracis AR16 strain was inoculated on 37 °C solid LB agar plate overnight, and the next day single colony was inoculated into liquid LB agar medium (30 mL, pH = 7.4) in a 37 °C incubator (200 rpm, 37 °C) until OD 600 0.3-0.5, and the initial culture was obtained.

[0076] Subculture: The initial culture (25 mL) was subcultured in liquid LB medium (225 mL, pH = 7.4) using an Erlenmyer flask with a gas-permeable membrane for sufficient aeration, and then incubated in a shaker (130 rpm, 37 °C) for 92 h.

[0077] The collected spores were washed in 1 × PPS, then centrifuged in a centrifuge with an isodensity percoll gradient; the bottom spores were repeatedly washed in 1 × PPS and stored at 4 °C; the spore quality was confirmed by aberration microscopy, and each strain was adjusted to the order of 10 9 CFU / mL using a plate count chamber.

[0078] (3) Spore heat activation The step (2) standby spores were heat activated (72 °C, 1 h) to promote spore germination. (4) Preparation of germination dye solution L-alanine (100 mM, 100 μL) and near-infrared fluorescent probe (compound 3) solution (1.5 mM, 400 μL) were mixed as a germination dye solution, and L-alanine was used as a spore germination agent. L- (5) Bacterial solution treatment and imaging

[0079] 50 μL of heat-activated bacterial solution was mixed with 450 μL of 1 × PPS to obtain a mixed solution; 10 μL of the mixed solution was loaded on a glass slide (4 °C, 1 h) for standby; the above bacterial solution was treated with a germination dye solution, and imaging was performed under a super-resolution microscope, and the imaging results are shown in FIG. . Figures 9 and 10

[0080] By Figure 9 ~Appendix Figure 10 ​It can be known from the data that, by observing the contrast before and after spore germination, it is more intuitive to show that with the increase of time, the spores gradually release DPA, and the fluorescence diffuses from the periphery to the core, and the results show that the near-infrared fluorescent probe (compound 3) of the dicyanoisophorone derivative of the present application can distinguish the dormant and germinated spores of the anthrax bacillus according to the different DPA contents. In the dormant spores, the DPA content is low, and the fluorescence signal is weak; while in the germinated spores, with the release of DPA, the fluorescence signal gradually increases and diffuses from the periphery to the core, which shows that the near-infrared fluorescent probe (compound 3) of the dicyanoisophorone derivative of the present application has high sensitivity and specificity, and can be used for real-time monitoring of the germination process of the anthrax bacillus spores.

[0081] The above is only to illustrate the technical idea of the present application, and cannot limit the protection scope of the present application. Any modification made according to the technical idea of the present application on the basis of the technical scheme falls within the protection scope of the claims of the present application.

Claims

1. Application of a near-infrared fluorescent probe of a dicyanoisophorone derivative in the preparation of a product for detecting anthrax Bacillus, characterized in that: The near-infrared fluorescent probe of the dicyanoisophorone derivative has a structural formula as shown in Formula 1: 。 2. The use according to claim 1, characterized in that The near-infrared fluorescent probe of the dicyanoisophorone derivative is prepared by the following steps: mixing compound 2 with 4-(diethylamino) salicylaldehyde and anhydrous sodium sulfate under nitrogen atmosphere, refluxing, and purifying to obtain the near-infrared fluorescent probe of the dicyanoisophorone derivative; The structural formula of compound 2 is ; The molar ratio of the compound 2 to 4-(diethylamino) salicylaldehyde and anhydrous sodium sulfate is 1:(1-2):(3-4).

3. The use according to claim 1, characterized in that The product for detecting Bacillus anthracis is used to measure, screen and detect the Bacillus anthracis biomarker DPA, and to achieve specific identification through hydrogen bonding and steric hindrance effects between a near-infrared fluorescent probe of a dicyanoisophorone derivative and DPA.

4. The use according to claim 1, characterized in that The product for detecting Bacillus anthracis is a test paper, a test kit, a chip or a reagent for detecting Bacillus anthracis in a biological sample.

5. The use according to claim 4, characterized in that The product is a product that uses the near-infrared luminescence of a dicyanoisophorone derivative as a near-infrared fluorescent probe to perform bacterial imaging.

6. The use according to claim 5, characterized in that The product is a product that uses near-infrared fluorescent probe cell imaging of dicyanoisophorone derivatives to distinguish between the dormant state and the germination state of anthrax Bacillus spores.

7. A kit for detecting Bacillus anthracis, characterized in that: The kit comprises the near-infrared fluorescent probe of the dicyanoisophorone derivative according to claim 1.

8. A kit for detecting Bacillus anthracis according to claim 7, characterized in that: The kit also includes reagents for preparing the probe solution.

9. A method for detecting DPA, a biomarker of Bacillus anthracis, based on a dicyanoisophorone derivative fluorescent probe, characterized in that: include: mixing the near-infrared fluorescent probe solution of the dicyanoisophorone derivative with the sample solution to be tested, incubating, and obtaining a mixture; The fluorescence signal of the mixture is detected to determine whether the sample to be tested contains DPA and its content.

10. The method for detecting the anthrax biomarker DPA based on a dicyanoisophorone derivative fluorescent probe according to claim 9, characterized in that: The incubation temperature is 24-26° C., and the incubation time is 20-40 min.

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