Fluorescent probe, preparation method and application thereof, bacterial detection kit and detection method thereof

Abstract

The invention provides a fluorescent probe, a preparation method and application thereof, a bacterium detection kit and a detection method, and belongs to the technical field of fluorescent probes. According to the method, the Cys is detected through the fluorescent probe, so that the bacterial metabolite Cys is detected, and the condition that the bacterial concentration changes along with the fluorescence intensity is obtained. Meanwhile, the fluorescent probe provided by the invention can realize colorimetric fluorescence double-signal response, that is, in the presence of bacteria (escherichia coli) of which the metabolite is Cys, a fluorescent probe solution is changed from light yellow to purple, and the fluorescence color is changed from orange yellow to red. The fluorescent probe provided by the invention has high sensitivity and selectivity on detection of bacteria, can realize rapid detection of the content of bacteria in food, and provides a new guide direction for detecting bacteria and improving the safety quality of food; in addition, the excellent colorimetric fluorescence double signals can be developed into a corresponding food bacteria content test kit, and the kit has practical application value in the fields of biochemistry, analysis and detection and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe and a preparation method and application thereof, a bacteria detection kit and a detection method. Background Art

[0002] With the rapid development of my country's economy, the quantity and variety of food are becoming increasingly rich, and food issues have become the focus of people's attention. Therefore, improving food quality and solving food safety issues are becoming more and more hot issues of common concern to the world. Among food contamination, microbial contamination is the most common and has the most serious harm, which can cause hazards such as food poisoning and infection.

[0003] Escherichia coli is the most numerous Gram-negative bacteria in the intestines of humans and animals. Generally, it does not cause harm to the human body, but eating food contaminated by E. coli can cause intestinal diseases and trigger various infectious diseases. Therefore, the development of advanced and effective rapid detection methods can avoid food safety contamination caused by E. coli and ensure the health of the people. Traditional methods for detecting E. coli are mainly multi-tube fermentation method and membrane filter method, but both methods have the disadvantages of cumbersome procedures, time-consuming and labor-intensive, and cannot meet the needs of rapid diagnosis of E. coli in food.

[0004] In recent years, fluorescent probes have attracted wide attention due to their good selectivity, high sensitivity, simple operation and ability to visualize the detection of biological molecules in living cells. For example, patent CN115490654A discloses the synthesis and application of a fluorescent probe for detecting Escherichia coli carrying Clb toxins, which can achieve a colorimetric and fluorescent dual signal response to Escherichia coli carrying Clb toxins, but has the defect of a complex synthesis route. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a fluorescent probe and a preparation method and application thereof, an Escherichia coli detection kit and a detection method thereof. The fluorescent probe provided by the present invention has a simple structure, a short synthesis route, and has high sensitivity and selectivity for the detection of bacteria (including Escherichia coli and / or Staphylococcus aureus).

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a fluorescent probe having a structure shown in Formula I:

[0008]

[0009] The present invention provides a method for preparing the fluorescent probe described in the above technical solution, comprising the following steps:

[0010] In the presence of a first organic base, 3-hydroxy-3-methyl-2-butanone and malononitrile undergo a ring-forming reaction to obtain 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene) malononitrile having a structure shown in Formula 1;

[0011]

[0012] Under the action of a catalyst, the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 1 undergoes an aldol condensation reaction with 3-chloro-4-hydroxybenzaldehyde to obtain (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 2;

[0013]

[0014] Under the action of the second organic base, the (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having the structure shown in Formula 2 undergoes a substitution reaction with acryloyl chloride to obtain a fluorescent probe having the structure shown in Formula I.

[0015] Preferably, the first organic base comprises sodium ethoxide;

[0016] The cyclization reaction comprises: first reacting at room temperature for 0.5 h, and then reacting at reflux for 2 to 4 h.

[0017] Preferably, the catalyst comprises piperidine;

[0018] The temperature of the condensation reaction is reflux temperature, and the time is 12 to 16 hours.

[0019] Preferably, the second organic base comprises triethylamine;

[0020] The time of the substitution reaction is 1 to 3 hours.

[0021] The present invention provides the use of the fluorescent probe described in the above technical solution or the fluorescent probe prepared by the preparation method described in the above technical solution in detecting cysteine.

[0022] The present invention provides the use of the fluorescent probe described in the above technical solution or the fluorescent probe prepared by the preparation method described in the above technical solution in detecting bacteria, wherein the bacteria are bacteria containing cysteine ​​in their metabolites.

[0023] Preferably, the bacteria include Escherichia coli and / or Staphylococcus aureus.

[0024] The present invention provides a bacteria detection kit, comprising the fluorescent probe described in the above technical solution or the fluorescent probe prepared by the preparation method described in the above technical solution.

[0025] The present invention provides a method for detecting bacterial content, comprising the following steps:

[0026] Perform ultrasonic lysis on the bacterial solution to be tested to obtain a bacterial lysate;

[0027] A fluorescent probe, acetonitrile, bacterial lysate and PBS buffer solution are mixed and incubated, and the fluorescence intensity of the obtained incubation solution at 620 nm is detected under an excitation wavelength of 583 nm; the fluorescent probe is the fluorescent probe described in the above technical solution or the fluorescent probe prepared by the preparation method described in the above technical solution;

[0028] Obtaining the content of bacteria in the bacterial solution to be tested according to the fluorescence intensity and the standard curve;

[0029] The standard curve is a linear standard curve of bacterial concentration versus fluorescence intensity at 620 nm.

[0030] The present invention provides a fluorescent probe having a structure shown in formula I. The fluorescent probe provided by the present invention has a simple structure and has the characteristics of red light emission. At the same time, the selective recognition group of the fluorescent probe of the structure shown in formula I provided by the present invention is the most common recognition group of cysteine-acryloyl chloride, so the fluorescent probe provided by the present invention can quickly and highly selectively detect cysteine ​​(Cys). Cysteine ​​is a common metabolite of bacteria such as Escherichia coli or Staphylococcus aureus. The present invention detects Cys through a fluorescent probe, and then detects the metabolite Cys of Escherichia coli or Staphylococcus aureus, and obtains the situation where the concentration of Escherichia coli or Staphylococcus aureus changes with the fluorescence intensity. At the same time, the fluorescent probe provided by the present invention can realize a colorimetric fluorescence dual signal response, that is, in the presence of Escherichia coli or Staphylococcus aureus, the fluorescent probe solution is from light yellow to purple, the fluorescence color is orange-yellow to red, and the fluorescence turns on the response signal at 620nm. The fluorescent probe provided by the present invention has high sensitivity and selectivity for the detection of Escherichia coli or Staphylococcus aureus, and can realize rapid detection of the content of Escherichia coli or Staphylococcus aureus in food, which provides a new guiding direction for detecting Escherichia coli or Staphylococcus aureus and improving food safety quality; in addition, its excellent colorimetric and fluorescent dual signals can be developed into corresponding food bacterial content measurement kits, which have great practical application value in the fields of biochemistry, analytical detection, etc.

[0031] The present invention provides a method for preparing the fluorescent probe, which has a short synthesis route, simple operation, low cost and is easy to realize industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A synthetic route for fluorescent probes;

[0033] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe of the present invention;

[0034] Figure 3 is the response of the fluorescent probe of the present invention to cysteine;

[0035] Figure 4 is the change of the fluorescence intensity of the fluorescent probe with the concentration of E. coli lysate;

[0036] Figure 5 is the change of the fluorescence intensity of the fluorescent probe with the concentration of Staphylococcus aureus lysate;

[0037] Figure 6 Flow chart of the fluorescent probe detection of Escherichia coli lysate. DETAILED DESCRIPTION

[0038] The present invention provides a fluorescent probe having a structure shown in Formula I:

[0039]

[0040] The present invention provides a method for preparing the above fluorescent probe, comprising the following steps:

[0041] In the presence of a first organic base, 3-hydroxy-3-methyl-2-butanone and malononitrile undergo a ring-forming reaction to obtain 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene) malononitrile having a structure shown in Formula 1;

[0042]

[0043] Under the action of a catalyst, the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 1 undergoes an aldol condensation reaction with 3-chloro-4-hydroxybenzaldehyde to obtain (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 2;

[0044]

[0045] Under the action of the second organic base, the (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having the structure shown in Formula 2 undergoes a substitution reaction with acryloyl chloride to obtain a fluorescent probe having the structure shown in Formula I.

[0046] In the present invention, in the presence of a first organic base, 3-hydroxy-3-methyl-2-butanone and malononitrile undergo a cyclization reaction to obtain 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene) malononitrile having a structure shown in Formula 1. In the present invention, the molar ratio of 3-hydroxy-3-methyl-2-butanone to malononitrile is preferably 1:2.

[0047] In the present invention, the first organic base preferably includes sodium ethoxide; the molar ratio of the 3-hydroxy-3-methyl-2-butanone to malononitrile to the first organic base is preferably 1:0.15.

[0048] In the present invention, the cyclization reaction is preferably carried out in an organic solvent, and the organic solvent is preferably ethanol.

[0049] In the present invention, the cyclization reaction preferably comprises: first reacting at room temperature for 0.5 h, and then reacting at reflux for 2 to 4 h.

[0050] After the cyclization reaction, the present invention preferably performs post-treatment on the obtained cyclization reaction solution, and the post-treatment preferably comprises the following steps:

[0051] The cyclization reaction solution is cooled to room temperature, and solid-liquid separation, washing and drying are performed in sequence.

[0052] In the present invention, the solid-liquid separation is preferably suction filtration; the detergent used in the washing is preferably ethanol, and the number of washings is preferably 3 times; and the drying is preferably vacuum drying.

[0053] After obtaining 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile having the structure of Formula 1, the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile having the structure of Formula 1 is subjected to a condensation reaction with 3-chloro-4-hydroxybenzaldehyde under the action of a catalyst to obtain (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile having the structure of Formula 2. In the present invention, the molar ratio of the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile having the structure of Formula 1 to 3-chloro-4-hydroxybenzaldehyde is preferably 1:1.

[0054] In the present invention, the catalyst preferably includes piperidine; the molar ratio of the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile having the structure shown in Formula 1 to the catalyst is preferably 1:1.

[0055] In the present invention, the condensation reaction is preferably carried out in an organic solvent, and the organic solvent is preferably ethanol.

[0056] In the present invention, the temperature of the condensation reaction is preferably reflux temperature, and the time is preferably 12 hours.

[0057] After the condensation reaction, the present invention preferably performs post-treatment on the obtained condensation reaction solution, and the post-treatment preferably comprises the following steps:

[0058] The condensation reaction liquid is cooled to room temperature, and solid-liquid separation, washing and drying are performed in sequence.

[0059] In the present invention, the solid-liquid separation is preferably suction filtration; the detergent used in the washing is preferably ethanol, and the number of washings is preferably 3 times; and the drying is preferably vacuum drying.

[0060] After obtaining (E)-2-(4-(3-chloro-4-hydroxystyryl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 2, the (E)-2-(4-(3-chloro-4-hydroxystyryl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 2 is subjected to a substitution reaction with acryloyl chloride under the action of a second organic base to obtain a fluorescent probe having a structure shown in Formula I. In the present invention, the molar ratio of the (E)-2-(4-(3-chloro-4-hydroxystyryl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 2 to acryloyl chloride is preferably 1:1.1-1.2.

[0061] In the present invention, the second organic base preferably includes triethylamine; the molar ratio of the (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having the structure shown in Formula 2 to the second organic base is preferably 1:1.2-1.3.

[0062] In the present invention, the substitution reaction is preferably carried out in an organic solvent, and the organic solvent is preferably N,N-dimethylformamide.

[0063] In the present invention, the temperature of the substitution reaction is preferably room temperature, and the time is preferably 1 to 3 hours, more preferably 2 hours.

[0064] After the substitution reaction, the present invention preferably performs post-treatment on the obtained substitution reaction solution, and the post-treatment preferably comprises the following steps:

[0065] The substitution reaction solution is mixed with petroleum ether to precipitate a solid;

[0066] The solid was recrystallized using a mixture of petroleum ether and dichloromethane.

[0067] In the present invention, in the mixed liquid of petroleum ether and dichloromethane, the volume ratio of petroleum ether to dichloromethane is preferably 10:1.

[0068] In the present invention, the synthetic route of the fluorescent probe is as follows Figure 1 shown.

[0069] The present invention provides application of the fluorescent probe in detecting cysteine.

[0070] The present invention provides the use of the above-mentioned fluorescent probe in detecting bacteria, wherein the bacteria are bacteria containing cysteine ​​in their metabolites. In the present invention, the bacteria preferably include Escherichia coli and / or Staphylococcus aureus, specifically Escherichia coli or Staphylococcus aureus. In the present invention, the fluorescent probe can realize colorimetric fluorescence dual signal detection of bacterial lysate (Escherichia coli lysate or Staphylococcus aureus lysate), the fluorescence color is orange-yellow to red, the solution is from light yellow to purple, and the fluorescence turn-on response signal is at 620nm.

[0071] The invention provides a bacteria detection kit, comprising the fluorescent probe.

[0072] The bacteria detection kit provided by the present invention also includes acetonitrile, which is used to dissolve and dilute the fluorescent probe.

[0073] The bacteria detection kit provided by the present invention also includes a PBS buffer solution. In the present invention, the pH value of the PBS buffer solution is preferably pH=7.4.

[0074] In the present invention, the bacteria detection kit is specifically an Escherichia coli and / or Staphylococcus aureus detection kit.

[0075] In the present invention, the fluorescent probe in the bacteria detection kit is preferably dissolved in acetonitrile to obtain an acetonitrile solution of the fluorescent probe.

[0076] In the present invention, when the bacterial kit is used, the present invention preferably dissolves the fluorescent probe in acetonitrile to obtain an acetonitrile solution of the fluorescent probe.

[0077] The present invention provides a method for detecting bacterial content, comprising the following steps:

[0078] Perform ultrasonic lysis on the bacterial solution to be tested to obtain a bacterial lysate;

[0079] The fluorescent probe, acetonitrile, bacterial lysate and PBS buffer solution were mixed and incubated, and the fluorescence intensity of the obtained incubation solution at 620 nm was detected under an excitation wavelength of 583 nm;

[0080] Obtaining the content of target bacteria in the bacterial solution to be tested according to the fluorescence intensity and the standard curve;

[0081] The standard curve is a linear standard curve of bacterial concentration versus fluorescence intensity at 620 nm.

[0082] The present invention performs ultrasonic lysis on a bacterial solution to be tested to obtain a bacterial lysate.

[0083] In the present invention, the power of the ultrasound is preferably 400 W; the time is preferably 10 min.

[0084] After obtaining the bacterial lysate, the present invention mixes the fluorescent probe, acetonitrile (ACN), the bacterial lysate and a PBS buffer solution, incubates the mixture, and detects the fluorescence intensity of the obtained incubation solution at 620 nm under an excitation wavelength of 583 nm.

[0085] The present invention preferably dissolves the fluorescent probe in part of acetonitrile to obtain an acetonitrile solution (stock solution) of the fluorescent probe. In the acetonitrile solution of the fluorescent probe, the molar concentration of the fluorescent probe is preferably 1 mM. Then, the present invention mixes the acetonitrile solution of the fluorescent probe, the remaining acetonitrile (for dilution), the bacterial lysate and the PBS buffer solution, and incubates to obtain an incubation solution; the fluorescence intensity of the obtained incubation solution at 620 nm is detected at an excitation wavelength of 583 nm.

[0086] In the present invention, the molar concentration (working concentration) of the fluorescent probe in the incubation solution is preferably 10 μM.

[0087] In the present invention, the pH value of the PBS buffer solution is preferably 7.4. In the present invention, when the acetonitrile solution of the fluorescent probe, the remaining acetonitrile, the bacterial lysate and the PBS buffer solution are mixed: the volume ratio of the acetonitrile solution (stock solution) of the fluorescent probe to the remaining acetonitrile is preferably 20:180: the volume ratio of the acetonitrile solution (stock solution) of the fluorescent probe to the bacterial lysate is preferably 20:1000; the volume ratio of the acetonitrile solution (stock solution) of the fluorescent probe to the PBS buffer solution is preferably 20:800. In the present invention, after the acetonitrile solution (stock solution) of the fluorescent probe, the remaining acetonitrile, the bacterial lysate and the PBS buffer solution are mixed, the volume ratio of the organic phase to the aqueous phase in the resulting mixed solution is preferably 1:9. Among them, the acetonitrile solution (stock solution) of the fluorescent probe and the remaining acetonitrile are organic phases, and the bacterial lysate and the PBS buffer solution are aqueous phases.

[0088] In the present invention, the incubation temperature is preferably 37° C., and the incubation time is preferably 30 min.

[0089] The present invention obtains the content of target bacteria in the bacterial solution to be tested according to the fluorescence intensity and the standard curve. In the present invention, the standard curve is a linear standard curve of the concentration of target bacteria versus the fluorescence intensity at 620nm. In the present invention, the concentration of the target bacteria is preferably expressed in terms of OD 600 In the present invention, the target bacteria are bacteria whose bacterial metabolites contain Cys. The target bacteria are preferably Escherichia coli and / or Staphylococcus aureus.

[0090] In the present invention, the detected temperature is preferably 4-37°C, more preferably 25°C.

[0091] In the present invention, the method for obtaining the standard curve preferably comprises the following steps:

[0092] Provide a gradient of bacterial solutions with known concentrations;

[0093] Performing ultrasonic lysis on bacterial solutions of known concentrations respectively to obtain bacterial lysates of gradient known concentrations corresponding to bacterial solutions of gradient known concentrations;

[0094] The organic solution of the fluorescent probe, acetonitrile, a gradient bacterial lysate with known concentrations, and a PBS buffer solution were mixed and incubated respectively. The fluorescence intensity of the obtained incubation solution at 620 nm was detected under an excitation wavelength of 583 nm to obtain the fluorescence intensity corresponding to the Escherichia coli solution of different concentrations;

[0095] A standard curve was drawn with the concentration of the E. coli solution as the horizontal axis and the fluorescence intensity as the vertical axis.

[0096] In the present invention, the detection limit of Escherichia coli is OD 600 : 0.0032, linear detection range is OD 600 : 0-1. The detection limit of Staphylococcus aureus is OD 600 : 0.024, linear detection range is OD 600 :0~1

[0097] The following is a detailed description of a fluorescent probe, a preparation method and application thereof, and an Escherichia coli detection kit provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0098] Example 1

[0099] according to Figure 1 The synthetic route to synthesize the fluorescent probe is as follows:

[0100] (1) Synthesis of 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile

[0101] 3-Hydroxy-3-methyl-2-butanone (2g, 19.58mmol) and malononitrile (2.59g, 39.16mmol) were dissolved in anhydrous ethanol (20mL), and sodium ethoxide (199.89mg, 2.94mmol) was added. The reaction was stirred at room temperature for 0.5h, and then heated under reflux for 2h until the solution turned golden yellow to stop the reaction. The mixture was filtered and washed with ethanol 3 times. The filter cake was vacuum dried to obtain 2.3g of 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene) malononitrile as a white solid with a yield of 59.0%.

[0102] (2) Synthesis of (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile

[0103] 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile (1 g, 5.02 mmol), 3-chloro-4-hydroxybenzaldehyde (785.92 mg, 5.02 mmol), and a catalytic amount of piperidine were heated to reflux in anhydrous ethanol (10 mL) for 12 h, and solids were precipitated after cooling to room temperature. The mixture was filtered and washed with ethanol three times. The filter cake was vacuum dried to obtain 1.4 g of (E)-2-(4-3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile as a purple solid with a yield of 82.6%.

[0104] (3) Synthesis of (E)-2-chloro-4-(2-(4-cyano-5-(dicyanomethylidene)-2,2-dimethyl-2,5-dihydrofuran-3-yl)vinyl)phenyl acrylate (i.e., fluorescent probe)

[0105] (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile (500 mg, 1.48 mmol) and triethylamine (179.76 mg, 1.78 mmol) were dissolved in N,N-dimethylformamide (10 mL) and stirred for 0.5 h. Acryloyl chloride (147.38 mg, 1.63 mmol) diluted in N,N-dimethylformamide (5 mL) was added. mmol) was added dropwise to the above solution and the reaction was continued for 1-3h; the mixed solution was poured into petroleum ether (25mL), a large amount of solid precipitated, and then recrystallized and purified with petroleum ether and dichloromethane (volume ratio of 10:1) to obtain (E)-2-chloro-4-(2-(4-cyano-5-(dicyanomethylidene)-2,2-dimethyl-2,5-dihydrofuran-3-yl)vinyl)phenyl acrylate as a ginger yellow solid 260mg, with a yield of 44.83%.

[0106] The nuclear magnetic resonance hydrogen spectrum (DMSO-d6, 500 MHz) of the obtained fluorescent probe is as follows: Figure 2 shown.

[0107] Test Example 1 Response of fluorescent probe to cysteine

[0108] The specific experimental process is as follows: accurately prepare 1.0mM probe acetonitrile solution as a stock solution for standby use. Take a certain amount of probe stock solution and an appropriate amount of analyte stock solution (the concentration of the analyte stock solution is 10mM), dilute it with PBS buffer (10mM, pH=7.4, containing 10% ACN) into 2mL of test solution, where the working concentration of the probe is 10μM and the concentration of the analyte is 100μM. Use a UV-2600 spectrophotometer to record its ultraviolet-visible spectrum, and use an F-7000 fluorescence spectrometer to obtain the fluorescence spectrum at an excitation wavelength of 583nm, with the slit width set to 5 / 5nm. In order to reduce errors and ensure the accuracy of the experiment, all analytical experiments were measured in parallel three times.

[0109] Figure 3 The response of the fluorescent probe prepared in Example 1 to cysteine, Figure 3 (A) is the UV absorption spectra of the probe (10 μM), the mother core, and the probe after responding with cysteine ​​(100 μM) in acetonitrile-PBS (pH=7.4, v / v=1:9) solution. Figure 3 (B) is the fluorescence spectrum of the probe, the mother core and the probe in acetonitrile-PBS (pH=7.4, v / v=1:9) solution after responding to Cys. Figure 3 In (A) and (B), "Fluorophore-OH" represents the parent compound, and its structural formula is Figure 3 In (A) and (B), "Probe-Cys" represents a fluorescent probe having a structure shown in Formula I, Figure 3 In (A) and (B), "Probe-Cys+Cys" represents the fluorescent probe of the structure shown in Formula I plus the analyte Cys.

[0110] Figure 3 (C) in the figure shows the fluorescence intensity after the probe (10 μM) responds to different analytes (100 μM). Figure 3 (D) is the fluorescence intensity of the probe and other analytes in the presence of Cys (λex / λem=583 / 615nm, slit: 5nm / 5nm). Figure 3 The substances 1 to 29 in (C) are: Cys, Hcy, GSH, Met, His, Trp, Gln, Ala, Gly, Lys, Thr, Ser, Pro, Phe, Leu, Arg, Val, Na +, Fe 3+ Mg 2+ , Ca 2+ , Ba 2+ , K + 、Al 3+ , Cl - , AC - 、CO3 2- 、NO2 - 、SO4 2- .in, Figure 3 (D) is the fluorescence intensity of the probe and 2 to 29 analytes in the presence of Cys. Figure 3 (D) has only 28 columns.

[0111] Depend on Figure 3 It can be seen that the fluorescent probe with the structure shown in Formula I provided by the present invention can detect cysteine ​​(Cys) rapidly and with high selectivity.

[0112] Test Example 2: Detection of Cys-containing Escherichia coli lysate by fluorescent probe colorimetric and fluorescent dual signals

[0113] The experiment of detecting the spectral properties of Cys in E. coli lysate by fluorescent probe is as follows:

[0114] The probe was dissolved in acetonitrile (CH3CN) to prepare a probe solution with a concentration of 1 mM (acetonitrile solution of fluorescent probe).

[0115] The preparation method of E. coli lysate is as follows: prepare different OD 600 coli solution (an E. coli solution prepared with PBS buffer), and then the E. coli solution was ultrasonically lysed (400W, 10min) to obtain an E. coli lysate containing Cys.

[0116] The specific test method is: 5% (i.e. 100 μL) of the total volume of 2 mL is added to tap water and urine, which is equivalent to a spiked experiment. The specific solution preparation is: PBS environment: 20 μL probe solution + 180 μL CH3CN + 1000 μL E. coli lysate + 800 μL PBS. Tap water or urine environment: 20 μL probe solution + 180 μL CH3CN + 1000 μL E. coli lysate + 700 μL PBS + 100 μL tap water or urine. The concentration of the probe in the prepared test solution is 10 μM.

[0117] After incubation at 37°C for 30 min, the fluorescence intensity at 620 nm was detected at an excitation wavelength of 583 nm. In a mixed system with a volume ratio of organic phase to aqueous phase of 1:9, the change of fluorescence intensity with the concentration of E. coli lysate is shown in Figure 2. Figure 4 shown.

[0118] Figure 4 (A) is the fluorescence spectrum of the mixed system formed by the E. coli lysate prepared with PBS buffer. Figure 4 (D) is the OD of E. coli in the E. coli solution prepared with PBS buffer. 600 Standard linear curve of the fluorescence intensity at 620 nm.

[0119] Figure 4 (B) is the fluorescence spectrum of the mixed system formed by the E. coli lysate prepared with tap water. Figure 4 (E) is the OD of E. coli in the E. coli solution prepared with tap water. 600 Standard linear curve of the fluorescence intensity at 620 nm.

[0120] Figure 4 (C) is the fluorescence spectrum of the mixed system formed by the E. coli lysate prepared with urine. Figure 4 (F) is the OD of E. coli in the E. coli solution prepared with urine 600 Standard linear curve of the fluorescence intensity at 620 nm.

[0121] Figure 4 (A) to (C) are the fluorescence spectra of the probe (10 μM) after adding different concentrations of E. coli lysate (OD600: 0 to 1) under different environments (PBS, water, urine). Figure 4 (D) to (F) are the linear fitting curves of the probe (10 μM) and different concentrations of E. coli lysate (OD600: 0 to 1). Figure 4 This indicates that the fluorescent probe provided by the present invention can still accurately detect Escherichia coli in a complex environment. Therefore, the fluorescent probe provided by the present invention is suitable for detecting bacteria in various complex environmental solutions.

[0122] Test Example 3: Fluorescent probe used to detect E. coli content in food

[0123] The method for detecting the content of E. coli in food is as follows: food samples purchased from supermarkets (such as fruits: oranges, apples or pears) are pre-treated, and the specific steps of the pre-treatment include: each food sample (5.00g) is dried and ground into powder. Subsequently, the powdered sample is extracted with 50ml PBS-DMSO-HCl (3:7:1, v / v / v), and the mixture is filtered after ultrasonication at room temperature for 30 minutes. The filtrate is diluted 20 times with PBS-CH3CN (9:1, v / v), and the pH is adjusted to 7.4 to obtain the sample to be tested. Subsequently, different concentrations of bacterial lysate (E. coli lysate, the preparation method of E. coli lysate is the same as that of Test Example 2, and the metabolite cysteine ​​of E. coli is introduced into the above-mentioned fruit sample) is added respectively, and its actual concentration is further determined by the fluorescence intensity of the probe (10μM). Table 1 shows the detection results of E. coli in actual samples by the fluorescent probe.

[0124] Table 1 Detection of Escherichia coli in actual samples by fluorescent probes

[0125]

[0126] Test Example 4 Fluorescent Probe Detection of Cys-Containing Staphylococcus aureus Lysate

[0127] The experiment of detecting the spectral properties of Cys in Staphylococcus aureus by fluorescent probe is as follows:

[0128] The probe was dissolved in acetonitrile (CH3CN) to prepare a probe solution with a concentration of 1 mM, and different OD 600 The Staphylococcus aureus solution (Staphylococcus aureus solution prepared with PBS buffer) was then ultrasonically lysed (400W for 10 min) to obtain a Cys-containing Staphylococcus aureus lysate. The specific test method was as follows: 20 μL of 1 mM probe solution, 180 μL of analytical grade CH3CN, 1 mL of Staphylococcus aureus lysate, and 800 μL of PBS buffer solution were placed in a sample tube (where the volume ratio of the organic phase to the aqueous phase was 1:9), where the working concentration of the probe was 10 μM, and after shaking and incubating at 37°C for 30 min, the fluorescence intensity at 620 nm was detected at an excitation wavelength of 583 nm. In a mixed system where the volume ratio of the organic phase to the aqueous phase was 1:9, the change in fluorescence intensity with the concentration of the Staphylococcus aureus lysate is shown in the figure. Figure 5 shown.

[0129] Figure 5 (A) Fluorescence spectra of the probe (10 μM) after adding different concentrations of Staphylococcus aureus lysate (OD600: 0-1). Figure 5(B) is the linear fitting curve of the fluorescence intensity at 620nm of the probe (10μM) and different concentrations of Staphylococcus aureus lysate (OD600: 0~1). Figure 5 It can be seen that the fluorescent probe provided by the present invention can also perform quantitative and accurate detection on Staphylococcus aureus.

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that It has the structure shown in formula I:

2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The following steps are involved: In the presence of a first organic base, 3-hydroxy-3-methyl-2-butanone and malononitrile undergo a ring-forming reaction to obtain 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene) malononitrile having a structure shown in Formula 1; Under the action of a catalyst, the 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 1 undergoes an aldol condensation reaction with 3-chloro-4-hydroxybenzaldehyde to obtain (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having a structure shown in Formula 2; Under the action of the second organic base, the (E)-2-(4-(3-chloro-4-hydroxyphenylvinyl)-3-cyano-5,5-dimethylfuran-2(5H)-methylene)malononitrile having the structure shown in Formula 2 undergoes a substitution reaction with acryloyl chloride to obtain a fluorescent probe having the structure shown in Formula I.

3. The preparation method according to claim 2, characterized in that: The first organic base comprises sodium ethoxide; The cyclization reaction comprises: first reacting at room temperature for 0.5 h, and then reacting at reflux for 2 to 4 h.

4. The preparation method according to claim 2, characterized in that: The catalyst includes piperidine; The temperature of the condensation reaction is reflux temperature, and the time is 12 to 16 hours.

5. The preparation method according to claim 2, characterized in that: The second organic base comprises triethylamine; The time of the substitution reaction is 1 to 3 hours.

6. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 5 in detecting cysteine.

7. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 5 in detecting bacteria, wherein the bacteria are bacteria containing cysteine ​​in their metabolites.

8. The use according to claim 7, characterized in that: The bacteria include Escherichia coli and / or Staphylococcus aureus.

9. A bacteria detection kit, characterized in that: The invention comprises the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 5.

10. A method for detecting bacterial content, characterized in that: The following steps are involved: Ultrasonic lysis is performed on the bacterial solution to be tested to obtain a bacterial lysate; A fluorescent probe, acetonitrile, bacterial lysate and PBS buffer solution are mixed and incubated, and the fluorescence intensity of the obtained incubation solution at 620 nm is detected under an excitation wavelength of 583 nm; the fluorescent probe is the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 5; Obtaining the content of bacteria in the bacterial solution to be tested according to the fluorescence intensity and the standard curve; The standard curve is a linear standard curve of bacterial concentration versus fluorescence intensity at 620 nm.

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