Application of fluorescent probe compound as fluorescent probe capable of specifically recognizing bacteria

Abstract

This invention relates to the field of fluorescent probe applications, and discloses the application of fluorescent probe compounds as fluorescent probes capable of specifically recognizing bacteria. The fluorescent probe compounds have the structure shown in formula (I). The application of fluorescent probe compounds as fluorescent probes capable of specifically recognizing bacteria provided by this invention enables in-situ visual detection of plant pathogens, greatly improving the efficiency of bacterial detection and possessing excellent market promotion and application value.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe applications, and more specifically to the application of a fluorescent probe compound as a fluorescent probe capable of specifically recognizing bacteria. Background Technology

[0002] Bacterial plant diseases are the second most numerous type of plant disease after fungal diseases, and are a common problem encountered in agricultural production.

[0003] Therefore, developing detection methods for plant bacterial diseases for diagnosis, monitoring, and research is crucial for crop protection.

[0004] Currently, many technologies can be used to detect plant pathogens, such as polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and chromatography-mass spectrometry.

[0005] However, these methods often involve cumbersome sample pretreatment processes, require expensive consumables and instruments, and cannot provide real-time visualization for detection.

[0006] Fluorescent probe technology often provides ultra-high spatiotemporal resolution, enabling real-time detection and tracking of specific targets, and also has advantages such as simple operation and low cost. Summary of the Invention

[0007] The purpose of this invention is to provide a new application of fluorescent probe compounds as fluorescent probes that can specifically identify bacteria.

[0008] To achieve the above objectives, a first aspect of the present invention provides an application of a fluorescent probe compound as a fluorescent probe capable of specifically recognizing bacteria, said fluorescent probe compound having the structure shown in formula (I):

[0009]

[0010] In equation (I),

[0011] R is selected from H and C. 1-6 alkyl, C 6-20 aryl;

[0012] X is selected from I and BF4.

[0013] A second aspect of the present invention provides a fluorescent probe reagent containing an active ingredient that can specifically identify an effective amount of bacteria, wherein the active ingredient is the fluorescent probe compound used in the application described in the first aspect above, and the content of the active ingredient is 1-99.99 wt%.

[0014] A third aspect of the present invention provides a fluorescent probe kit containing the reagents described in the second aspect above.

[0015] The application of fluorescent probe compounds as fluorescent probes that can specifically identify bacteria, provided by this invention, enables in-situ visual detection of plant pathogens, greatly improving the efficiency of bacterial detection and possessing excellent market promotion and application value. Attached Figure Description

[0016] Figure 1 This is a concentration-dependent graph showing the reaction of the small molecule fluorescent probe compound involved in this invention with different concentrations of nitro reductase.

[0017] Figure 2 This is a fluorescence imaging image of the small molecule fluorescent probe compound involved in this invention after co-incubation with two bacteria;

[0018] Figure 3 The small molecule fluorescent probe compound involved in this invention is used to detect the fluorescence imaging of plants infected by bacteria. Detailed Implementation

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] As previously stated, a first aspect of the present invention provides the application of a fluorescent probe compound as a fluorescent probe capable of specifically recognizing bacteria, said fluorescent probe compound having the structure shown in formula (I):

[0021]

[0022] In equation (I),

[0023] R is selected from H and C. 1-6 alkyl, C 6-20 aryl;

[0024] X is selected from I and BF4.

[0025] The C of this invention 1-6 The alkyl groups include straight-chain alkyl groups and branched alkyl groups with a total number of carbon atoms of 1-6, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0026] Preferably, in formula (I), R is selected from H, methyl, ethyl, n-propyl, isopropyl, and phenyl; X is selected from I and BF4.

[0027] More preferably, in equation (I), R is H and X is I.

[0028] This invention does not impose any particular requirements on the specific method for preparing the aforementioned compounds. Those skilled in the art can determine a suitable synthetic route by combining the structural features of this invention with known knowledge in the field to obtain the aforementioned compounds. However, in order to obtain compounds of this invention with higher purity and yield, this invention provides a method for preparing the compounds of this invention.

[0029] As previously stated, the present invention provides a method for preparing the compound described in the first aspect, the method comprising: reacting the compound of formula (II) with the compound of formula (III) in the presence of a solvent.

[0030]

[0031] In equation (II), the definitions of R and X are the same as those described in the first aspect.

[0032] Preferably, the conditions for the contact reaction include a temperature of 40-120°C and a time of 2-24 hours. More preferably, the conditions for the contact reaction include a temperature of 60-100°C and a time of 4-12 hours.

[0033] Preferably, the molar ratio of the compound represented by formula (II) to the compound represented by formula (III) is 1:1-2. More preferably, the molar ratio of the compound represented by formula (II) to the compound represented by formula (III) is 1:1-1.2.

[0034] Preferably, the solvent is selected from at least one of ethanol, acetonitrile, and dioxane.

[0035] The preparation method of the present invention may further include purification or post-treatment of the product obtained after the reaction, such as column chromatography. The details will not be elaborated here, and those skilled in the art should not understand it as a limitation of the present invention.

[0036] This invention does not impose any particular limitation on the source of the compounds represented by formula (II) and formula (III). Those skilled in the art can determine suitable synthetic routes using methods known in the art, or the compounds can be obtained commercially. An exemplary method for obtaining these compounds is provided below, and should not be construed as a limitation of this invention.

[0037] According to a particularly preferred embodiment, the method for synthesizing the compound represented by formula (II) includes: reacting substance A with an iodide in the presence of a solvent (preferably acetonitrile); the structural formula of substance A is...

[0038] Preferably, the bacteria are plant pathogens.

[0039] Preferably, the bacteria are selected from at least one of Staphylococcus aureus, Xanthomonas campestris, Streptomyces cerevisiae, and Erwinia amyloliquefaciens.

[0040] The fluorescent probe compounds synthesized in this invention exhibit excellent self-fluorescence quenching, effectively avoiding interference from their own fluorescence. In vitro, they show an increase in fluorescence signal after being catalyzed and reduced by nitroreductase. In in vivo experiments, they can clearly illuminate bacteria invading plants, enabling visual detection of bacterial plant diseases.

[0041] A second aspect of the present invention provides a fluorescent probe reagent containing an active ingredient that can specifically identify an effective amount of bacteria, wherein the active ingredient is the fluorescent probe compound used in the application described in the first aspect above, and the content of the active ingredient is 1-99.99 wt%.

[0042] Preferably, the content of the active ingredient is 20-90 wt%.

[0043] A third aspect of the present invention provides a fluorescent probe kit containing the reagents described in the second aspect above.

[0044] Therefore, the solution of the present invention can conveniently, quickly, in real time, and visually monitor the occurrence and development of plant bacterial diseases, and has excellent market promotion and application value.

[0045] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, the raw materials used are all common commercially available products.

[0046] Example 1: Synthesis of fluorescent probe compound NP

[0047]

[0048] 1) Synthesis of intermediate 1: Add substance A (10 mmol) and 1 ml of iodomethane to 10 mL of acetonitrile solution, reflux for 8 h, filter the reaction solution, and then wash slightly with acetonitrile. The resulting solid is intermediate 1.

[0049] 2) Synthesis of probe NP: Intermediate 1 (1 mmol) and p-nitrobenzaldehyde (1 mmol) were added to 10 ml of ethanol and refluxed for 8 h; then, the ethanol was removed by vacuum distillation and the residue was purified by column chromatography to obtain 157 mg of fluorescent probe compound NP.

[0050] Fluorescent probe compound NP: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.58 (d, J = 16.4Hz, 1H), 8.52 (d, J = 8.9Hz, 3H), 8.45 (d, J = 8.6Hz, 2H), 8.37 (d, J = 9.1Hz, 1H), 8.28 (d, J = 8.3Hz, 1H), 8.20 (d, J = 8.0Hz, 1H), 7.95 (d, J = 16.6Hz, 1H), 7.86 (d, J = 8.1Hz, 1H), 7.81 (d, J = 7.8Hz, 1H), 4.43–4.35 (m, 3H), 2.11–2.01 (m, 6H). The structure and purity of the fluorescent probe compound NP were verified by ESI-MS spectroscopy and HPLC. The calculated value for electrospray high-resolution mass spectrometry m / z: C23H21N2O2+(M+) is 357.1597; the detected value is 357.1594.

[0051] Test Example 1

[0052] To verify the targeting ability and detection capability of the fluorescent probe compound synthesized in this invention, a series of experiments were conducted using the fluorescent probe compound NP. The specific procedures are as follows:

[0053] Experiment 1: Reaction of fluorescent probe compounds with different concentrations of nitroreductase in PBS buffer

[0054] 10 μM of the fluorescent probe compound NP was added to PBS buffer containing 500 μM reduced nicotinamide adenine dinucleotide (NADH) and different concentrations of nitroreductase (purchased from Sigma, catalog number N9284). After incubation at 30 °C for 4 min, the fluorescence emission spectrum was scanned.

[0055] Figure 1 The fluorescence emission spectra of the fluorescent probe compound NP before and after reaction with different concentrations of nitroreductase are shown.

[0056] Depend on Figure 1 It can be seen that the fluorescence of the fluorescent probe compound NP is well quenched. After reacting with nitroreductase, a significant increase in fluorescence signal was observed at 595 nm under excitation of 495 nm light. Furthermore, the intensity of the fluorescence signal and the concentration of nitroreductase showed a good linear relationship.

[0057] The above experiments demonstrate that the fluorescent probe compound of the present invention can be activated by nitroreductase to generate a fluorescent signal.

[0058] Experiment 2: Fluorescent probe compounds for bacterial imaging

[0059] Staphylococcus aureus and Xanthomonas campestris were cultured in LB medium at 37°C (Staphylococcus aureus) or 28°C (Xanthomonas campestris) until OD. 600 The concentration reached 0.8. Afterward, the normal group was left untreated and incubated for 2 hours, while the inhibitor group was incubated for 2 hours with 100 μM nitroreductase inhibitor (dicoumarin). Then, 10 μM of the fluorescent probe compound NP was added to both the normal and inhibitor groups, and incubation was performed for 1 hour. The bacteria were then imaged using an inverted fluorescence microscope (Olympus IX-71).

[0060] The results are as follows Figure 2 As shown, in the normal group without inhibitor treatment, obvious red fluorescence was observed and overlapped well with bacteria in the bright field, while fluorescence in the inhibitor group was significantly reduced. This confirms that the fluorescent probe compound of the present invention is specifically activated by nitroreductase and can be used for imaging bacteria.

[0061] Experiment 3: Fluorescent probe compounds were used for imaging a bacterial infection model of plants.

[0062] Sow rapeseed seeds in culture pots filled with seedling substrate (soil moisture content above 65%), and cover the pots with plastic wrap. After seed germination, place the rapeseed in a container at 25℃, 50% humidity, and 150 μmol / m² light intensity. 2 The bacteria were cultured in a greenhouse at a temperature of / s for 15 days. Then, *Xanthomonas brasiliensis* was inoculated onto wounds on rapeseed leaves, and the wounds were covered with plastic wrap. Once the bacterial plaques were visible, the leaves were immersed in a 10 μM solution of the fluorescent probe compound NP for 1 hour, and the fluorescence was imaged using a Cy5-channel (GE Amersham Typhoon 5, USA) multispectral laser imager.

[0063] The results are as follows Figure 3 As shown in the figure, compared with the uninoculated control group, the bacterial-inoculated experimental group showed obvious fluorescence signals, and the fluorescence signals highly overlapped with the location of the bacterial plaques, demonstrating good spatial resolution.

[0064] In summary, the results show that the fluorescent probe compound provided by this invention can visualize and image bacteria infecting plants, and has the potential to diagnose plant bacterial diseases at an early stage and monitor the development of plant bacterial diseases.

[0065] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Use of a fluorescent probe compound as a fluorescent probe capable of specifically recognizing bacteria, the fluorescent probe compound having a structure shown in formula (I): Formula (I), wherein In formula (I), R is selected from H, C 1-6 alkyl; X is I; the bacteria are at least one selected from the group consisting of Staphylococcus aureus, Xanthomonas campestris, Streptomyces rimosus and Erwinia amylovora.

2. The use according to claim 1, wherein, In formula (I), R is selected from H, methyl, ethyl, n-propyl, isopropyl; X is I.

3. The use according to claim 2, wherein, In formula (I), R is H; X is I.

4. A fluorescent probe reagent characterized in that, The reagent contains an effective amount of an active ingredient capable of specifically recognizing bacteria, the active ingredient being the fluorescent probe compound used in the use according to any one of claims 1-3, and the content of the active ingredient being 1-99.99 wt%.

5. The reagent according to claim 4, wherein the content of the active ingredient is 20-90 wt%.

6. A fluorescent probe kit characterized in that, The kit contains the reagent according to claim 4 or 5.

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