A fluorescent probe for detecting bacterial infection in a dual channel and a preparation method and application thereof

Abstract

The present application relates to a kind of double-channel fluorescence probe for detecting bacterial infection and its preparation method and application, belong to the detection and treatment technical field of bacterial infection, specifically a kind of double mechanism response double-channel precision detection bacterial infection and photodynamic therapy fluorescence probe preparation and application.The present application has prepared a kind of double mechanism response fluorescence probe, can realize double emission channel fluorescence detection without crosstalk under single excitation light, simultaneously, probe has single targeting to bacteria and does not enter mammalian cell, therefore its photodynamic effect can carry out precision photodynamic killing to bacteria, to realize double-channel precision detection bacterial infection and photodynamic therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to a dual-channel fluorescent probe for detecting bacterial infection and its preparation method and application, and belongs to the technical field of detection and treatment of bacterial infection, in particular to a preparation and application of a dual-mechanism responding dual-channel fluorescent probe for precisely detecting bacterial infection and photodynamic therapy. BACKGROUND

[0002] Bacterial infection poses a serious threat to public health. In the treatment of bacterial infection in clinic, accurate and early detection of bacteria and identification of pollution sources are very important for the diagnosis of infectious diseases and the treatment of antibiotics. In recent years, with the continuous progress of laser technology and fluorescent dyes, fluorescence imaging assisted by fluorescent probes can detect and reveal the physiological and pathological functions of the analyte of interest at a non-invasive, longitudinal molecular level, and is a simple, low-cost, high-sensitivity and spatially resolved imaging diagnostic method, which has been widely used in the fields of biology, physiology, pharmacology and medicine. Among various types of probes, activatable probes are particularly interesting because they are initially in the "off state and can only be activated to the "on state to emit fluorescence signals in the presence of certain target enzymes, and these fluorescence signals exhibit measurable changes in spectral characteristics, thereby better understanding and diagnosing bacterial infection. However, in a complex biological environment, unwanted targets usually have moderate concentrations of biomarkers, allowing the probe to be non-specifically activated during transport, thus easily producing "false positive" signals, and the probe or generated intermediates are easily diffused and changed in position in a highly heterogeneous and dynamic biological environment, which can pose a challenge to avoiding phototoxicity to normal tissues. SUMMARY

[0003] The technical problem solved by the present application is to overcome the shortcomings of the prior art and provide a fluorescent probe for double-channel detection of bacterial infection and a preparation method and application thereof.The probe produces non-crosstalk fluorescent signals through two different response mechanisms to perform double-channel accurate bacterial infection detection, has excellent sensitized oxygen ROS generation capability, can successfully attach or insert bacteria without entering mammalian cells, provides the possibility of precise pathogenic microorganism killing while serving as an imaging material, and has broad-spectrum bacterial killing capability.The positive charge from the cationic methylpyridine part of the probe enables the probe to target the negatively charged bacterial surface through electrostatic interaction, but not to enter mammalian cells, and the complex spatial structure of the bacterial surface induces red fluorescence due to restricted intramolecular rotation (RIM).In addition, the nitro group in the probe can be reduced to an amino group by the ubiquitous NTR in bacteria, destroys the PET effect, releases the green fluorescence of naphthalimide, and realizes non-crosstalk double-channel bacterial infection fluorescence detection.Meanwhile, the single selectivity of Nap-CefTTPy to bacteria enables the ROS generated thereby to accurately kill bacteria without affecting cells.

[0004] To achieve the above object, the present application adopts the following technical solutions:

[0005] The fluorescent probe for double-channel detection of bacterial infection has a chemical structure as shown in formula (I):

[0006]

[0007] The preparation method of the fluorescent probe for double-channel detection of bacterial infection comprises the following steps:

[0008] (1) Compound 1 is subjected to a substitution reaction with an iodine salt in a solvent I, and then subjected to a substitution reaction with compound 2 in an organic solvent II under the action of a base, and after the reaction is completed, compound 3 is obtained through extraction and silica gel chromatography purification, wherein the molar ratio of compound 1 to compound 2 is 1:1-3, the reaction temperature is 0-25℃, the reaction time is 1-24h, the solvent used for silica gel chromatography purification is dichloromethane and methanol, the volume ratio of dichloromethane to methanol is 5-60:1, the ratio of the iodine salt to compound 1 is 1:1-4, the volume of the organic solvent I to the mass of compound 1 is 0.6-5mL:1mg, and the ratio of compound 2 to sodium bicarbonate is 1:1-10;

[0009] (2) under ice bath condition, compound 3 is added into organic solvent III, a protecting group is removed under the action of an acid, then the obtained solution is added into organic solvent IV for recrystallization, the precipitate is collected and washed for 3 times to obtain compound Nap-CefTTPy; the ratio of the volume of organic solvent III to the mass of compound 3 is 1-10 ml: 1 mg; the acid is trifluoroacetic acid, and the ratio of the acid to compound 3 is 0.5-10: 1;

[0010] The chemical structural formula of the compound 1 is as follows:

[0011]

[0012] The chemical structural formula of the compound 2 is as follows:

[0013]

[0014] The chemical structural formula of the compound 3 is as follows:

[0015]

[0016] In the above step (1), the organic solvent I is at least one selected from methanol, ethanol, acetonitrile, diethyl ether and acetone; the organic solvent II is at least one selected from toluene, DMSO, DMF and acetonitrile;

[0017] In the above step (2), the organic solvent III is at least one selected from methanol, ethanol, dichloromethane and dichloroethane; the organic solvent IV is at least one selected from methanol, ethanol, acetonitrile, diethyl ether and acetone.

[0018] The application of the fluorescent probe for detecting bacterial infection in two channels, the fluorescent probe is incubated with the to-be-detected substance for 30 min, under the irradiation of 488 nm laser, when the green and red two fluorescence channels are not bright, it indicates that there is no bacteria in the to-be-detected substance, and when the green and red two fluorescence channels are both bright, it indicates that there is bacteria in the to-be-detected substance.

[0019] The application of the fluorescent probe for detecting bacterial infection in two channels, the fluorescent probe is incubated with the to-be-detected substance after bacterial infection, and then photodynamic sterilization is carried out, and it can be seen from the bacterial spread plate method that the fluorescent probe has good photodynamic sterilization effect.

[0020] The advantages and beneficial effects of the present application are as follows:

[0021] The present application prepares a fluorescent probe with double mechanism response, which can realize fluorescence detection in two emission channels without crosstalk under single excitation light, and the probe has single targeting property for bacteria and does not enter mammalian cells, so that the photodynamic effect can accurately kill bacteria, thereby realizing accurate detection of bacterial infection and photodynamic treatment in two channels.

[0022] The application provides a compound with a structure shown in formula (I) as a dual mechanism response dual-channel precise detection bacterial infection and photodynamic therapy fluorescent probe.

[0023] The fluorescent probe Nap-CefTTPy provided by the application has typical AIE characteristics, and when the compound is in a DMSO and toluene mixed solvent, when the toluene component is 0% (100% DMSO), the compound shows weak luminescence, when the toluene component is higher than 60%, the fluorescent intensity rapidly increases, and the fluorescent intensity slowly moves from 710 nm to 650 nm; when the toluene component is between 60% and 90%, the fluorescent intensity rapidly increases, and when the toluene component reaches 95%, the fluorescent intensity is the strongest.

[0024] The fluorescent probe Nap-CefTTPy provided by the application is a bacterial targeting probe, does not enter mammalian cells, and the complex spatial structure of the bacterial surface induces restricted intramolecular rotation (RIM) to make the Nap-CefTTPy produce red fluorescence; in addition, the nitro group in the probe can be reduced to an amino group by the NTR widely existing in bacteria, the PET effect is destroyed, the green fluorescence of naphthalimide is released, and therefore, the fluorescent probe Nap-CefTTPy can realize precise dual-channel fluorescence detection of pathogenic microorganisms.

[0025] In addition, the structure of the fluorescent probe Nap-CefTTPy provided by the application can interact with beta-lactamase (Bla) to realize recognition of Bla in vitro.

[0026] The application provides application of the fluorescent probe Nap-CefTTPy in precise dual-channel fluorescence detection of pathogenic microorganisms, bacteria are dyed or incubated with the fluorescent probe Nap-CefTTPy, then a dual-channel imaging experiment is carried out, the fluorescent images of the Nap-CefTTPy are captured by CLSM respectively, and different imaging capabilities of the fluorescent probe on bacteria and cells are evaluated.

[0027] The application provides that the precise detection of pathogenic microorganisms by the fluorescent probe Nap-CefTTPy is single targeting recognition of the probe on bacteria, and the probe does not enter mammalian cells.

[0028] The application provides application of the fluorescent probe Nap-CefTTPy in photodynamic sterilization, incubates the fluorescent probe Nap-CefTTPy with a bacterial liquid and places the fluorescent probe Nap-CefTTPy under white light irradiation, so as to kill pathogenic microorganisms, and evaluates the sterilization capacity of the fluorescent probe Nap-CefTTPy by counting the number of colonies of the bacterial liquid after plate culture.

[0029] The target pathogenic microorganism for the pathogenic microorganism detection or the photodynamic antibiosis is gram-negative bacteria or gram-positive bacteria.

[0030] The target pathogenic microorganism for the pathogenic microorganism or the photodynamic antibiosis is Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a mechanism of the fluorescent probe Nap-CefTTPy;

[0032] Figure 2 is a synthesis route of the fluorescent probe Nap-CefTTPy;

[0033] Figure 3 is a characterization diagram of experimental results of examples; wherein, A) is a fluorescence spectrum of a glycerol component (f gly ) of the fluorescent probe Nap-CefTTPy (10 µM) in a glycerol / water mixed solvent from 0% to 99%, λex = 488 nm; B) is a fluorescence spectrum of a toluene component (f tol ) of the fluorescent probe Nap-CefTTPy (10 µM) in a dimethyl sulfoxide / toluene mixed solvent from 0% to 95%, λex = 480 nm; C) is a fluorescence spectrum of Nap-CefTTPy (10 µM) in the presence of NTR (10 4 U / ml) and both enzymes, λex = 480 nm; D) is a fluorescence response of Nap-CefTTPy (10 µM) in PBS to various analytes, 1.520 nm: NTR (15 µg / ml) + NADH (200 µM); 650nm: f gly(vol %)= 96%,2.Blank, 3.NaCl (10 mM), 4.KCl(10 mM), 5.CaCl2(10 mM), 6.MgCl2(10 mM), 7.H2O2(10 mM), 8.HCIO (10 mM), 9.GSH (1 mM), 10.Cys (1 mM), 11.Hcy (1 mM), 12.Arg(1 mM), 13.DTT (1 mM), 14.V C (1 mM), 15. glucose (10 mM), 16. sucrose (10 mM), 17. AchE (1 mM), 18. NADH (1 mM); E) is the fluorescence spectrum of Nap-CefTTPy after incubation with NTR (10 µg / mL) for 2 hours in the absence or absence of the NTR inhibitor dicoumarol (200 µM); F) is the fluorescence spectrum of Nap-CefTTPy in response to NTR at different pH values; G) is the fluorescence spectrum of Nap-CefTTPy (10 μM) after incubation with different proteins (proteinase K, lipase, AchE, BSA and Bla).

[0034] Figure 4 The MTT assay is used to estimate the viability (%) of BV2 cells.

[0035] Figure 5 The images show a comparison of simultaneous imaging of bacteria and cells using Nap-CefTTPy in dual-channel fluorescence. A) Confocal fluorescence imaging of Staphylococcus aureus, GL261, and BV2 cells incubated with Nap-CefTTPy (10 µM) in PBS (10 mM, pH 7.4) at 37°C for 1 h. B) Relative fluorescence intensities of Staphylococcus aureus and mammalian cells (BV2 and GL261).

[0036] Figure 6 This is the result of Nap-CefTTPy generating reactive oxygen species (ROS) under illumination; A) Reaction mechanism of total ROS detection by DCFH. B) Fluorescence spectra of DCFH (10µM) and Nap-CefTTPy (25µM) after irradiation at 488nm for different times. C) Relationship between relative fluorescence intensity (I / I0) at 525 nm and irradiation time for the fluorescence spectra of DCFH (10µM) and Nap-CefTTPy (25µM). D) ABDA detection. 1Reaction mechanism of O2. E) Absorption spectra of ABDA (75 µM) and Nap-CefTTPy (25 µM) at different irradiation times. F) Plot of relative absorption intensity (A / A0) at 378 nm versus irradiation time for ABDA (75 µM) and Nap-CefTTPy (25 µM).

[0037] Figure 7 Bacterial imaging of Nap-CefTTPy in fluorescence dual channels. A) Confocal fluorescence imaging of bacterial cells (C. coccoides, S. aureus, E. faecalis, K. pneumoniae and P. aeruginosa) with Nap-CefTTPy (10 µM) in PBS (10 mM, pH 7.4) at 37 °C for 1 h; B) Relative fluorescence intensity of the five bacteria in two fluorescence channels of green and red.

[0038] Figure 8 Photodynamic bactericidal results of Nap-CefTTPy; A) Photographs of agar plates of bacteria treated with / without Nap-CefTTPy (50 µM) and white light. B) Statistics of the number of bacteria on agar plates under different treatments.

[0039] Figure 9 is the nuclear magnetic resonance hydrogen spectrum of compound 3.

[0040] Figure 10 is the nuclear magnetic resonance hydrogen spectrum of fluorescent probe Nap-CefTTPy. DETAILED DESCRIPTION

[0041] The present application is further illustrated in conjunction with the accompanying drawings and specific examples, but the embodiments of the present application are not limited thereto. For the process parameters not specifically mentioned, refer to the conventional techniques. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0042] Example 1: Synthesis of fluorescent probe Nap-CefTTPy

[0043] Step (1): 100 mg of compound 1 was dissolved in 6 mL of acetone, then 212 mg of sodium iodide was added, and the mixture was reacted at room temperature for 1 hour to obtain mixture a, which was concentrated under reduced pressure and diluted with 5 ml of water. The suspension was extracted with 25 ml of ethyl acetate, and the organic phase was washed with 10% sodium thiosulfate (5 ml), water (5 ml), and brine (5 ml), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain a slightly orange powder.

[0044] A mixture of the micro orange powder was dissolved in 0.4 mL DMF followed by the addition of 38.6 mg sodium bicarbonate to obtain mixture b, then 52 mg (0.12 mmol) of compound 2 was dissolved in 0.4 mL DMF to obtain mixture c, mixture b and mixture c were mixed to obtain mixture d; mixture d was stirred at room temperature for 2 hours. Then mixture d was diluted with water (10 mL) and extracted with ethyl acetate (25 mL). The organic phase was washed with brine (10 mL) and water (10 mL) and dried over anhydrous sodium sulfate. The solvent was removed and the residue was purified by flash chromatography on silica gel (dichloromethane:methanol = 20:1 v / v) to obtain 55 mg of compound 3. 1 H NMR (400MHz, CDCl3) δ 8.78 – 8.47 (m, 4H), 8.32 – 8.07 (m, 2H), 7.96 – 7.62 (m, 4H),7.52 – 7.22 (m, 17H), 7.18 - 6.94 (m, 10H), 6.78 (s, 1H), 6.62 (d, J = 15.32Hz, 1H), 5.62 (d, J = 4.3 Hz, 2H), 5.25 – 5.10 (m, 2H), 4.87 (s, 1H), 4.44(s, 2H), 2.92 (d, J = 29.4 Hz, 2H). HRMS calculated C 65 H 49 N6O8S2 + ([M]+) of 1105.3053; the corresponding value was found at 1105.3045.

[0045] Step (2): Compound 3 (55 mg, 0.05 mmol) was dissolved in 10 mL of anhydrous dichloromethane, then trifluoroacetic acid (600 µL) was slowly added under ice bath conditions, then the mixed solution continued to react under ice bath conditions for 1 h. After the reaction was monitored by TLC, the reaction solution was poured into ethyl ether (20 mL) to produce a wine red precipitate, which was filtered to obtain a wine red solid, then washed with ethyl ether (10 mL x 3), and finally 30 mg (65%) of compound Nap-CefTTPy was obtained. 1H NMR (400 MHz, DMSO) δ 9.03 (d, J = 7.20 Hz, 1H), 8.81 - 8.51 (m, 5H), 8.27 - 8.06 (m, 3H), 7.68 - 7.46 (m, 3H), 7.41 - 6.87 (m, 16H), 5.59 (d, J = 4.56 Hz, 1H), 5.35 (dd, J = 7.32, 3.76 Hz, 1H), 5.08 - 5.30 (m, 3H), 4.29 (td, J = 12.88, 5.68 Hz, 2H), 2.64 (t, J = 7.54 Hz, 2H); 13 C NMR (400 MHz, DMSO) δ 171.17, 168.72, 163.90, 163.33, 162.53, 153.69, 149.59, 148.04, 147.00, 144.31, 132.19, 130.72, 130.58, 130.23, 130.11, 129.93, 129.47, 128.89, 127.31, 127.16, 126.76, 125.26, 125.04, 124.73, 124.41, 123.65, 123.29, 123.21, 120.91, 114.32, 61.07, 55.77, 55.37, 53.28, 36.99, 33.15; HRMS calculated C 52 H 39 N6O8S2 + (M+H)+: 939.2271; found 939.2232.

[0046] Example 2: Optical performance test of fluorescent probe Nap-CefTTPy:

[0047] The optical properties of fluorescent probe Nap-CefTTPy were measured in PBS (10 mM, pH 7.4) at room temperature. Nap-CefTTPy was first prepared into a stock solution with a concentration of 10 mM using DMSO, and then the fraction of glycerol was increased to limit the molecular motion to different viscosities (0.1, 0.3, 0.5, 0.7, 0.9, 1.0). Figure 3A), which demonstrated the AIE property. Here, with the increase of viscosity (glycerol content from 0 to 99%), the fluorescence intensity of Nap-CefTTPy (10 µM) gradually increased by 33.4 folds, indicating that Nap-CefTTPy maintained the AIE property of triphenylaminothienopyridine salt. Subsequently, the AIE property of Nap-CefTTPy in different dimethyl sulfoxide (DMSO) and toluene mixtures was further tested. As shown in Fig. 1B, Nap-CefTTPy exhibited very weak fluorescence emission in pure DMSO. With the increase of toluene content in the mixed solvent to 80%, the fluorescence intensity gradually increased. Then, with the continuous increase of toluene content to 95%, the luminescence intensity of Nap-CefTTPy increased sharply and reached a maximum value. The luminescence peak ranged between 660 and 700 nm, which was about 5 times that of the pure DMSO solution, and had a typical AIE characteristic. The emission enhancement was due to the restriction of rotation motion after the formation of aggregates. Figure 3

[0048] Example 3: Response test of fluorescent probe Nap-CefTTPy to nitroreductase (NTR):

[0049] The response of Nap-CefTTPy to NTR was tested in PBS (10 mM, pH 7.4) at room temperature. Nap-CefTTPy was prepared into a solution with a concentration of 10 µM ([NADPH] = 200 µM) using PBS (10 mM, pH 7.4). After the addition of NTR and incubation at 45 ℃ for 30 min, the results are shown in Fig. 2C. Under 480 nm excitation, Nap-CefTTPy showed about 27-fold fluorescence enhancement at 20 nm, while it showed very low background fluorescence without NTR, which was due to its intramolecular photoinduced electron transfer (PeT) mechanism. This indicated that NTR successfully reduced the nitro group to an amino group, which turned off the original PET effect of naphthalimide. Figure 3

[0050] Example 4: Response test of fluorescent probe Nap-CefTTPy to β-lactamase (Bla):

[0051] Considering the cephalosporin structure in Nap-CefTTPy, the response of Nap-CefTTPy to Bla was tested in PBS (10 mM, pH 7.4) at room temperature. When Bla was added, a nearly 100-fold fluorescence enhancement was produced at 650 nm (Fig. 3A and Fig. 3B). Figure 3 Figure 3 ​​​B), which should be due to the interaction of the AIE moiety attached to the pyridine salt in Nap-CefTTPy structure with the enzyme cavity of Bla, limiting its own rotation to generate fluorescence. When NTR and Bla were added simultaneously, probe NB produced a dual-channel fluorescence response to both enzymes without crosstalk and mutual interference Figure 3 C) in FIG. 6. The above results show that Nap-CefTTPy can detect the coexistence of NTR and Bla in dual channels.

[0052] Example 5: Anti-interference ability test of fluorescent probe Nap-CefTTPy:

[0053] The selectivity of Nap-CefTTPy in the presence of various potential interference substances was tested, including inorganic salts (Na + , K + , Ca 2+ , Mg 2+ ), amino acids (Arg), various active oxygen / nitrogen / sulfur (H2O2, HOCl, GSH, Cys, Hcy), reducing agents (DTT), vitamins (Vc), carbohydrates (glucose and sucrose), and enzymes (acetylcholinesterase, NADH). As shown in D of FIG. 6, the response of Nap-CefTTPy to NTR was much stronger than that to other analytes, indicating that Nap-CefTTPy has high selectivity for NTR. Figure 3

[0054] The specificity of the response of Nap-CefTTPy to NTR was tested, and the response of Nap-CefTTPy to NTR was significantly decreased after the addition of Dihydroxycoumarin, an effective inhibitor of NTR activity, which showed competitive inhibition with NADH, and the results are shown in E of FIG. 6. Figure 3

[0055] The effect of pH on the fluorescence intensity of probe Nap-CefTTPy was tested, and the fluorescence of Nap-CefTTPy remained constant in phosphate buffer at pH 6.0 - pH 8.0 Figure 3 F of FIG. 6, indicating that the response of probe Nap-CefTTPy is not affected by pH.

[0056] The response specificity of Nap-CefTTPy to Bla was tested. Nap-CefTTPy remained silent in the presence of acetylcholinesterase, lipase, protease K, and bovine serum albumin, and only produced specific responses to NTR and Bla, indicating that the probe has good specificity for the two enzymes in vitro Figure 3 G of FIG. 6.

[0057] ​​Example 6: In vitro cell dark toxicity test of fluorescent probe Nap-CefTTPy;

[0058] The dark toxicity of Nap-CefTTPy in living BV2 cells was evaluated by standard CCK-8 assay. Figure 4 After incubating BV2 cells with different concentrations of Nap-CefTTPy for 24 h, standard CCK-8 assay was used to evaluate the cell viability. The results showed that the cell viability was still above 90% when the concentration of Nap-CefTTPy was 80 µM, indicating that the cytotoxicity of the probe could be ignored.

[0059] Example 7: Discrimination test of fluorescent probe Nap-CefTTPy for bacteria and mammalian cells:

[0060] BV2 cells and GL261 cells were selected for simultaneous imaging with S. aureus. After incubating 10 µM of Nap-CefTTPy with BV2 cells and GL261 cells with S. aureus at 37 ℃ for 30 min, confocal fluorescence imaging was performed in the green channel (λex= 488 nm; λem= 510 - 580 nm) and the red channel (λex= 488 nm; λem= 620- 800 nm). As shown in Fig. 7, BV2 and GL261 did not show fluorescence in the green and red fluorescence channels, while S. aureus showed fluorescence in both fluorescence channels, indicating that the probe selectively imaged bacteria and was not affected by NTRs in tumor cells. This may be due to the relatively large molecular weight of the probe, making it difficult for the probe to enter the cells. Figure 5

[0061] Example 8: ROS production ability test of fluorescent probe Nap-CefTTPy:

[0062] The ROS production ability of Nap-CefTTPy was investigated by ROS indicator agent. The fluorescence spectrum of dichlorofluorescein (DCFH, 10 uM) and the UV / visible spectrum of ABDA (9,10-anthracenediyl-bis(methylene)dipionic acid) (100 yM) were used to monitor the total ROS and singlet oxygen (1O2) production ability of Nap-CefTTPy (30 µM) under 488 nm (50 mW cm −2 ) irradiation.

[0063] The total content of various ROS production abilities of Nap-CefTTPy was evaluated by monitoring the enhanced fluorescence intensity of dichlorofluorescein (DCFH) at 525 nm, as shown in Fig. 8. The total ROS production ability of Nap-CefTTPy was 0.23 ± 0.01, and the singlet oxygen (1O2) production ability was 0.02 ± 0.01, indicating that the probe had good ROS production ability. Figure 6 −2 ​​) The increase of light irradiation time, in the presence of Nap-CefTTPy, the fluorescence intensity of DCFH rapidly increased, reaching 12-fold enhancement within 30 min, indicating that Nap-CefTTPy can efficiently generate ROS; and the absorption intensity of ABDA at around 419 nm gradually decreased under white light irradiation, while there was almost no obvious degradation in the absence of PSs, indicating that Nap-CefTTPy can effectively generate singlet oxygen (1O2) to consume ABDA. 1 O2) to consume ABDA.

[0064] Application of a dual-channel fluorescent probe for detecting bacterial infection:

[0065] Five strains of live bacteria containing gram-positive and gram-negative bacteria were selected and incubated with 10 µM of Nap-CefTTPy at 37 ℃ for 30 min, and then green channel confocal fluorescence imaging (λex = 488 nm; λem = 510 - 580 nm) and red channel (λex = 488 nm; λem = 620 - 800 nm) were performed. Figure 7 It was shown that after incubation with 10 µM of Nap-CefTTPy for 30 min, both gram-positive bacteria (E. coli and S. aureus) and gram-negative bacteria (B. subtilis, K. pneumoniae, and P. aeruginosa) could be turned on, indicating that Nap-CefTTPy could successfully attach or insert into the cell wall and plasma membrane of gram-positive and gram-negative bacteria, and was triggered by the structural constraint of the bacterial surface restriction molecule rotation. At the same time, it could also be further triggered by intracellular nitroreductase (NTR).

[0066] Gram-negative and gram-negative bacteria were used as representative pathogenic bacteria, including S. aureus, B. subtilis, K. pneumoniae, and P. aeruginosa, to study the in vitro bactericidal activity of Nap-CefTTPy. After mixing different bacterial solutions with Nap-CefTTPy (50 µM) and white light irradiation for 1 h, the bacterial solution was plated, then incubated at 37 ℃ in the dark for 12 h, and the bactericidal effect of Nap-CefTTPy on the four bacteria was evaluated by colony-forming unit (CFU) plating method. The results are shown in Figure 8 As shown, without Nap-CefTTPy treatment or only with Nap-CefTTPy dark treatment, the four bacteria grew vigorously and maintained similar viability, while the experimental group with 50 µM Nap-CefTTPy and white light irradiation produced obvious bactericidal effect, with a significant reduction in CFU number, and even complete bactericidal effect in the groups of S. aureus and P. aeruginosa, indicating that Nap-CefTTPy has good photodynamic bactericidal effect.

[0067] To sum up, the above is only the preferred embodiment of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A dual-channel fluorescent probe for detecting bacterial infection, characterized in that... The chemical structural formula of the fluorescent probe is: 。 2. The method for preparing a dual-channel fluorescent probe for detecting bacterial infection according to claim 1, characterized in that, The approach of this method is as follows: 。 3. The application of the dual-channel fluorescent probe for detecting bacterial infection according to claim 1 in the preparation of reagents for detecting bacterial infection, characterized in that: The fluorescent probe was incubated with the analyte for 30 minutes. Under 488nm laser irradiation, if the green and red fluorescent channels were not lit, it indicated that there were no bacteria in the analyte. If both the green and red fluorescent channels were lit, it indicated that there were bacteria in the analyte.

4. The application of the dual-channel fluorescent probe for detecting bacterial infection according to claim 1 in the preparation of photodynamic bactericides, characterized in that: The fluorescent probes were incubated with the bacterial-infected test samples and then subjected to photodynamic sterilization. The results showed that the fluorescent probes had a good photodynamic sterilization effect by the bacterial plating method.

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