Single-molecule fluorescent probe based on phenothiazine structure as well as preparation method and application of single-molecule fluorescent probe
By integrating multiple reaction sites with a single-molecule fluorescent probe based on the phenothiazine structure, the problem of existing fluorescent probes being unable to distinguish different analytes in complex systems is solved, and a highly sensitive fluorescent response to phosgene, copper ions and hypochlorite is achieved, making it suitable for environmental and biosensing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- PUTIAN UNIV
- Filing Date
- 2026-03-09
- Publication Date
- 2026-05-19
AI Technical Summary
Existing fluorescent probes typically rely on a single reaction mechanism, making it difficult to generate specific optical signals for different analytes in complex chemical systems. This is especially true when detecting trace amounts of phosgene and copper ions, where they face detection challenges. Furthermore, the accuracy of the signals is easily affected by competitive electronic effects and cross-reactivity.
By employing a single-molecule fluorescent probe based on a phenothiazine structure, multiple reaction sites are integrated into the same molecular backbone to achieve selective fluorescence response to phosgene, copper ions, and hypochlorite ions. Different chemical action mechanisms are used to generate significantly distinguishable fluorescence response signals, such as redshift effect, fluorescence quenching, and ratiometric spectral conversion.
This multi-purpose molecular probe achieves high sensitivity, rapid response kinetics, and excellent selective fluorescence visualization for phosgene, copper ions, and hypochlorite under near-physiological conditions. The signals do not overlap, making it suitable for complex analytical systems and expanding its applications in environmental and biosensing fields.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to single-molecule fluorescent probes based on phenothiazine structures, their preparation methods, and applications. Single-molecule fluorescent probes based on phenothiazine structures are molecular probes capable of generating specific optical signals based on reaction mechanisms for different analytes within the same molecular framework. Background Technology
[0002] Fluorescence sensing, due to its high sensitivity, non-invasiveness, and real-time spatial resolution for analyte detection, has become an indispensable tool in modern analytical chemistry, widely used in chemical analysis, environmental monitoring, food safety, and bioimaging. Despite significant progress, existing probes typically rely on a single dominant reaction mechanism, producing only one mode of optical response. This limits their performance in complex chemical systems and often lacks the flexibility required to handle diverse analytical scenarios or maintain stability under varying experimental conditions. Therefore, developing molecular probes capable of generating reaction-mechanism-based specific optical signals for different analytes within the same molecular framework remains a pressing technological goal. However, integrating multiple reactive groups into the same molecular backbone is inherently challenging, as competitive electronic effects, cross-reactivity, or signal crosstalk can easily impair the accuracy of the detection response. Solving this problem will significantly broaden the application scope of fluorescence molecular sensing and enhance its applicability for monitoring multiple analytes in complex chemical, environmental, and biological systems.
[0003] This challenge is particularly pronounced when a single probe is required to selectively respond to analytes with varying chemical reactivity. For example, phosgene (COCl2), a highly electrophilic acyl chloride widely used in industrial synthesis, is also a highly toxic, colorless gas that, through its action on the respiratory system, can cause irreversible alveolar damage, pulmonary edema, and respiratory failure, even at low exposure levels. Trace detection of phosgene is extremely challenging due to its readily hydrolytic properties under humid conditions, its nonspecific reactions with ubiquitous nucleophiles in the environment, and the difficulty in analyte enrichment at the sensing interface caused by its high volatility. In contrast, copper ions (CuCl2) offer a more effective response. 2+ As a transition metal cofactor essential for various enzymatic reactions and redox processes in organisms, abnormal accumulation of copper ions within cells can trigger cytotoxicity, leading to oxidative stress and cell damage. Achieving accurate detection of copper ions also faces significant challenges due to intense competition from other metal ions, complex coordination balances, and interference from endogenous ligands in the biological matrix. Summary of the Invention
[0004] To address the shortcomings of the existing technologies, this invention provides a single-molecule fluorescent probe based on a phenothiazine structure, its preparation method, and its applications. The single-molecule fluorescent probe based on a phenothiazine structure prepared by this invention is compatible with orthogonal chemical reactivity and maintains controllable electron transfer to convert different chemical events into specific optical signals.
[0005] Based on the above technical objectives, the present invention adopts the following technical solution: This invention protects a method for preparing a single-molecule fluorescent probe based on a phenothiazine structure, comprising the following steps: by and C1~C 12 Using haloalkanes as raw materials, an N-alkylation reaction is carried out under an alkaline environment to achieve... By introducing an alkyl chain onto the nitrogen atom, we obtain... , where n is 0~19.
[0006] by Using phosphorus oxychloride and N,N-dimethylformamide as raw materials, a Vilsmeier-Haack reaction was carried out under the conditions of phosphorus oxychloride and N,N-dimethylformamide, with phosphorus oxychloride and N,N-dimethylformamide being the reactants, to achieve the desired effect. Regioselective formylation yields aldehyde intermediates. .
[0007] Will O-demethylation was achieved by treatment with aluminum trichloride to obtain the salicylaldehyde intermediate. .
[0008] Will An intramolecular cyclization reaction was carried out with 1H-benzimidazole-2-acetonitrile under piperidine catalysis. 1H-benzimidazole-2-acetonitrile provides a molecular recognition site for analyte detection, yielding… This refers to a single-molecule fluorescent probe based on the phenothiazine structure.
[0009] Preferred, With C1~C 12 The molar ratio of the haloalkanes is 1:1 to 5.
[0010] Preferably, the N-alkylation reaction is carried out at 60°C to 90°C for 6 to 16 hours.
[0011] Preferably, the Vilsmeier-Haack reaction conditions are: reaction at 0℃~70℃ for 3h~6h.
[0012] Preferably, AlCl3 and The molar ratio is 2~20:1.
[0013] Preferably, the conditions for O-demethylation are: stirring at room temperature for 12-24 hours.
[0014] Preferably, 1H-benzimidazole-2-acetonitrile and The molar ratio is 1~3:1.
[0015] Preferably, the conditions for the intramolecular cyclization reaction are: stirring at room temperature for 12-24 hours.
[0016] This invention also protects a single-molecule fluorescent probe based on a phenothiazine structure, which is prepared using the above-described method.
[0017] This invention also protects the use of single-molecule fluorescent probes based on phenothiazine structures in the preparation of phosgene detectors, copper ion detectors, or hypochlorite ion detectors.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a fluorescent probe PTBI based on a phenothiazine-iminocoumarin structure, which integrates multiple reaction sites into a single molecular platform, achieving the detection of phosgene and copper ions (Cu). 2+ The selective and mechanism-specific fluorescence responses of phosgene and hypochlorite ions are based on different chemical interactions—electrophilic acylation (phosgene), metal coordination (Cu) and... 2+ Through oxidation and transformation (hypochlorite), the fluorescent probe can produce significantly distinct fluorescence response signals, including a marked redshift effect, fluorescence quenching, and ratiometric spectral conversion. PTBI exhibits high sensitivity, rapid response kinetics, and excellent selectivity for all the target analytes under near-physiological conditions.
[0019] 2. In addition to solution systems, this fluorescent probe can also achieve effective fluorescence visualization of copper ions and hypochlorite ions in plant tissues, living cells, and zebrafish, and exhibits good membrane permeability, signal contrast, and stability in various biological models. Of particular note is the consistency of fluorescence response behavior observed from in vitro to in vivo systems, confirming the reliability of the employed molecular design strategy. This demonstrates a rational design strategy for distinguishing analytes with different chemical properties on a single molecular framework through orthogonal reaction pathways. This not only expands the application of metal ions and reactive oxygen species in environmental and biosensing fields but also provides design principles for the future development of multi-purpose molecular probes suitable for complex analytical systems.
[0020] 3. Considering that competitive electronic effects, cross-reactivity, or signal crosstalk can easily impair the accuracy of the detection response, this invention integrates multiple reactive groups into the same molecular framework. Using phenothiazine as the fluorescent group, this invention constructs a multifunctional molecular probe PTBI. This probe exhibits orange emission at 605 nm, and upon interaction with analytes of different properties, it produces optically distinct optical responses: a nucleophilic substitution reaction with phosgene causes the emission peak to redshift to 675 nm; and a reaction with Cu... 2+ Coordination leads to efficient fluorescence quenching; selective oxidation with hypochlorous acid causes the emission peak to shift to 495 nm, exhibiting green fluorescence. These highly spectrally separated signals originate from different chemical transformation pathways within a single molecular platform, thus clearly distinguishing electrophiles, metal ions, and oxidants, with no signal overlap. Attached Figure Description
[0021] The PTBI in the attached figures are all samples from Example 1.
[0022] Figure 1 This is a PTBI synthesis path diagram.
[0023] Figure 2 This is a diagram illustrating the detection mechanism of triphosgene by PTBI.
[0024] Figure 3 This is a diagram illustrating the detection mechanism of copper ions by PTBI.
[0025] Figure 4 This is a diagram illustrating the detection mechanism of hypochlorous acid by PTBI.
[0026] Figure 5 The image shows the UV-Vis absorption spectra before and after the reaction of PTBI with triphosgene.
[0027] Figure 6 The graph shows the fluorescence titration curves of PTBI on triphosgene of different concentrations.
[0028] Figure 7 The graph shows the relationship between the fluorescence intensity ratio and the triphosgene concentration.
[0029] Figure 8 This is a time-dependent fluorescence response curve of PTBI to triphosgene.
[0030] Figure 9 This is a graph showing the fluorescence lifetime decay of PTBI before and after its reaction with triphosgene.
[0031] Figure 10 The fluorescence spectra of PTBI solutions after the addition of different analytes are shown.
[0032] Figure 11 This is a graph showing the fluorescence response of PTBI to triphosgene in the presence of different coexisting analytes.
[0033] Figure 12 The image shows the fluorescence response of the PTBI-loaded test strip to triphosgene.
[0034] Figure 13 This is a graph showing the red / green (R / G) ratio extracted from the fluorescence image of the test strip.
[0035] Figure 14 For PTBI, add Cu 2+ UV-Vis absorption spectra before and after.
[0036] Figure 15 PTBI for different concentrations of Cu 2+ The fluorescence titration curve.
[0037] Figure 16 The fluorescence intensity of PTBI at 605 nm and Cu 2+ Linear relationship between concentrations.
[0038] Figure 17 This is a graph showing the fluorescence response of PTBI to various analytes.
[0039] Figure 18 For PTBI in the presence of different coexisting analytes, the analysis of Cu 2+ The fluorescence response diagram.
[0040] Figure 19 For PTBI in Cu 2+ Fluorescence lifetime decay curves before and after the reaction.
[0041] Figure 20 Adding ClO to PTBI - UV-Vis absorption spectra before and after.
[0042] Figure 21 For PTBI to ClO - The fluorescence titration spectrum.
[0043] Figure 22 The fluorescence intensity ratio of PTBI at 495 nm and 605 nm compared to ClO - Linear relationship graph of concentration.
[0044] Figure 23 This is a graph showing the change in the fluorescence intensity ratio of PTBI as a function of solution pH.
[0045] Figure 24 For PTBI, different ClO - Concentration fluorescence intensity versus time curve.
[0046] Figure 25For PTBI and ClO - Fluorescence lifetime decay curves before and after the reaction. Figure 26 This is a graph showing the changes in fluorescence signal ratio of PTBI in the presence of multiple analytes.
[0047] Figure 27 This is a fluorescence image of Arabidopsis thaliana with PTBI. Detailed Implementation
[0048] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention is further described below with reference to specific embodiments. However, the embodiments are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.
[0049] The target probe PTBI was synthesized from 2-methoxy-10H-phenthiazine (S1, purchased) via a four-step reaction. First, S1 underwent N-alkylation to introduce an alkyl chain (or other alkane chain) onto the nitrogen atom to increase solubility and improve stability, yielding intermediate S2. Subsequently, S2 underwent regioselective formylation via the Vilsmeier-Haack reaction to generate the aldehyde intermediate S3. S3 was then treated with aluminum trichloride (a Lewis acid) to achieve O-demethylation, yielding the corresponding salicylaldehyde intermediate S4. Finally, S4 reacted with 1H-benzimidazole-2-acetonitrile in ethanol under piperidine catalysis, resulting in condensation and intramolecular cyclization, yielding probe PTBI in good overall yield. The purpose of S-3 through S-5 was to introduce an iminocoumarin structure; detailed preparation procedures are provided in the examples.
[0050] Example 1 The preparation method of single-molecule fluorescent probes based on phenothiazine structure includes the following steps: Synthesis of compound S-2: Compound S-1 (5.0 g, 21.8 mmol) and potassium tert-butoxide (3.7 g, 33.0 mmol) were dissolved together in N,N-dimethylformamide (40 mL), stirred, and heated under reflux for 2 h. Then, iodoethane (10.2 g, 65.4 mmol) was added to the reaction system, and the reaction was continued at 65 °C with stirring for 10 h. After the reaction was complete, the reaction solution was poured into water and stirred continuously. After standing, a solid precipitated, which was filtered and dried to obtain the crude product. Further purification was performed by silica gel column chromatography using petroleum ether / ethyl acetate as eluent to obtain compound S-2, a pale pink solid (3.6 g, yield 64%). Its 1H NMR spectrum (…) 1The ¹H NMR (CDCl₃) data are as follows: δ = 7.16 (t, J = 8.0 Hz, 1H), 7.04 (d, J = 8.8 Hz, 1H), 6.92 (t, J = 7.6 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.51–6.49 (m, 2H), 3.93 (q, J = 7.2 Hz, 2H), 3.81 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H).
[0051] Synthesis of compound S-3: Phosphorus oxychloride (0.72 mL, 8.6 mmol) was placed in an ice bath at 0 °C, and N,N-dimethylformamide (2.0 mL) was slowly added dropwise. The mixture was stirred for 15 min to obtain a pre-cooled reagent. Subsequently, compound S-2 (1.9 g, 7.2 mmol, dissolved in 5 mL of anhydrous DMF) was added dropwise to the pre-cooled reagent, and stirring continued. The reaction system was then heated to 60 °C and stirred for 4 h. After the reaction was completed, the reaction solution was poured into ice water (100 mL), and the resulting clear solution was neutralized to neutral with 10% sodium bicarbonate solution. The solution was extracted with dichloromethane (3 × 50 mL), and the organic phases were combined. The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain compound S-3 as an orange powder (1.7 g, yield 82%). Its 1H NMR spectrum (… 1 The ¹H NMR (CDCl₃) data are as follows: δ = 10.23 (s, 1H), 7.56 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 6.98 (t, J = 7.2 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.41 (s, 1H), 4.02 (q, J = 6.8 Hz, 2H), 3.95 (s, 3H), 1.50 (t, J = 6.8 Hz, 3H). The electrospray mass spectrometry (ESI-MS) data are as follows: m / z [M⁺ H]⁺ calculated value: 286.0902; measured value: 286.0903.
[0052] Synthesis of compound S-4: Under nitrogen protection, AlCl3 (1.38 g, 10 mmol) was added to a double-necked round-bottom flask and thoroughly suspended in anhydrous CH2Cl2 (20 mL) to obtain a mixture. Compound S-3 (1.00 g, 3.5 mmol) was dissolved in anhydrous CH2Cl2 (5 mL) and added dropwise to the stirred mixture at room temperature. The reaction system was stirred for 20 h under nitrogen protection. After the reaction was completed, the mixture was slowly poured into ice water (50 mL), and then dilute hydrochloric acid (1 M HCl, 20 mL) was added to quench the excess AlCl3. The mixture was stirred for another 1 h, during which a large amount of solid precipitated. The solid was collected by vacuum filtration, thoroughly washed with water, and dried under reduced pressure to obtain 0.6 g of the target product, an orange solid, with a yield of 90%. ¹H NMR (400 MHz, CDCl3) δ 11.39(s, 1H), 9.60 (s, 1H), 7.21–7.11 (m, 2H), 7.10 (dd, J = 7.6, 1.6 Hz, 1H), 6.97 (td, J = 7.5, 1.2 Hz, 1H), 6.92 (dd, J = 8.8, 1.6 Hz, 1H), 6.39 (s, 1H), 3.95 (q, J = 7.2 Hz, 2H), 1.45 (t, J = 7.6 Hz, 3H).
[0053] Synthesis of a phenothiazine-based single-molecule fluorescent probe (PTBI): Under nitrogen protection, compound S-4 (100 mg, 0.37 mmol) and 1H-benzimidazole-2-acetonitrile (58 mg, 0.37 mmol) were dissolved together in anhydrous ethanol (10 mL), followed by the addition of piperidine (32 mg, 0.37 mmol). The reaction mixture was stirred at room temperature for 24 h, during which an orange-red solid precipitate gradually formed. After the reaction was complete, the solid was collected by filtration and washed with cold ethanol (3 × 5 mL). The crude product was purified by recrystallization from a methanol / dichloromethane mixed solvent to obtain PTBI as an orange-red solid (138 mg, yield 91%). Its 1H NMR spectrum (…) 1The ¹H NMR (CDCl₃) data are as follows: δ = 12.61 (s, 1H), 8.57 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.61 (s, 1H), 7.53 (d, J = 6.0 Hz, 1H), 7.31–7.29 (m, 2H), 7.20 (t, J = 7.6 Hz, 1H), 7.18 (s, 1H), 7.15 (d, J = 7.2 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 3.99 (q, J = 6.8 Hz, 2H), 1.50 (t, J = 6.8 Hz, 3H). Its carbon nuclear magnetic resonance spectrum ( 13 The C NMR (DMSO-d6) data are as follows: δ = 149.16, 148.89, 148.07, 147.95, 142.96, 138.42, 133.42, 130.10, 128.38, 127.58, 124.90, 123.64, 122.53, 122.46, 117.86, 116.58, 114.54, 113.16, 112.37, 102.94, 102.59, 42.31, 12.83.
[0054] Its electrospray high-resolution mass spectrometry (ESI-HRMS) data are as follows: m / z Calculated value C 24 H 19 N4OS [M + H]+:411.1280; Measured value: 411.1278.
[0055] The fluorescent probe PTBI can be obtained using the above method, and the compound can be characterized by nuclear magnetic resonance and mass spectrometry. The prepared fluorescent probe PTBI is studied below, and the research methods and results are shown in the figure. 1. Example of triphosgene detection: Add triphosgene of different concentrations to a 10 μM, PTBI dichloromethane solution (probe solution) to gradually increase the triphosgene concentration in the system. After mixing, gently shake and react directly at room temperature for 3 min to 10 min without additional catalyst or heating. After the reaction is complete, measure its UV-Vis absorption spectrum or fluorescence emission spectrum.
[0056] Ultraviolet-visible absorption spectroscopy determination: Using a UV-visible spectrophotometer, with a blank sample as a reference, the absorption spectrum of the above-mentioned sample was measured, and its maximum absorption peak was located at 450 nm. The detection mechanism of triphosgene is as follows: Figure 2 As shown.
[0057] Figure 5 The images show the UV-Vis absorption spectra before and after the reaction of PTBI with triphosgene. The results indicate that the maximum absorption peak in the UV-Vis absorption spectrum increases from 450 nm to 525 nm after the addition of triphosgene.
[0058] Figure 6 The figure shows the fluorescence titration curves of PTBI with different concentrations of triphosgene. The results show that with the gradual addition of triphosgene, the emission of PTBI gradually shifts from 600 nm to the near-infrared region of 675 nm, and the fluorescence gradually quenches.
[0059] Figure 7 This is a graph showing the relationship between the fluorescence intensity ratio and the triphosgene concentration. The results indicate that the intensity ratio (I... 675 / I 600 The relationship between phosgene concentration and phosgene concentration is linear.
[0060] Figure 8 The graph shows the time-dependent fluorescence response of PTBI to triphosgene. The results indicate that, over time, the fluorescence response plateaus within 5 minutes after the addition of triphosgene, demonstrating a rapid reaction.
[0061] Figure 9 The graph shows the fluorescence lifetime decay curves of PTBI before and after the reaction with triphosgene. The results indicate that the fluorescence lifetime is significantly shortened after the reaction with triphosgene.
[0062] Figure 10 The images show the fluorescence spectra of PTBI solutions after the addition of different analytes. The results indicate that these potential interfering analytes cause minor changes in the fluorescence spectrum, while phosgene causes a significant redshift accompanied by emission attenuation.
[0063] Figure 11 The images show the fluorescence spectra of PTBI solutions after the addition of different analytes. Competition experiments further confirmed that the presence of coexisting species does not interfere with phosgene detection.
[0064] Figure 12 This is a visualization of the fluorescence response of the PTBI-loaded test strip to triphosgene. The results show that the PTBI-loaded filter paper exhibits distinct and easily distinguishable fluorescence colors, and the filter paper gradually changes from yellow to red as the triphosgene vapor concentration increases.
[0065] Figure 13 This is a graph showing the red / green (R / G) ratio extracted from the fluorescence image of the test strip. The R / G ratio gradually increases with increasing triphosgene concentration.
[0066] 2. Example of copper ion detection: 100 μL of Tween 20 and 54.8 μL of probe solution were added together to a glass bottle containing 1000 μL of LMF, followed by 9200 μL of PBS buffer solution. The mixture was shaken thoroughly to obtain the probe stock solution. Preparation of reference and test samples: (1) Blank control sample: 300 μL of LMF was placed in a quartz cuvette, and 2700 μL of PBS buffer solution was added. The mixture was stirred thoroughly. (2) Sample 1 (background control): 3000 μL of probe stock solution was placed in a quartz cuvette without adding any other substances. (3) Sample 2 (copper ion test sample): 3000 μL of probe stock solution was placed in another quartz cuvette, and 50 μL of 1 mM copper ion solution was added. The mixture was stirred thoroughly.
[0067] Ultraviolet-Visible Absorption Spectroscopy Measurement: Using a UV-Vis spectrophotometer, with a blank sample as a reference, the absorption spectrum of the copper-free sample 1 was measured, with its maximum absorption peak located at 450 nm; sample 2 was then placed in the sample, and the absorption spectrum of the copper-containing sample 2 was measured, with its maximum absorption peak located at 500 nm. The detection mechanism for copper ions is as follows: Figure 3 As shown.
[0068] Figure 14 For PTBI, add Cu 2+ The UV-Vis absorption spectra before and after the addition of Cu. The results show that the addition of Cu... 2+ Subsequently, the main absorption band shifted from 450 nm to 500 nm, and the solution color changed from yellow to brown.
[0069] Figure 15 PTBI for different concentrations of Cu 2+ The fluorescence titration curves are shown. The results indicate that as Cu... 2+ As the concentration gradually increases, the fluorescence at 605 nm is gradually quenched.
[0070] Figure 16 The fluorescence intensity of PTBI at 605 nm and Cu 2+ Linear relationship between concentration. Quantitative analysis shows that fluorescence intensity is related to Cu. 2+ There is an excellent linear relationship between concentrations.
[0071] Figure 17 This is a graph showing the fluorescence response of PTBI to various analytes. The results indicate that only Cu... 2+ It causes significant fluorescence quenching, while the changes caused by other analytes are negligible.
[0072] Figure 18 For PTBI in the presence of different coexisting analytes, the analysis of Cu 2+ The fluorescence response diagram shows that the addition of Cu in the presence of competing ions...2+ Afterwards, PTBI was applied to Cu. 2+ It can still maintain a stable response.
[0073] Figure 19 For PTBI and Cu 2+ Fluorescence lifetime decay curves before and after the reaction. The results show that the addition of Cu... 2+ After the reaction, the time-resolved fluorescence decay curve remained essentially unchanged.
[0074] 3. Example of hypochlorous acid detection: 100 μL of Tween 20 and 54.8 μL of probe solution were added together to a glass bottle containing 1000 μL of LDMF, followed by 9200 μL of PBS buffer solution. The mixture was shaken thoroughly to obtain the probe stock solution. Preparation of reference and test samples: (1) Blank control sample: 300 μL of LDMF was placed in a quartz cuvette, and 2700 μL of PBS buffer solution was added. The mixture was stirred thoroughly. (2) Sample 1 (background control): 3000 μL of probe stock solution was placed in a quartz cuvette without adding any other substances. (3) Sample 2 (hypochlorous acid test sample): 3000 μL of probe stock solution was placed in another quartz cuvette, and 30 μL of 1 mM hypochlorous acid solution was added. The mixture was stirred thoroughly.
[0075] Ultraviolet-Visible Absorption Spectroscopy: Using a UV-Vis spectrophotometer, with a blank sample as a reference, the absorption spectrum of the above-mentioned sample 1 without hypochlorite ions was measured, and its maximum absorption peak was located at 450 nm; sample 2 was then placed in the sample, and the absorption spectrum of the above-mentioned sample 2 containing hypochlorite was measured, and its maximum absorption peak was located at 420 nm. The detection mechanism for hypochlorite ions is as follows: Figure 4 As shown.
[0076] Figure 20 Adding ClO to PTBI - The UV-Vis absorption spectra before and after are shown. The results indicate that the main absorption peak of PTBI shifts from 450 nm to 420 nm (blue shift), and the visible color changes from yellow to pale green.
[0077] Figure 21 For PTBI to ClO - The fluorescence titration spectrum was obtained. The results showed that the original emission at 605 nm gradually decreased with increasing hypochlorite concentration, while a new emission band centered at 495 nm appeared and was enhanced.
[0078] Figure 22 The fluorescence intensity ratio of PTBI at 495 nm and 605 nm compared to ClO - Linearity graph of concentration. The results show that the proportional calibration graph of NaClO concentration exhibits excellent linearity in the range of 2 μM to 12 μM.
[0079] Figure 23 This is a graph showing the fluorescence intensity ratio of PTBI as a function of solution pH. The proportional response of PTBI was measured over a wide pH range, and the results indicate that the strongest response was observed at neutral pH.
[0080] Figure 24 For PTBI, different ClO - The fluorescence intensity curves after concentration change over time are shown. The results indicate that NaClO reacts rapidly within 5 seconds at different concentrations.
[0081] Figure 25 For PTBI and ClO - Fluorescence lifetime decay curves before and after the reaction. The results show that the addition of ClO... - After the reaction, the time-resolved fluorescence decay curve remained essentially unchanged.
[0082] Figure 26 This is a graph showing the relative fluorescence signal changes of PTBI in the presence of multiple analytes. Adding NaClO to a solution containing other substances resulted in a change in its proportional fluorescence signal (IL). 495 / I605) The enhancement is comparable to that observed in NaClO alone.
[0083] 4. Plant Imaging Test: Arabidopsis thaliana seeds (Columbia-0 ecotype) were sterilized under aseptic conditions with 10% sodium hypochlorite solution and then thoroughly rinsed several times with sterile distilled water. The surface-sterilized seeds were inoculated onto solid plates containing half-strength Murashige and Skoog (MS) medium (containing MS basal salts, vitamins, and sucrose, prepared with distilled water, solidified on agar, and autoclaved before being poured into sterile Petri dishes). The Petri dishes were sealed and placed in the dark for vernalization for 2 days to promote uniform seed germination. The Petri dishes were then transferred to a growth chamber for cultivation under controlled conditions of 22°C and a 16-hour light / 8-hour dark photoperiod. Seedlings were used for subsequent imaging experiments after 7 days of vertical growth.
[0084] Take 1000 μL of DMF into a glass bottle, add 200 μL of Tween 20 and 54.8 μL of FSQ-5 probe, then add 9200 μL of PBS buffer solution, shake thoroughly to mix, and obtain the probe stock solution.
[0085] Fluorescence imaging experiment of copper ions in Arabidopsis thaliana: The prepared probe stock solution was divided into four equal portions, 1 mL each, and labeled as Sample 1 to Sample 4. 0 μL, 2 μL, 4 μL, and 8 μL of copper ion solution were added to Samples 1 to 4, respectively. After mixing, the samples were immersed in the roots of Arabidopsis thaliana plants (healthy seven-day-old seedlings were used for exogenous analyte detection) and incubated for 30 minutes. Fluorescence images were acquired using a fluorescence microscope. First, a blank image of untreated Arabidopsis thaliana was captured, showing no fluorescence signal. Subsequently, the incubated samples were observed and photographed sequentially using bright-field and red-channel imaging (excitation at 488 nm, emission at 570 nm–630 nm).
[0086] Figure 27 This is a fluorescence imaging image of Arabidopsis thaliana. The results show that PTBI-loaded roots in Cu-free... 2+ Under these conditions, it exhibits a strong fluorescence signal in the red channel, with Cu 2+ The concentration gradually decreased with increasing concentration, while the corresponding bright-field image showed no obvious morphological changes. Next, hypochlorous acid imaging of Arabidopsis thaliana was performed. The remaining solution prepared above was divided into four equal parts, each 1 mL, labeled 1, 2, 3, and 4. Then, 0 μL, 4 μL, 8 μL, and 12 μL of hypochlorous acid were added sequentially, and Arabidopsis thaliana was incubated for about 2 hours. Using a fluorescence microscope, a blank image of Arabidopsis thaliana without any added substances was first taken, which was dark and without fluorescence. Next, the incubated Arabidopsis thaliana was observed and photographed sequentially in bright-field, red channel, and green channel. When exposed to increasing concentrations of ClO⁻ (0 μM~24 μM), the fluorescence signal in the red channel gradually weakened, accompanied by fluorescence in the green channel. The combined image clearly showed the transition between red and green fluorescence.
[0087] The imaging steps for copper ions and hypochlorous acid in rice are the same as described above.
[0088] Example 2 The preparation method of single-molecule fluorescent probes based on phenothiazine structure includes the following steps: Synthesis of compound S-2: Compound S-1 (5.0 g, 21.8 mmol) and potassium tert-butoxide (3.7 g, 33.0 mmol) were dissolved together in N,N-dimethylformamide (40 mL), stirred and heated under reflux for 2 h. Then, iodoethane (3.4 g, 21.8 mmol) was added to the reaction system, and the reaction was continued to be stirred at 90 °C for 6 h. After the reaction was completed, the reaction solution was poured into water and stirred continuously. After standing, a solid precipitated out. The crude product was obtained by filtration and drying, and then further purified by silica gel column chromatography with petroleum ether / ethyl acetate as eluent to obtain compound S-2, which was a light pink solid.
[0089] Synthesis of compound S-3: Phosphorus oxychloride (0.72 mL, 8.6 mmol) was placed in an ice bath at 0 °C, and N,N-dimethylformamide (2.0 mL) was slowly added dropwise. The mixture was stirred for 15 min to obtain a pre-cooled reagent. Subsequently, compound S-2 (1.9 g, 7.2 mmol, dissolved in 5 mL of anhydrous DMF) was added dropwise to the pre-cooled reagent, and the mixture was stirred for 6 h. After the reaction was completed, the reaction solution was poured into ice water (100 mL), and the resulting clear solution was neutralized to neutral with 10% sodium bicarbonate solution. The solution was extracted with dichloromethane (3 × 50 mL), and the organic phases were combined. The solution was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain compound S-3 as an orange powder.
[0090] Synthesis of compound S-4: Under nitrogen protection, AlCl3 (1.0 g, 7 mmol) was added to a double-necked round-bottom flask and suspended in anhydrous CH2Cl2 (20 mL) to obtain a mixture. Compound S-3 (1.00 g, 3.5 mmol) was dissolved in anhydrous CH2Cl2 (5 mL) and added dropwise to the stirred mixture at room temperature. The reaction system was stirred for 12 h under nitrogen protection. After the reaction was completed, the mixture was slowly poured into ice water (50 mL), and then dilute hydrochloric acid (1 M HCl, 20 mL) was added to quench the excess AlCl3. The mixture was stirred for another 1 h, during which a large amount of solid precipitated. The solid was collected by vacuum filtration, washed thoroughly with water, and dried under reduced pressure to obtain the target product, an orange solid.
[0091] Synthesis of a phenothiazine-based single-molecule fluorescent probe (PTBI): Under nitrogen protection, compound S-4 (100 mg, 0.37 mmol) and 1H-benzimidazole-2-acetonitrile (116 mg, 0.74 mmol) were dissolved together in anhydrous ethanol (10 mL), followed by the addition of piperidine (32 mg, 0.37 mmol). The reaction mixture was stirred at room temperature for 18 h, during which an orange-red solid precipitate gradually formed. After the reaction was complete, the solid was collected by filtration and washed with cold ethanol (3 × 5 mL). The crude product was purified by recrystallization from a methanol / dichloromethane mixed solvent to obtain PTBI, which was an orange-red solid.
[0092] Example 3 The preparation method of single-molecule fluorescent probes based on phenothiazine structure includes the following steps: Synthesis of compound S-2: Compound S-1 (5.0 g, 21.8 mmol) and potassium tert-butoxide (3.7 g, 33.0 mmol) were dissolved together in N,N-dimethylformamide (40 mL), stirred and heated under reflux for 2 h. Then, iodoethane (17 g, 109 mmol) was added to the reaction system, and the reaction was continued to be stirred at 60 °C for 16 h. After the reaction was completed, the reaction solution was poured into water and stirred continuously. After standing, a solid precipitated out. The crude product was obtained by filtration and drying, and then further purified by silica gel column chromatography with petroleum ether / ethyl acetate as eluent to obtain compound S-2, which was a light pink solid.
[0093] Synthesis of compound S-3: Phosphorus oxychloride (0.72 mL, 8.6 mmol) was placed in an ice bath at 0 °C, and N,N-dimethylformamide (2.0 mL) was slowly added dropwise while stirring for 15 min to obtain a pre-cooled reagent. Subsequently, compound S-2 (1.9 g, 7.2 mmol, dissolved in 5 mL of anhydrous DMF) was added dropwise to the pre-cooled reagent while stirring continued. The reaction system was then heated to 70 °C and stirred for 3 h. After the reaction was completed, the reaction solution was poured into ice water (100 mL), and the resulting clear solution was neutralized to neutral with 10% sodium bicarbonate solution. The solution was extracted with dichloromethane (3 × 50 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether / ethyl acetate as the eluent to obtain compound S-3 as an orange powder.
[0094] Synthesis of compound S-4: Under nitrogen protection, AlCl3 (10.0 g, 70 mmol) was added to a double-necked round-bottom flask and thoroughly suspended in anhydrous CH2Cl2 (20 mL) to obtain a mixture. Compound S-3 (1.00 g, 3.5 mmol) was dissolved in anhydrous CH2Cl2 (5 mL) and added dropwise to the stirred mixture at room temperature. The reaction system was stirred for 24 h under nitrogen protection. After the reaction was completed, the mixture was slowly poured into ice water (50 mL), and then dilute hydrochloric acid (1 M HCl, 20 mL) was added to quench the excess AlCl3. The mixture was stirred for another 1 h, during which a large amount of solid precipitated. The solid was collected by suction filtration, thoroughly washed with water, and dried under reduced pressure to obtain the target product, an orange solid.
[0095] Synthesis of a phenothiazine-based single-molecule fluorescent probe (PTBI): Under nitrogen protection, compound S-4 (100 mg, 0.37 mmol) and 1H-benzimidazole-2-acetonitrile (174 mg, 0.111 mmol) were dissolved together in anhydrous ethanol (10 mL), followed by the addition of piperidine (32 mg, 0.37 mmol). The reaction mixture was stirred at room temperature for 12 h, during which an orange-red solid precipitate gradually formed. After the reaction was complete, the solid was collected by filtration and washed with cold ethanol (3 × 5 mL). The crude product was purified by recrystallization from a methanol / dichloromethane mixed solvent to obtain PTBI, which was an orange-red solid.
[0096] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A method for preparing a single-molecule fluorescent probe based on a phenothiazine structure, characterized in that, Includes the following steps: by and C1~C 12 Using haloalkanes as raw materials, an N-alkylation reaction is carried out under an alkaline environment to achieve... By introducing an alkyl chain onto the nitrogen atom, we obtain... Where n is 0~19; by Using phosphorus oxychloride and N,N-dimethylformamide as raw materials, a Vilsmeier-Haack reaction was carried out to achieve the desired effect. Regioselective formylation yields aldehyde intermediates. ; Will O-demethylation was achieved by treatment with aluminum trichloride to obtain the salicylaldehyde intermediate. ; Will Intramolecular cyclization reaction with 1H-benzimidazole-2-acetonitrile under piperidine catalysis yields This refers to a single-molecule fluorescent probe based on the phenothiazine structure.
2. The method for preparing a single-molecule fluorescent probe based on a phenothiazine structure according to claim 1, characterized in that, With C1~C 12 The molar ratio of the haloalkanes is 1:1 to 5.
3. The method for preparing a single-molecule fluorescent probe based on a phenothiazine structure according to claim 1, characterized in that, The conditions for the N-alkylation reaction are: reaction at 60℃~90℃ for 6h~16h.
4. The method for preparing a single-molecule fluorescent probe based on a phenothiazine structure according to claim 1, characterized in that, The conditions for the Vilsmeier-Haack reaction are: reaction at 0℃~70℃ for 3h~6h.
5. The method for preparing a single-molecule fluorescent probe based on a phenothiazine structure according to claim 1, characterized in that, The conditions for O-demethylation are: stirring at room temperature for 12-24 hours.
6. The method for preparing a single-molecule fluorescent probe based on a phenothiazine structure according to claim 1, characterized in that, The conditions for the intramolecular cyclization reaction are: stirring at room temperature for 12-24 hours.
7. A single-molecule fluorescent probe based on a phenothiazine structure, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 6.
8. The application of the single-molecule fluorescent probe based on the phenothiazine structure as described in claim 7 in the preparation of phosgene detectors.
9. The application of the single-molecule fluorescent probe based on the phenothiazine structure as described in claim 7 in the preparation of a copper ion detection agent.
10. The application of the single-molecule fluorescent probe based on the phenothiazine structure as described in claim 7 in the preparation of hypochlorite ion detection reagent.