A single-excitation visible and near-infrared dual-emission small molecule fluorescent probe and a preparation method and application thereof

Abstract

The application provides a single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe and a preparation method and application thereof, and belongs to the field of organic fluorescent molecular probes. In the application, a single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe coupled by stilbene and a macrocyclic polyamine group is prepared, wherein R is selected from any one of the groups. The fluorescent probe provided in the application has low preparation cost and good solubility, under single-wavelength excitation, the fluorescent emission spectrum of the fluorescent probe contains two emission peaks, the maximum emission wavelengths of the two emission peaks are in a visible region of about 380 nm and a near-infrared region of about 730 nm respectively, the spectrum has no overlap, the maximum emission wavelength interval is about 350 nm, the influence of probe concentration, instruments and external environmental factors can be effectively eliminated, and the detection signal-to-noise ratio is improved. According to the response of the fluorescence intensity ratio at the two maximum emission wavelengths, the fluorescent probe provided in the application can be used for ratio detection of Fe 3+ .

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of organic fluorescent molecular probes, and particularly relates to a single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe as well as a preparation method and application thereof. BACKGROUND

[0002] Small-molecule fluorescent probes have great application potential in many fields such as chemical sensing, biomolecular labeling, cell imaging, environmental detection and organic light-emitting diodes due to their controllable preparation, good repeatability, high sensitivity, good biocompatibility and other advantages, and have attracted extensive attention and research of scientific researchers. The currently reported small-molecule fluorescent probes can be generally divided into single-emission and ratio fluorescent response types. The single-emission fluorescent probe can only emit fluorescence of a single wavelength, and recognition and detection are achieved by changes in fluorescence intensity, but it lacks self-calibration capability and is easily disturbed by background interference, and has insufficient quantitative capability. In comparison, the ratio fluorescent probe can emit fluorescence peaks of two or more wavelengths, and changes in the fluorescence intensity ratio of the two emission peaks eliminate systematic errors caused by background interference or fluctuations in external factors, thereby improving the signal-to-noise ratio and achieving quantitative detection.

[0003] The construction of ratio-type small-molecule fluorescent probes usually adopts a mechanism-oriented molecular design strategy, mainly including donor-acceptor structures of intramolecular charge transfer effect, structures of excited-state intramolecular proton transfer effect, double-fluorophore systems of fluorescence resonance energy transfer mechanism and molecular aggregation effect structures and the like. However, the existing ratio-type fluorescent probes generally have small wavelength interval and low separation degree between the two emission peaks, resulting in high spectral overlap degree and affecting the signal-to-noise ratio. Therefore, it is urgent to develop a kind of double-emission ratio-type small-molecule fluorescent probe with large wavelength interval between the emission peaks. SUMMARY

[0004] The primary purpose of the present application is to overcome the deficiencies of the prior art, and provide a single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe.

[0005] Another purpose of the present application is to provide a preparation method of the single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe.

[0006] Still another purpose of the present application is to provide an application of the single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe.

[0007] To achieve the above technical purposes, the technical solutions adopted by the present application are as follows:

[0008] A single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe has the following general structure:

[0009]

[0010] wherein R is selected from -Br, Any one of the groups.

[0011] The preparation method of the single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe comprises the following preparation steps:

[0012] Step a: preparation of intermediate 1:

[0013] 3-methoxybenzaldehyde and 4-bromobenzyl diethyl phosphite are dissolved in N,N-dimethylacetamide (DMAc), and a DMAc solution of potassium tert-butoxide is added dropwise under ice bath, and stirred at room temperature. After reaction, ultrapure water is added under ice bath to obtain intermediate 1.

[0014] Step b: preparation of intermediate 2:

[0015] Intermediate 1 is dissolved in dichloromethane (DCM), and a DCM solution of boron tribromide is added dropwise under ice bath. After reaction, ice water is added, and stirred at room temperature overnight. Ultrapure water and DCM are added for extraction and purification to obtain intermediate 2.

[0016] Step c: preparation of intermediate 3:

[0017] Intermediate 2 and potassium carbonate are dissolved in N,N-dimethylformamide (DMF), and a DMF solution of 1,3-dibromopropane is added dropwise, and stirred at room temperature. After reaction, ultrapure water and diethyl ether are added for extraction and purification to obtain intermediate 3.

[0018] Step d: preparation of intermediate 4:

[0019] Under a nitrogen atmosphere, tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate and potassium carbonate are dissolved in anhydrous acetonitrile, and an acetonitrile solution of intermediate 3 is added dropwise, and heated to reflux. After reaction, ultrapure water and ethyl acetate are added for extraction and purification, and separated and purified by column chromatography to obtain intermediate 4.

[0020] Step e: preparation of intermediates 5 and 6:

[0021] Intermediate 4, potassium carbonate, 3-aminobenzeneboronic acid or 3-(N,N-dimethylamino)benzeneboronic acid, and dichlorobis(triphenylphosphine)palladium are dissolved in a mixed solution of 1,4-dioxane and water, and heated to reflux under a nitrogen atmosphere. After reaction, ultrapure water and ethyl acetate are added for extraction and purification, and separated and purified by column chromatography to obtain intermediate 5 or 6.

[0022] Step f: preparation of the target fluorescent probe:

[0023] The intermediate 4, the intermediate 5 and the intermediate 6 are respectively dissolved in dichloromethane (DCM), and a DCM solution of trifluoroacetic acid is added dropwise under ice bath, and after reaction, the trifluoroacetate is precipitated out with ether to obtain the fluorescent probes TAT, TBT and TCT.

[0024] The reaction process is as follows:

[0025]

[0026] Further, in step a, the molar ratio of the 3-methoxybenzaldehyde, 4-bromobenzyl diethyl phosphite and potassium tert-butoxide is 1:1:1.2, and the stirring time at room temperature is 24 h. After reaction, ice bath is added and stirred for 5 h.

[0027] Further, in step b, the molar ratio of the intermediate 1 and boron tribromide is 1:10, and the reaction time is 4 h.

[0028] Further, in step c, the molar ratio of the intermediate 2, 1,3-dibromopropane and potassium carbonate is 1:5:5, and the reaction time is 5 h.

[0029] Further, in step d, the molar ratio of the intermediate 3, tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate and potassium carbonate is 1:1:2, and the reaction time is 48 h, and the purification is carried out by column chromatography with petroleum ether / ethyl acetate (5 / 1, v / v).

[0030] Further, in step e, the molar ratio of the intermediate 4, 3-aminobenzene boronic acid or 3-(N,N-dimethylamino)benzene boronic acid, potassium carbonate and palladium tetrakis(triphenylphosphine)dichloride is 1:1.5:2:0.1, the volume ratio of 1,4-dioxane to water is 10:1, the reaction time is 10 h, and the purification is carried out by column chromatography with petroleum ether / ethyl acetate (3 / 1, v / v).

[0031] Further, in step f, the reaction time is 12 h, and the post-treatment is to evaporate the reaction solution under reduced pressure, dissolve the obtained solid with a small amount of dichloromethane, then add a large amount of ether to precipitate the target fluorescent probe.

[0032] The single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe is used for detecting Fe 3+ application in changes, specifically, 1 μL of Fe 3+ aqueous solution (100 μM) is titrated into 1 mL of the small molecule fluorescent probe aqueous solution (30 μM) in sequence, and the fluorescence emission spectrum is scanned.

[0033] In addition, in the selective experiment, other metal cations, anions, glycerol and other interference substances are used to investigate the probe for Fe3+ The selective response to change is analyzed by the same method as the above.

[0034] Further, the fluorescence parameters of the probe TAT are as follows: the excitation wavelength is 300 nm, and the maximum emission wavelengths are 381 nm and 730 nm respectively. The fluorescence parameters of the probe TBT are as follows: the excitation wavelength is 302 nm, and the maximum emission wavelengths are 385 nm and 733 nm respectively. The fluorescence parameters of the probe TCT are as follows: the excitation wavelength is 302 nm, and the maximum emission wavelengths are 385 nm and 733 nm respectively. The slit width is 20 nm, and the voltage is 400 V.

[0035] Further, the other metal cations are Al 3+ , Ba 2+ , Ca 2+ , Co 2+ , Cu 2+ , Fe 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Zn 2+ , Na + , K + , and the anions are ClO2 - , CO3 2- , HPO4 2- , S2O3 2- , SO3 2- , SO4 2- , H2PO4 2- , HCO3 - , HSO3 2- , and NO2 2- .

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] The present application couples a stilbene group to a macrocyclic polyamine 1,4,7,10-tetraazacyclododecane (cyclen) to obtain a single-excitation dual-emission small-molecule fluorescent probe, the two fluorescent emission peaks have maximum emission wavelengths of about 380 nm and about 730 nm respectively, the wavelength interval is about 350 nm, and the wavelength interval of the two emission peaks is large, the separation degree is high, and the spectrum has no overlap.

[0038] The single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe provided by the present application has the advantages of simple preparation process, low price of raw materials, easy-to-adjust structure, good solubility and the like.

[0039] The single-excitation visible and near-infrared dual-region emission small-molecule fluorescent probe provided by the present application can be used for ratio detection of Fe 3+The method has the advantages of good selectivity, rapid response, high sensitivity, and the ratio response mode can effectively eliminate the influence of probe concentration, instrument and external environment factors, and improve the detection signal-to-noise ratio. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 A general synthesis route map of the single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe in the examples is shown in the following figure, and preparation of the probe TAT is taken as an example:

[0041] Figure 2 Absorption spectrum graphs of the fluorescent probes TAT, TBT and TCT in the examples are shown in the following figure:

[0042] Figure 3 Fluorescence emission spectrum graphs of the fluorescent probes TAT, TBT and TCT in the examples under different concentration conditions are shown in the following figure:

[0043] Figure 4 Fluorescence emission spectrum graphs of the fluorescent probes TAT, TBT and TCT in the examples to different concentrations of Fe 3+ are shown in the following figure:

[0044] Figure 5 Two maximum emission wavelength fluorescence intensity and ratio change column graphs of the fluorescent probes TAT, TBT and TCT in the examples in response to different metal cations are shown in the following figure:

[0045] Figure 6 Two maximum emission wavelength fluorescence intensity and ratio change column graphs of the fluorescent probes TAT, TBT and TCT in the examples in response to different anions and glycerol are shown in the following figure: DETAILED DESCRIPTION

[0046] The present application can be better understood according to the following examples. However, it is easily understood by those skilled in the art that the content described in the examples is only for illustrating the present application, and should not and will not limit the present application described in detail in the claims.

[0047] Example 1:

[0048] Figure 1 A general synthesis route map of the single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe in the examples is shown in the following figure, and preparation of the probe TAT is taken as an example:

[0049] 1. Preparation of intermediate 1

[0050] To two flasks, 10 mL of N,N-dimethylacetamide (DMAc) was added and 3-methoxybenzaldehyde (120 μL, 1 mmol) and 4-bromobenzyl diethyl phosphite (220 μL, 1 mmol) were dissolved in DMAc and stirred in an ice bath for 0.5 h. A solution of potassium tert-butoxide (133 mg, 1.2 mmol) in DMAc (5 mL) was added dropwise and stirred at room temperature for 24 h. Work-up: 20 mL of ultra-pure water was added under ice bath and white precipitate appeared. After stirring for 5 h, the precipitate was filtered and dried in vacuum to give intermediate 1 (204 mg, yield: 70%). 1 H NMR (400 MHz, CDC13) δ 7.46 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 8.6 Hz, 2H), 7.26 (t, J = 7.9 Hz, 1H), 7.10-7.07 (m, 1H), 7.02 (d, J = 5.7 Hz, 3H), 6.82 (dd, J = 8.2, 2.6 Hz, 1H), 3.83 (s, 3H).

[0051] 2. Preparation of intermediate 2

[0052] Intermediate 1 (289 mg, 1 mmol) was dissolved in 20 mL of dichloromethane (DCM) and a solution of boron tribromide (1 mL) in DCM (5 mL) was added dropwise under ice bath. After stirring for 4 h, the reaction was quenched by adding ice water carefully and stirred at room temperature overnight. The reaction was partitioned by adding ultra-pure water and DCM and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to give intermediate 2 (232 mg, yield: 84%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.46 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 8.6 Hz, 2H), 7.26 (t, J = 7.9 Hz, 1H), 7.10-7.07 (m, 1H), 7.02 (d, J = 5.7 Hz, 3H), 6.82 (dd, J = 8.2, 2.6 Hz, 1H), 3.83 (s, 3H).

[0053] 3. Preparation of intermediate 3

[0054] Intermediate 2 (100 mg, 0.34 mmol), potassium carbonate (235 mg, 1.7 mmol) were dissolved in 15 mL of dry N,N-dimethylformamide (DMF) and a solution of 1,3-dibromopropane (0.5 mL) in DMF (5 mL) was added dropwise. After stirring at room temperature for 5 h, the mixture was filtered and the DMF phase was added with ultrapure water and diethyl ether. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was evaporated under reduced pressure to give intermediate 3 (56 mg, yield: 45%) as an orange oil. 1 H NMR (400 MHz, CDC13) δ 7.48 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 3.8 Hz, 3H), 6.84 (dd, J = 8.7, 2.0 Hz, 1H), 4.15 (t, J = 5.8 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.34 (p, J = 6.1 Hz, 2H).

[0055] 4. Preparation of intermediate 4

[0056] Tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylate (473 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were dissolved in dry acetonitrile (30 mL) and heated under nitrogen atmosphere for 0.5 h. After cooling to room temperature, a solution of intermediate 3 (396 mg, 1 mmol) in acetonitrile was added dropwise and the mixture was heated under reflux for 48 h. The filtrate was added with ultrapure water and ethyl acetate and the organic phases were separated, dried over anhydrous sodium sulfate, filtered and the filtrate was evaporated under reduced pressure and dried. The product was purified by column chromatography on silica gel with petroleum ether / ethyl acetate (5 / 1, v / v) to give intermediate 4 (260 mg, yield: 33%) as a white solid. 1 H NMR (400 MHz, CDC13) δ 7.48 (d, J = 8.6 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 7.7 Hz, 1H), 7.04 (d, J = 3.8 Hz, 3H), 6.84 (dd, J = 8.7, 2.0 Hz, 1H), 4.15 (t, J = 5.8 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.34 (p, J = 6.1 Hz, 2H).

[0057] 5. Preparation of fluorescent probe TAT

[0058] Intermediate 4 (78 mg, 0.1 mmol) was dissolved in 3 mL of dichloromethane (DCM), and a solution of trifluoroacetic acid (1 mL) in DCM was added dropwise under ice bath. After stirring overnight at room temperature, the product was precipitated from the solution by adding ether to obtain the fluorescent probe TAT as a yellowish brown solid. 1 H NMR (400 MHz, D20) δ 7.12 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 7.0 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 6.71 - 6.57 (m, 5H), 3.62 (s, 2H), 3.03 (s, 16H), 2.62 (s, 2H), 2.56 (s, 2H).

[0059] Example Two:

[0060] Figure 1 A general synthetic route for the single-excitation visible and near-infrared dual-emission small molecule fluorescent probe in the examples is shown below, and the preparation of probe TBT is exemplified:

[0061] 1. Preparation of Intermediate 5

[0062] Intermediate 4 (787 mg, 1 mmol), potassium carbonate (276 mg, 2 mmol), 3- aminobenzeneboronic acid (205 mg, 1.5 mmol), and bis(triphenylphosphine)palladium dichloride (70 mg, 0.1 mmol) were dissolved in a mixed solution of 1,4-dioxane / water (10 / 1, v / v) and heated to reflux under nitrogen atmosphere for 10 h. To the filtrate, ultrapure water and ethyl acetate were added, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was evaporated under reduced pressure and the residue was separated and purified by column chromatography using petroleum ether / ethyl acetate (3 / 1, v / v) to obtain Intermediate 5 (250 mg, yield: 31%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 6.8 Hz, 4H), 7.42 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 9.0 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H), 7.12 (d, J = 6.4 Hz, 2H), 7.06 - 7.00 (m, 2H), 6.92 (d, J = 12.0 Hz, 1H), 6.80 (d, J = 6.5 Hz, 1H), 6.69 (d, J = 7.6 Hz, 1H), 4.02 (t, J = 5.9 Hz, 2H), 3.51 (s, 2H), 3.57 (s, 2H), 3.29 (s, 6H), 3.42 (s, 2H), 2.83 - 2.57 (m, 6H), 2.01 - 1.94 (m, 2H), 1.45 (s, 27H).

[0063] 2. Preparation of fluorescent probe TBT

[0064] Intermediate 5 (250 mg, 0.31 mmol) was dissolved in 3 mL of dichloromethane (DCM), and a solution of trifluoroacetic acid (1 mL) in DCM was added dropwise under ice bath. After stirring at room temperature overnight, the trifluoroacetate salt was precipitated with ether as a brown-yellow solid fluorescent probe TBT. 1 H NMR (400 MHz, D20) δ 7.65 (d, J = 7.7 Hz, 1H), 7.59 (d, J = 2.4 Hz, 5H), 7.51 (t, J = 7.8 Hz, 1H), 7.35 - 7.29 (m, 2H), 7.18 - 7.09 (m, 4H), 6.88 (dd, J = 8.4, 2.4 Hz, 1H), 4.11 - 4.05 (m, 2H), 3.06 (d, J = 5.5 Hz, 8H), 2.88 (t, J = 15.0 Hz, 10H), 1.96 (t, J = 7.2 Hz, 2H).

[0065] Example Three:

[0066] Figure 1 A general synthetic route map of the single-excitation visible and near-infrared dual-region emitting small molecule fluorescent probe in the examples is shown below, and the preparation of probe TCT is exemplified:

[0067] 1. Preparation of Intermediate 6

[0068] Intermediate 4 (787 mg, 1 mmol), potassium carbonate (276 mg, 2 mmol), 3- dimethylaminophenylboronic acid (247.5 mg, 1.5 mmol), and bis(triphenylphosphine) palladium dichloride (70 mg, 0.1 mmol) were dissolved in a mixed solution of 1,4- dioxane / water (10 / 1, v / v) under nitrogen atmosphere, and heated to reflux for 10 h. To the filtrate, ultrapure water and ethyl acetate were added, and the organic phase was dried over anhydrous sodium sulfate, filtered, and then evaporated under reduced pressure. The residue was separated and purified by column chromatography using petroleum ether / ethyl acetate (3 / 1, v / v) to obtain Intermediate 6 (230 mg, yield: 28%) as a white solid. 1H NMR (400 MHz, CDC13) δ 7.63-7.55 (m, 4H), 7.37-7.27 (m, 2H), 7.15-7.11 (m, 3H), 7.07 (d, J = 7.6 Hz, 1H), 6.99-6.94 (m, 2H), 6.80 (dd, J = 7.8, 3.0 Hz, 1H), 6.75 (dd, J = 7.9, 3.1 Hz, 1H), 4.03 (t, J = 6.1 Hz, 2H), 3.60-3.28 (m, 12H), 3.02 (s, 6H), 2.78-2.67 (m, 4H), 2.03-1.94 (m, 2H), 1.66 (d, J = 8 Hz, 2H), 1.46 (d, J = 2.8 Hz, 27H).

[0069] 2. Preparation of fluorescent probe TCT

[0070] Intermediate 6 (230 mg, 0.27 mmol) was dissolved in 3 mL of dichloromethane (DCM), and a solution of trifluoroacetic acid (1 mL) in DCM was added dropwise under ice bath. After stirring at room temperature overnight, the trifluoroacetate salt was precipitated with ether to obtain fluorescent probe TCT as a brown-yellow solid. 1 H NMR (400 MHz, D20) δ 7.49 (s, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.24-7.19 (m, 3H), 7.14 (d, J = 7.2 Hz, 1H), 7.06-6.99 (m, 2H), 6.97 (d, J = 1.8 Hz, 1H), 6.83 (s, 1H), 6.72 (d, J = 8.4 Hz, 3H), 3.85 (s, 2H), 3.18 (s, 6H), 3.05 (s, 12H), 2.89 (s, 2H), 2.77 (s, 2H), 2.72 (d, J = 7.0 Hz, 2H), 1.81 (s, 2H).

[0071] Example Four:

[0072] Absorption and fluorescence spectra of single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe and determination of fluorescence response to different concentrations of Fe 3+ Determination of fluorescence response:

[0073] Prepare aqueous solutions of probes TAT, TBT, and TCT respectively to have a final concentration of 30 μΜ, and determine their ultraviolet absorption spectra. The absorption spectra show that their maximum absorption wavelengths are respectively: abs (TAT) = 303 nm, λ abs (TBT) = 321 nm, λ abs(TCT) = 326 nm, and the maximum absorbance was A(TAT) = 0.35, A(TBT) = 0.34, A(TCT) = 0.48, respectively. Figure 2

[0074] The aqueous solutions of the probes TAT, TBT and TCT were prepared with different concentrations (5, 10, 15, 20, 30, 40, 50 μM), respectively. The fluorescence parameters were as follows: the excitation wavelength of the probe TAT was 300 nm, the excitation wavelength of the probe TBT was 302 nm, and the excitation wavelength of the probe TCT was 302 nm, the filter was 320 nm, the slit width was 20 nm, and the voltage was 400 V. The fluorescence emission spectrum was scanned, respectively. The results showed that the maximum emission wavelength of the double emission peaks of the probe was λ em (TAT) = 381 nm, 730 nm, λ em (TBT) = 385 nm, 733 nm, λ em (TCT) = 385 nm, 733 nm Figure 3 .

[0075] The aqueous solutions of the probes TAT, TBT and TCT were prepared with a final concentration of 30 μM, respectively. Then 1 μL of Fe 3+ aqueous solution (100 μM) was titrated into 1 mL of the small molecule fluorescent probe aqueous solution. The fluorescence parameters were as follows: the excitation wavelength of the probe TAT was 300 nm, the excitation wavelength of the probe TBT was 302 nm, and the excitation wavelength of the probe TCT was 302 nm, the filter was 320 nm, the slit width was 20 nm, and the voltage was 400 V. The fluorescence response of the probe to Fe 3+ was measured. The results showed that with the increase of the concentration of Fe 3+ , the fluorescence intensity of the maximum emission wavelength of the double emission peaks of the probe gradually increased. The probe still showed excellent fluorescence response to Fe -7 at a low concentration (10 -6 -10 3+ M). The double emission wavelength of the probe TAT did not change, while the long wavelength emission peak of TBT and TCT was red-shifted by 22 nm and 27 nm, respectively Figure 4 .

[0076] Example Five:

[0077] In order to investigate the ratio selectivity of the single excitation visible and near-infrared double region emission small molecule fluorescent probe to Fe 3+ , the fluorescence response of the probe to different metal cations, anions and glycerol was determined.

[0078] The aqueous solutions of the probes TAT, TBT and TCT were prepared with a final concentration of 30 μM, respectively. Then 30 μM of different metal cations Al 3+ , Ba 2+ ​, Ca 2+ , Co 2+ , Cu 2+ , Fe 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Zn 2+ , Na + , K + . Fluorescence parameters: the excitation wavelength of probe TAT is 300 nm, the excitation wavelength of probe TBT is 302 nm, the excitation wavelength of probe TCT is 302 nm, the filter is 320 nm, the slit width is 20 nm, and the voltage is 400 V. The fluorescence emission spectrum is scanned respectively. The experimental results show that, compared with different metal cations, the fluorescence intensity of the two maximum emission wavelengths of the probes TAT, TBT and TCT and their ratio are the highest in the presence of Fe 3+ , which are 4.77, 8.10 and 8.44 respectively. Figure 5 This indicates that the ratio selectivity of the probe to Fe 3+ is high compared with other metal ions.

[0079] The aqueous solutions of TAT, TBT and TCT are respectively prepared to have a final concentration of 30 μM, and then 30 μM of different anions ClO2 - , CO3 2- , HPO4 2- , S2O3 2- , SO3 2- , SO4 2- , H2PO4 2- , HCO3 - , HSO3 2- , NO2 2- and glycerol with a volume ratio of 80% are added. Fluorescence parameters: the excitation wavelength of probe TAT is 300 nm, the excitation wavelength of probe TBT is 302 nm, the excitation wavelength of probe TCT is 302 nm, the filter is 320 nm, the slit width is 20 nm, and the voltage is 400 V. The fluorescence emission spectrum is scanned respectively. The experimental results show that, after the probes TAT, TBT and TCT respond to different anions and glycerol, the fluorescence intensity of the two maximum emission wavelengths and their ratio have little difference, which are significantly lower than the fluorescence intensity of the two maximum emission wavelengths of the probes TAT, TBT and TCT and their ratio in the presence of Fe 3+ . Figure 6 This indicates that the probe is less affected by anions, solution viscosity and other interference factors.

[0080] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the spirit of the present application should also be within the scope of the present application.

Claims

1. A single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe, characterized in that, The general formula of the single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe is as follows: Wherein, R is selected from -Br, Any one of the groups.

2. A method for preparing a single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe according to claim 1, characterized in that, Includes the following steps: Step 1, Intermediate 1 Preparation: 3-Methoxybenzaldehyde and 4-bromobenzyl phosphite diethyl ester were dissolved in N,N-dimethylacetamide (DMAc), and a DMAc solution of potassium tert-butoxide was added dropwise under an ice bath. The mixture was stirred at room temperature, and after the reaction, ultrapure water was added under an ice bath to obtain intermediate 1. Step 2, Intermediate 2 Preparation: Intermediate 1 was dissolved in dichloromethane (DCM), and a DCM solution of boron tribromide was added dropwise under an ice bath. After the reaction, ice water was added, and the mixture was stirred overnight at room temperature. Ultrapure water and DCM were added for extraction and purification to obtain intermediate 2. Step 3, Intermediate 3 Preparation: Intermediate 2 and potassium carbonate were dissolved in N,N-dimethylformamide (DMF), and a DMF solution of 1,3-dibromopropane was added dropwise. The mixture was stirred at room temperature, and after the reaction, ultrapure water and diethyl ether were added for extraction and purification to obtain intermediate 3. Step 4, Intermediate 4 Preparation: Under a nitrogen atmosphere, tritert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid ester and potassium carbonate were dissolved in anhydrous acetonitrile. An acetonitrile solution of intermediate 3 was added dropwise, and the mixture was heated under reflux. After the reaction, ultrapure water and ethyl acetate were added for extraction and purification. The mixture was then purified by column chromatography to obtain intermediate 4. Step 5, Intermediate 5 and 6 Preparation: Intermediate 4, potassium carbonate, 3-aminophenylboronic acid or 3-(N,N-dimethylamino)phenylboronic acid, and bis(triphenylphosphine)palladium dichloride were dissolved in a mixed solution of 1,4-dioxane and water. The mixture was heated to reflux under a nitrogen atmosphere. After the reaction, ultrapure water and ethyl acetate were added for extraction and purification. The mixture was then separated and purified by column chromatography to obtain intermediate 5 or 6. Step 6, Preparation of the target fluorescent probe: Intermediates 4, 5, and 6 were dissolved in dichloromethane (DCM), and a DCM solution of trifluoroacetic acid was added dropwise under ice bath conditions. After the reaction, the trifluoroacetic acid salt was precipitated with diethyl ether to obtain the fluorescent probe TAT. TBT and TCT 3. The method for preparing a single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe according to claim 2, characterized in that, In the preparation of intermediate 1 in step one, the molar ratio of 3-methoxybenzaldehyde, diethyl 4-bromobenzyl phosphite, and potassium tert-butoxide is 1:1:1.2, the stirring time is 24 h at room temperature, and after the reaction, ultrapure water is added under ice bath and the stirring time is 5 h.

4. The method for preparing a single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe according to claim 2, characterized in that, In step two, the molar ratio of intermediate 1 to boron tribromide in the preparation of intermediate 2 is 1:10, and the reaction time is 4 hours.

5. The method for preparing a single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe according to claim 2, characterized in that, In step three, the molar ratio of intermediate 2, 1,3-dibromopropane, and potassium carbonate in the preparation of intermediate 3 is 1:5:5, and the reaction time is 5 hours.

6. The method for preparing a single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe according to claim 2, characterized in that, In the preparation of intermediate 4 in step four, the molar ratio of intermediate 3, tritert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7-tricarboxylic acid ester, and potassium carbonate is 1:1:2, the reaction time is 48 h, and the intermediate is purified by column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5:

1.

7. The method for preparing a single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe according to claim 2, characterized in that, In step five, the preparation of intermediates 5 and 6 involves the following steps: the molar ratio of intermediate 4, 3-aminophenylboronic acid or 3-(N,N-dimethylamino)phenylboronic acid, potassium carbonate, and palladium dichloride is 1:1.5:2:0.1; the volume ratio of 1,4-dioxane to water is 10:1; the reaction time is 10 h; and the mixture is purified by column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 3:

1.

8. The method for preparing a single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe according to claim 2, characterized in that, In step six, the reaction time for preparing the target fluorescent probe is 12 hours. The post-treatment involves evaporating the reaction solution under reduced pressure, dissolving the obtained solid with a small amount of dichloromethane, and then adding a large amount of diethyl ether to precipitate the target fluorescent probe.

9. The single-excitation visible and near-infrared dual-region emission small molecule fluorescent probe according to claim 1 is used in the preparation of ratio detection of Fe. 3+ Its application in reagents, characterized in that, The probe TAT The fluorescence parameters are: excitation wavelength of 300 nm, and maximum emission wavelengths of 381 nm and 730 nm, respectively. The probe TBT... The fluorescence parameters are: excitation wavelength of 302 nm, maximum emission wavelengths of 385 nm and 733 nm, and the probe TCT... The fluorescence parameters are: excitation wavelength of 302nm, maximum emission wavelengths of 385nm and 733nm, slit width of 20nm, and voltage of 400V.

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