阅读说明：本技术 一种跨键能量转移型Hg2+荧光探针、制备方法及其应用 (Cross-bond energy transfer type Hg2+Fluorescent probe, preparation method and application thereof ) 是由 母晓玥 祁美蓉 于 2021-09-16 设计创作，主要内容包括：一种跨键能量转移型Hg～(2+)荧光探针、制备方法及其应用,属于荧光探针技术领域。本发明公开的跨键能量转移型Hg～(2+)荧光探针,以有机胺为供体,以罗丹明B为受体,通过硫代双酰肼对Hg～(2+)的响应实现了荧光探针对溶液中Hg～(2+)的检测。该跨键能量转移型Hg～(2+)荧光探针的结构式如下所示,其可以用于检测水溶液中微量Hg～(2+)浓度,还可以在Hela细胞中痕量检测Hg～(2+)、制备检测纸检测溶液中Hg～(2+)方面得到应用。(Cross-bond energy transfer type Hg 2+ A fluorescent probe, a preparation method and application thereof belong to the technical field of fluorescent probes. The invention discloses a cross-bond energy transfer type Hg 2+ The fluorescent probe takes organic amine as a donor, rhodamine B as an acceptor and Hg is subjected to thiobis-hydrazide 2+ The response of the probe realizes that the fluorescent probe is used for detecting Hg in the solution 2+ Detection of (3). The cross-bond energy transfer type Hg 2+ The structural formula of the fluorescent probe is shown as follows, and the fluorescent probe can be used for detecting trace Hg in an aqueous solution 2+ The concentration of Hg in Hela cells can be detected in trace amount 2+ And preparing Hg in the detection paper detection solution 2+ The method is applied.)

1. Cross-bond energy transfer type Hg²⁺The fluorescent probe has a structural formula as follows:

wherein the structural formula of Y is shown as follows:

wherein R is₁、R₂、R₃、R₄Independently selected from hydrogen, fluorine, cyano, C₁～C₆Alkyl of (C)₆～C₂₀Aryl of (2)C containing 1 to 5 hetero atoms₄～C₂₀The heteroaryl of (a), the heteroatom of N, O, S, Si; m, n, x and y are integers of 1-3.

2. The cross-bonding energy transfer type Hg of claim 1²⁺The fluorescent probe has a structural formula shown as one of the following formulas:

3. hg as claimed in claim 1 or 2²⁺The preparation method of the fluorescent probe comprises the following steps:

s1: mixing 3-hydroxy-N, N-diethylaniline and 4-bromophthalic anhydride, reacting in a propionic acid solution for 20-30 h, and then distilling to remove a solvent propionic acid to obtain p-bromorhodamine B (p-Rho-Br); wherein the dosage proportion of the 3-hydroxy-N, N-diethylaniline, the 4-bromophthalic anhydride and the propionic acid is 1 mmol: 1 mmol: (3-10) mL;

s2: dissolving p-Rho-Br in ethanol, adding hydrazine hydrate with the concentration of 15-20 mol/L, heating to 75-85 ℃ under stirring for reflux reaction for 2-4 h, then carrying out reduced pressure distillation to remove the solvent ethanol, and purifying by using a neutral alumina chromatographic column to obtain p-bromorhodamine B hydrazide (p-Rho-Br-NH)₂) (ii) a Wherein the dosage proportion of the p-Rho-Br, the hydrazine hydrate and the ethanol is 1 mmol: 3mL of: (15-25) mL; an eluent in the chromatographic purification process of the neutral alumina column is petroleum ether and ethyl acetate, and the volume ratio is (4-6): 1;

s3: adding phenothiazine, phenoxazine, 9, 10-dihydro-9, 9-dimethylacridine, 9-diphenyl-9, 10-dihydroacridine, phenylphenol oxazine, 1-naphthylaminobenzene or N-phenyl-2-naphthylamine and p-bromoiodobenzene into a dried flask, adding an ultra-dry tetrahydrofuran solution, dropwise adding N-butyllithium for lithiation for 1-2h at-78 ℃, adding trimethyl borate at-78 ℃, stirring for 20-30 h at 20-30 ℃, adding a saturated ammonium chloride solution, continuously stirring for 2-4 h, separating after the reaction is finished, collecting an organic phase, spin-drying, and crystallizing methanol to obtain a donor Y); wherein the dosage ratio of phenothiazine, phenoxazine, 9, 10-dihydro-9, 9-dimethylacridine, 9-diphenyl-9, 10-dihydroacridine, phenylphenolazine, 1-naphthylaminobenzene or N-phenyl-2-naphthylamine, p-bromoiodobenzene, tetrahydrofuran, N-butyl lithium, trimethyl borate and ammonium chloride is 1 mmol: 1 mmol: (40-60) mL: 1 mmol: 3 mmol: (30-60) mL;

s4: reacting p-Rho-Br-NH₂Donor Y, tetrabutylammonium bromide (TBAB), tetrakis (triphenylphosphine) palladium (Pd (PPh)₃)₄) And potassium carbonate (K)₂CO₃) Mixing, vacuumizing, introducing nitrogen, adding toluene and H₂Heating the mixture to 105-120 ℃ under stirring, carrying out reflux reaction for 8-12 h, then carrying out reduced pressure distillation to remove toluene, then carrying out liquid separation extraction on ethyl acetate and a saturated saline solution, removing residual potassium carbonate, and simultaneously extracting the product into ethyl acetate; drying the organic phase ethyl acetate with anhydrous sodium sulfate, concentrating to remove ethyl acetate, and purifying the residue obtained after removing ethyl acetate with neutral alumina chromatographic column to obtain p-Y-rhodamine B hydrazide (p-Rho-Y-NH)₂) (ii) a Wherein, p-Rho-Br-NH₂Donors Y, TBAB, Pd (PPh)₃)₄、K₂CO₃The ratio of water to toluene was 1 mmol: 1 mmol: 1 mmol: 0.03 mmol: 3 mmol: (7-10) mL: (1.5-2) mL; the eluent for the chromatographic purification of the neutral alumina column is ethyl acetate and petroleum ether, and the volume ratio is 1: (4-6);

s5: p-Rho-Y-NH₂Mixing and dissolving phenyl isothiocyanate and triethylamine in N, N-dimethylformamide, stirring for 4-8h at 20-25 ℃ under the atmosphere of nitrogen, and then removing the solvent N, N-dimethylformamide through reduced pressure distillation; purifying the residue obtained after removing the solvent by a neutral alumina chromatographic column to obtain solid p-RSP-Y; wherein, p-Rho-Y-NH₂The dosage ratio of phenyl isothiocyanate, triethylamine and N, N-dimethylformamide is 1 mmol: (2-6) mmol: 0.05 mmol: (5-15) mL; the eluent for the chromatographic purification of the neutral alumina column isEthyl acetate and petroleum ether in a volume ratio of 1: (4-5);

wherein, the chemical formula of the p-bromorhodamine B (p-Rho-Br) is as follows:

para-bromorhodamine B hydrazide (p-Rho-Br-NH)₂) The chemical formula of (A) is:

para-Y-rhodamine B hydrazide (p-Rho-Y-NH)₂) The chemical formula of (A) is:

the chemical formula of p-Y-rhodamine B hydrazide-phenyl isothiocyanate (p-RSP-Y) is as follows:

4. the cross-bonding energy transfer type Hg of claim 1 or 2²⁺Fluorescent probe for detecting trace Hg in aqueous solution²⁺The application in concentration.

5. The cross-bonding energy transfer type Hg of claim 1 or 2²⁺Trace detection of Hg in Hela cells by fluorescent probe²⁺Application of the aspect.

6. The cross-bonding energy transfer type Hg of claim 1 or 2²⁺Method for preparing and detecting Hg in paper detection solution by using fluorescent probe²⁺Application of the aspect.

Technical Field

The invention belongs to the technical field of fluorescent probes, and particularly relates to a cross-bond energy transfer type Hg²⁺Fluorescent probe, preparation method and application thereof in detecting trace Hg in aqueous solution²⁺The application in concentration.

Background

The pollution of heavy metal ions in the environment poses great threat to human health. Wherein the mercury ion (Hg)²⁺) It is considered to be one of the most harmful metal ions due to its high toxicity to organisms such as human beings and its accumulation in aquatic ecosystems.

It is possible. Hg detection with traditional atomic absorption spectrum, atomic fluorescence spectrum and inductively coupled plasma mass spectrum²⁺Compared with the method, the fluorescent probe has the advantages of simple operation, good selectivity, high sensitivity, fast response, small damage to cells and the like, and the fluorescent probe is used for detecting Hg in recent years²⁺The method of (a) has been rapidly developed. To date, various irreversible reactions have been used to synthesize fluorescent chemodosimeters for the detection of Hg²⁺. However, the fluorescence chemodosimeter is paired with Hg²⁺The quantitative detection of (2) has limitations because the fluorescence-on detection using one emission band cannot be Hg²⁺Providing a self-calibrated fluorescent signal. Therefore, proportional-type fluorescence chemodosimeters are strongly recommended for Hg²⁺Because the ratio between the two emission bands allows to self-calibrate the fluorescence signal and to correct the fluorescence probe concentration. The synthesis of Hg has the advantages of high selectivity, high sensitivity, strong fluorescence signal, easy operation in aqueous solution, etc²⁺Proportional fluorescence dosimeters, have high challenges.

One of the main ideas for constructing the proportional fluorescent chemical dosimeter fluorescent probe is as follows: the energy transfer process between fluorophores is utilized to construct ratiometric fluorescent probes, such as Fluorescence Resonance Energy Transfer (FRET) type and cross-bond energy transfer (TBET) type fluorescent probes.

The fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor of the fluorescence resonance energy transfer type fluorescence probe have certain overlap, and the efficiency of energy transfer is influenced by factors such as the degree of overlap of the emission spectrum of the donor and the absorption spectrum of the acceptor, the relative orientation of the jump dipoles of the donor and the acceptor, the distance between the donor and the acceptor, the fluorescence quantum yield of the acceptor part, and the like. Therefore, when such a fluorescent probe is constructed, the selection of the fluorophore is greatly limited.

Similar to the structure of a fluorescence resonance energy transfer type fluorescent probe, the cross-bond energy transfer type fluorescent probe also has two fluorophores, energy is directly transferred from a donor to an acceptor through a conjugate bond, and the fluorescent probe has no requirement on the spectral overlapping of the fluorophores and has higher energy transfer efficiency. Thus, the choice of fluorophore allows greater flexibility in constructing this type of fluorescent probe.

In conclusion, the invention takes the organic amine as a donor, the rhodamine B as an acceptor and the thiodihydrazide structure as Hg²⁺A series of cross-bonding energy transfer type Hg is constructed²⁺A fluorescent probe.

Disclosure of Invention

The purpose of the present invention is to provide a cross-bond energy transfer type Hg²⁺Fluorescent probe, preparation method and application thereof in detecting trace Hg in aqueous solution²⁺The application in concentration.

The invention discloses a cross-bond energy transfer type Hg²⁺The fluorescent probe takes organic amine as a donor, rhodamine B as an acceptor, and Hg is subjected to thiobis-hydrazide²⁺The response of the probe realizes that the fluorescent probe is used for detecting Hg in the solution²⁺Detection of (3).

The invention relates to a cross-bond energy transfer type Hg²⁺The fluorescent probe has a structural formula as follows:

wherein the structural formula of Y is shown as follows:

wherein R is₁、R₂、R₃、R₄Independently selected from hydrogen, fluorine, cyano, C₁～C₆Alkyl of (C)₆～C₂₀Aryl group of (1) to (5) hetero atom-containing C₄～C₂₀The heteroaryl of (a), the heteroatom of N, O, S, Si; m, n, x and y are integers of 1-3.

Preferably, the invention relates to a cross-bond energy transfer type Hg²⁺The fluorescent probe has a structural formula as follows:

hg according to the invention²⁺The preparation method of the fluorescent probe comprises the following steps:

s1: mixing 3-hydroxy-N, N-diethylaniline and 4-bromophthalic anhydride, reacting in a propionic acid solution for 20-30 h, and then distilling to remove a solvent propionic acid to obtain p-bromorhodamine B (p-Rho-Br); wherein the dosage proportion of the 3-hydroxy-N, N-diethylaniline, the 4-bromophthalic anhydride and the propionic acid is 1 mmol: 1 mmol: (3-10) mL;

s2: dissolving p-Rho-Br in ethanol, adding hydrazine hydrate with the concentration of 15-20 mol/L, heating to 75-85 ℃ under stirring for reflux reaction for 2-4 h, then carrying out reduced pressure distillation to remove the solvent ethanol, and purifying by using an alumina chromatographic column to obtain p-bromorhodamine B hydrazide (p-Rho-Br-NH)₂) (ii) a Wherein the dosage proportion of the p-Rho-Br, the hydrazine hydrate and the ethanol is 1 mmol: 3mL of: (15-25) mL; an eluent in the chromatographic purification process of the neutral alumina column is petroleum ether and ethyl acetate, and the volume ratio is (4-6): 1;

s3: adding phenothiazine, phenoxazine, 9, 10-dihydro-9, 9-dimethylacridine, 9-diphenyl-9, 10-dihydroacridine, phenylphenol oxazine, 1-naphthylaminobenzene or N-phenyl-2-naphthylamine and p-bromoiodobenzene into a dried flask, adding an ultra-dry tetrahydrofuran solution, dropwise adding N-butyllithium for lithiation for 1-2h at-78 ℃ (liquid nitrogen + acetone), adding trimethyl borate at-78 ℃, stirring for 20-30 h at-20 ℃, adding a saturated ammonium chloride solution, continuously stirring for 2-4 h, separating after the reaction is finished, collecting an organic phase, then spin-drying, and crystallizing methanol to obtain a donor Y); wherein the dosage ratio of phenothiazine, phenoxazine, 9, 10-dihydro-9, 9-dimethylacridine, 9-diphenyl-9, 10-dihydroacridine, phenylphenolazine, 1-naphthylaminobenzene or N-phenyl-2-naphthylamine, p-bromoiodobenzene, tetrahydrofuran, N-butyl lithium, trimethyl borate and ammonium chloride is 1 mmol: 1 mmol: (40-60) mL: 1 mmol: 3 mmol: (30-60) mL;

wherein compounds D1-1 and D4-1 from donor Y were purchased and the remaining compounds were obtained from the above protocol;

s4: reacting p-Rho-Br-NH₂Donor Y, tetrabutylammonium bromide (TBAB), tetrakis (triphenylphosphine) palladium (Pd (PPh)₃)₄) And potassium carbonate (K)₂CO₃) Mixing, vacuumizing, introducing nitrogen, adding toluene and H₂Heating the mixture to 105-120 ℃ under stirring, carrying out reflux reaction for 8-12 h, then carrying out reduced pressure distillation to remove toluene, then carrying out liquid separation extraction on ethyl acetate and a saturated saline solution, removing residual potassium carbonate, and simultaneously extracting the product into ethyl acetate; drying the organic phase ethyl acetate with anhydrous sodium sulfate, concentrating to remove ethyl acetate, and purifying the residue obtained after removing ethyl acetate with neutral alumina chromatographic column to obtain p-Y-rhodamine B hydrazide (p-Rho-Y-NH)₂) (ii) a Wherein, p-Rho-Br-NH₂Donors Y, TBAB, Pd (PPh)₃)₄、K₂CO₃The ratio of water to toluene was 1 mmol: 1 mmol: 1 mmol: 0.03 mmol: 3 mmol: (7-10) mL: (1.5-2) mL; the eluent for the chromatographic purification of the neutral alumina column is ethyl acetate and petroleum ether, and the volume ratio is 1: (4-6);

s5: p-Rho-Y-NH₂Phenyl isothiocyanate and triethylamine are mixed and dissolved in N, N-dimethylformamide, and stirred for 4-8h at the temperature of 20-25 ℃ in the nitrogen atmosphere,then removing the solvent N, N-dimethylformamide by reduced pressure distillation; purifying the residue obtained after removing the solvent by a neutral alumina chromatographic column to obtain yellow solid p-RSP-Y; wherein, p-Rho-Y-NH₂The dosage ratio of phenyl isothiocyanate, triethylamine and N, N-dimethylformamide is 1 mmol: (2-6) mmol: 0.05 mmol: (5-15) mL; the eluent for the chromatographic purification of the neutral alumina column is ethyl acetate and petroleum ether, and the volume ratio is 1: (4-5);

wherein, the chemical formula of the p-bromorhodamine B (p-Rho-Br) is as follows:

para-bromorhodamine B hydrazide (p-Rho-Br-NH)₂) The chemical formula of (A) is:

para-Y-rhodamine B hydrazide (p-Rho-Y-NH)₂) The chemical formula of (A) is:

the chemical formula of p-Y-rhodamine B hydrazide-phenyl isothiocyanate (p-RSP-Y) is as follows:

the p-RSP-Y fluorescent probe can be used for detecting trace Hg in an aqueous solution²⁺The concentration comprises the following specific steps:

t1: preparing a p-RSP-Y fluorescent probe mother solution: weighing 83.4mg of p-RSP-Y fluorescent probe, dissolving the p-RSP-Y fluorescent probe in DMSO, fixing the volume to a scale mark in a 10mL volumetric flask by using DMSO to prepare 1mM fluorescent probe mother solution, and storing the fluorescent probe mother solution in a refrigerator at 4 ℃;

HgCl₂preparing a solution to be detected: with deionized waterAdding HgCl₂Preparing 1mM solution to be tested, and storing in a refrigerator at 4 ℃;

t2: the fluorescent probe mother liquor in T1 is indirectly added into HgCl₂Adding the fluorescent probe into the solution to be detected, wherein the concentration of the fluorescent probe in the solution is 10 mu M; compared with the method before adding the fluorescent probe, after adding the fluorescent probe, the water solution to be detected has an absorption peak at 570nm, and the absorption peak at 276nm is weakened. The ratio of the absorption peak intensity at 570nm to that at 276nm, namely A, is calculated₅₇₀/A₂₇₆It can be found that HgCl₂The ratio of the concentration to the absorption peak intensity has a good linear relation in the range of 0-5 mu M.

Because the fluorescent probe is insoluble in HgCl₂Detecting HgCl in the solution to be detected₂HgCl in the solution to be tested₂When the concentration is high, the fluorescent probe needs to be dissolved in an organic solvent which is mutually soluble with water and then added into the solution to be detected. The organic solvent which is mutually soluble with water is one or a mixture of more of ethanol, dimethyl sulfoxide and tetrahydrofuran. And the volume ratio of the organic solvent to the water in the mixed solution after the fluorescent probe solution and the liquid to be tested are mixed is determined again according to each test actual condition.

The invention also provides the cross-bond energy transfer type Hg²⁺Fluorescent probe for detecting trace Hg in Hela cells²⁺Application of the aspect.

The aforementioned bonding-spanning energy transfer type Hg of the present invention²⁺Trace Hg of fluorescent probe in Hela cells²⁺Taking the probe prepared in example 1 as an example, the following tests are carried out, including the following steps:

r1: HeLa cells were stained with 10. mu.M fluorescent probe solution for two hours and then with different Hg²⁺Culturing the solution with the concentration for half an hour; among them, 10. mu.M of the fluorescent probe solution was prepared by diluting 1mM of the probe solution prepared in T1 in high-sugar cell culture medium (DMEM). Different Hg²⁺The solution at concentration was 1mM HgCl made up of T1₂The test solution is obtained by diluting with high-glucose cell culture medium (DMEM).

Wherein the Hela cells are obtained from Shanghai cell bank of Chinese academy of sciences, and high-sugar medium (DMEM) is obtained from Hyclone, and HeLa cells are cultured beforeFree of Hg²⁺Interfering with subsequent experiments.

R2: adopting a laser scanning confocal microscope to shoot a fluorescence confocal microscope image of the dyed HeLa cell, and detecting the existence of Hg in the cell according to the fluorescence confocal microscope image²⁺Realize the aim on Hg²⁺And (4) qualitative detection.

The invention also provides the cross-bond energy transfer type Hg²⁺Hg in detection paper detection solution prepared by fluorescent probe²⁺Application of the aspect.

The above-mentioned cross-bonding energy transfer type Hg of the present invention²⁺Method for preparing fluorescent probe to detect whether Hg is contained in paper detection solution²⁺The method comprises the following steps:

d1: 1.0g of polyvinyl alcohol PVA (molecular weight of 75000), 1.0g of polyethylene glycol PEG (molecular weight of 20000) and 5g of a Polyacrylamide (PAM) aqueous solution with the mass fraction of 10% (molecular weight of 400000-800000) are dissolved in 20mL of water to obtain a mixed solution with the mass fractions of 3.7% of PVA, 3.7% of PEG and 18.5% of PAM.

D2: spreading filter paper on a culture dish, coating the mixed solution prepared in the step D1, and drying at the temperature of 55-65 ℃;

d3: coating the filter paper obtained in the step D2 with a p-RSP-Y (10 mu M, solvent is absolute ethyl alcohol) fluorescent probe solution, and drying at 55-65 ℃;

in the technical scheme, the preparation method of the 10 mu M p-RSP-Y fluorescent probe solution comprises the following steps: weighing a p-RSP-Y fluorescent probe, dissolving the p-RSP-Y fluorescent probe in DMSO, and fixing the volume to a scale mark by using a 10mL volumetric flask to prepare 1mM fluorescent probe mother solution; then 100. mu.L of 1mM fluorescence probe mother liquor is added into a 10mL volumetric flask, and the volume is fixed to the scale mark by absolute ethyl alcohol solution.

D4: dipping Hg-containing material with sponge²⁺The solution is contacted with the filter paper dried in the step D3 for 3-5 seconds, the change of fluorescence of the filter paper is observed under the excitation of a 365nm ultraviolet lamp, and compared with the solution to be detected in which other metal ions are added, the Hg-containing solution is dipped²⁺The filter paper fluoresces red after the solution (2).

In the technical scheme, Hg²⁺The preparation method of the solution comprises the following steps: 2.8g HgCl were weighed₂Dissolving the solid in deionized water, transferring the dissolved solid to a 10mL volumetric flask, and fixing the volume to the scale mark with the deionized water to obtain 1mM HgCl₂A solution; then, 10. mu.L of 1mM HgCl was taken₂Adding 3990 muL deionized water into the solution in a 10mL volumetric flask, and then fixing the volume to the scale mark by using absolute ethyl alcohol to obtain 1 muM HgCl₂The volume ratio of absolute ethyl alcohol to water in the solution is 3: 2.

In all the above protocols, the reagents used were analytically pure.

Drawings

The invention will be further described with reference to the accompanying drawings and examples:

FIG. 1 shows that p-RSP-TPA fluorescent probe prepared in example 1 of the invention is excited by 355nm light at different ratios of THF-H₂A fluorescence spectrum in the O mixed solution;

FIG. 2 shows the fluorescence probe of p-RSP-TPA prepared in example 1 of the present invention excited by 355nm light at different pH THF-H₂Fluorescence spectra in O (v/v, 1/1) mixed solution;

FIG. 3 shows that the p-RSP-TPA fluorescent probe prepared in example 1 of the invention is excited by 355nm light and is in THF-H₂Different concentrations of Hg were added to O (v/v, 2/3) solution²⁺Fluorescence spectrogram of the post-mixed solution;

FIG. 4 shows the fluorescence of p-RSP-TPA fluorescent probe prepared in example 1 of the present invention in THF-H₂Different concentrations of Hg were added to O (v/v, 2/3) solution²⁺Ultraviolet absorption spectrogram of the rear mixed solution;

FIG. 5 shows the fluorescence of p-RSP-TPA fluorescent probe prepared in example 1 of the present invention in THF-H₂Detection of Hg in O (v/v, 2/3) solution²⁺Hg of mercury²⁺Concentration and solution ultraviolet absorption spectrum A₅₇₀/A₂₇₆The linear relation curve of (1);

FIG. 6 shows that the p-RSP-TPA fluorescent probe prepared in example 1 of the present invention is excited at 355nm under THF-H₂Adding metal chloride (Cu) with the same concentration into O (v/v, 2/3) solution²⁺、Zn²⁺、K⁺、Mg²⁺Etc.) the fluorescence spectrum of the post solution;

FIG. 7 shows an embodiment of the present invention1 the p-RSP-TPA fluorescent probe prepared by the method stains HeLa cells and then reacts with Hg²⁺Confocal microscopy after reaction;

FIG. 8 is a schematic structural diagram of detection paper prepared by the p-RSP-TPA fluorescent probe prepared in example 1 of the present invention;

FIG. 9 is an optical image of detection paper prepared by the p-RSP-TPA fluorescent probe prepared in example 1 of the present invention after contacting different metal cations.

Detailed Description

Example 1: synthesis of Compound 1

3-hydroxy-N, N-diethylaniline (4.93g, 21.8mmol) and 4-bromophthalic anhydride (7.2g, 43.6mmol) were mixed in a three-necked flask, dissolved in 150mL of propionic acid solution, stirred and refluxed for 24h at 150 ℃, and after the reaction was completed, the propionic acid solution in the reaction system was evaporated to dryness by atmospheric distillation to obtain p-Rho-Br (which was not purified and used directly in the next reaction). The p-Rho-Br obtained above was dissolved in 250mL of an ethanol solution, and 80% (concentration: 15.98mol/L) of a hydrazine hydrate solution (2mL, 4mmol) was added to the system, followed by heating, stirring and refluxing at 80 ℃ for 4 hours. Distilling under reduced pressure to remove solvent, separating and purifying the residue with neutral alumina chromatographic column using ethyl acetate-petroleum ether (v/v-1/4) as developing agent to obtain p-Rho-Br-NH₂The yield was 17%, and the mass of the molecular ions determined by mass spectrometry was: 535.89 (calculated 534.16).

¹HNMR(500MHz，Chloroform-d)δ7.82(d，J＝8.1Hz，1H)，7.60(d，J＝8.0Hz，1H)，7.27(s，1H)，6.49(d，J＝8.7Hz，2H)，6.45(s，2H)，6.35(d，J＝8.8Hz，2H)，3.63(s，2H)，3.38(q，J＝7.1Hz，8H)，1.21(t，J＝7.0Hz，12H).

Reacting p-Rho-Br-NH₂(0.534g，1mmol)、TPA-B(OH)₂(0.289g，1mmol)、TBAB(0.322g，1mmol)、Pd(PPh₃)₄(35mg, 0.03mmol) and K₂CO₃(0.414g，3mmol) were mixed and toluene (7.36mL, 1mmol) and H were added₂O (1.5mL, 0.08mmol) was reacted at 110 ℃ under nitrogen at reflux for 8 h. After the reaction is finished, removing toluene in the solution by reduced pressure distillation, extracting the solution by ethyl acetate and saturated sodium chloride solution for three times, drying an organic phase by anhydrous sodium sulfate, removing an organic solvent by reduced pressure distillation, separating and purifying a residue after the solvent is removed by a neutral alumina chromatographic column, and obtaining a compound p-Rho-TPA-NH, wherein a developing agent is ethyl acetate-petroleum ether (v/v ═ 1/4)₂The yield is: 77%, mass spectrometry analysis determined the molecular ion mass as: 699.84 (calculated 699.36).

¹HNMR(500MHz，Chloroform-d)δ7.99(d，J＝8.0Hz，1H)，7.73–7.66(m，2H)，7.49(dt，J＝9.9，5.0Hz，1H)，7.40(d，J＝8.4Hz，2H)，7.25(d，J＝7.7Hz，3H)，7.10(d，J＝7.9Hz，4H)，7.07–7.02(m，4H)，6.55(d，J＝8.8Hz，2H)，6.45(s，2H)，6.33(d，J＝9.1Hz，2H)，3.65(s，2H)，3.37(q，J＝7.1Hz，8H)，1.20(t，J＝7.0Hz，12H).

p-Rho-TPA-NH₂(174.3mg, 0.25mmol), phenylisothiocyanate (135mg, 1mmol) and triethylamine (1mL) were added to the 10mL DMAF solution, and the mixture was stirred at room temperature for 8 hours under a nitrogen atmosphere. After the reaction is finished, the solvent in the reaction system is removed by reduced pressure distillation, the residue is separated and purified by a neutral alumina column chromatography column, and a developing agent is ethyl acetate-petroleum ether (1/4), so that the compound 1 p-triphenylamine rhodamine phenyl isothiocyanate (p-RSP-TPA) is obtained, wherein the yield is as follows: 78%, mass spectrometry determined the molecular ion mass as: 832.59 (calculated 833.38).

¹HNMR(500MHz，Chloroform-d)δ8.07(d，J＝8.0Hz，1H)，7.82(d，J＝8.2Hz，1H)，7.57(s，1H)，7.46(d，J＝8.6Hz，3H)，7.27(d，J＝7.8Hz，3H)，7.21(t，J＝7.7Hz，2H)，7.14–7.05(m，12H)，6.96(s，1H)，6.69–6.58(m，2H)，6.50(d，J＝13.9Hz，2H)，6.34(s，2H)，3.41–3.33(m，8H)，1.19(t，J＝7.0Hz，12H)。

Example 2: synthesis of Compound 2.

According to the synthetic route, the donor D2-1 is synthesized by the following steps: p-bromoiodobenzene (2.83g,10mmol) and phenothiazine (1.99g,10mmol) are added into a dry 100mL flask, 40mL of ultra-dry tetrahydrofuran is added, n-butyllithium (1 equivalent) is added dropwise for lithiation for 1-2h under-78 ℃ (liquid nitrogen + acetone), trimethyl borate (3 equivalent) is added under-78 ℃, the mixture is stirred for 24h at room temperature of 20-30 ℃, a saturated ammonium chloride solution is added, an organic phase is retained after liquid separation, a residue obtained after the organic phase is removed is crystallized by methanol to obtain D2-1, and the D2-1 is directly put into the next step without other treatment.

Compound 2 was synthesized according to the same procedure as compound 1, following the above synthetic route. The yield was 55%. The mass of the molecular ions determined by mass spectrometry was: 831.11 (calculated 831.37). Theoretical element content: c₅₄H₅₃N₆O₂S₂C, 73.52; h, 6.06; n, 9.53; o, 3.63; s,7.27, actually measuring the element content C, 73.32; h, 6.10; n, 9.43; o, 3.33; and S, 7.47.

Example 3: synthesis of Compound 3.

According to the above synthetic route, the procedure for synthesizing the donor D3-1 is the same as that for synthesizing D2-1, and according to the synthesis of the compound 1, the compound 3 is synthesized by the same procedure. The yield was 64%. The mass of the molecular ions determined by mass spectrometry was: 865.39 (calculated: 866.12). Theoretical element content: c₅₄H₅₃N₆O₃S, C, 74.89; h, 6.17; n, 9.70; o, 5.54; s,3.70 actually measuring the element content C, 73.62; h, 6.10; n, 9.43; o, 3.40; and S, 7.40.

Example 4: synthesis of Compound 4.

Donor D4-1 was purchased and synthesized according to the same procedure as for the synthesis of Compound 1 to give Compound 4. The yield was 64%. The mass of the molecular ions determined by mass spectrometry was: 831.40 (calculated 832.36). Theoretical element content: c₅₄H₅₃N₆O₂S, C, 76.29; h, 6.28; n, 9.89; o, 3.76; s,3.77 actually measuring the element content C, 76.62; h, 6.10; n, 9.63; o, 3.40; and S, 3.56.

Example 5: synthesis of Compound 5.

According to the above synthetic route, the procedure for synthesizing the donor D5-1 is the same as that for synthesizing D2-1, and according to the synthesis of the compound 1, the same procedure is followed to synthesize the compound 5. The yield was 54%. The mass of the molecular ions determined by mass spectrometry was: 891.44 (calculated 892.20). Theoretical element content: c₅₇H₅₉N₆O₂S, C, 76.73; h, 6.67; n, 9.42; o, 3.59; s,3.59 actually measuring the element content C, 76.62; h, 6.50; n, 9.43; o, 3.40; and S, 7.40.

Example 6: synthesis of Compound 6.

According to the above synthetic route, the procedure for synthesizing the donor D6-1 is the same as that for synthesizing D2-1, and according to the synthesis of the compound 1, the same procedure is followed to synthesize the compound 6. The yield was 63%. The mass of the molecular ions determined by mass spectrometry was: 923.40 (calculated 924.18). Theoretical element content: C59H53N7O2S, C, 76.68; h, 5.78; n, 10.61; o, 3.46; s,3.47, actually measuring the element content: c, 76.30; h, 5.88; n, 10.21; o, 3.66; and S, 3.30.

Example 7: synthesis of Compound 7.

According to the above synthetic route, the procedure for synthesizing the donor D7-1 is the same as that for synthesizing D2-1, and according to the synthesis of the compound 1, the same procedure is followed to synthesize the compound 7. The yield was 43%. The mass of the molecular ions determined by mass spectrometry was: 884.39 (calculated 885.14). Theoretical element content: c₅₇H₅₂N₆O₂S, C, 77.35; h, 5.92; n, 9.49; o, 3.62; s,3.62 actually measured element content: c, 77.33; h,5.50N, 9.20; o, 3.33; s,3.15

Example 8: synthesis of Compound 8.

According to the above synthetic route, the procedure for synthesizing the donor D8-1 is the same as that for synthesizing D2-1, and according to the synthesis of the compound 1, the same procedure is followed to synthesize the compound 8. The yield was 43%. The mass of the molecular ions determined by mass spectrometry was: 884.39 (calculated 885.14). Theoretical element content: c₅₇H₅₂N₆O₂S, C, 77.35; h, 5.92; n, 9.49; o, 3.62; s,3.62 actually measured element content: c, 77.52; h,5.10N, 9.45; o, 3.48; and S, 3.66.

Example 9: synthesis of Compound 9.

According toIn the above synthetic route, the procedure for synthesizing the donor D9-1 is the same as that for synthesizing D2-1, and the compound 9 is synthesized in accordance with the synthesis of the compound 1. The yield was 63%. The mass of the molecular ions determined by mass spectrometry was: 998.43 (calculated value: 999.29. theoretical element content: C)₆₆H₅₈N₆O₂S, C, 79.33; h, 5.85; n, 8.41; o, 3.20; s,3.21 actually measured element content: c, 77.23; h,5.30N, 9.35; o, 3.55; and S, 3.52.

Example 10:

taking the compound 1 as an example, a series of properties of the fluorescent probe p-RSP-TPA are measured.

Preparing a fluorescent probe mother solution: 83.4mg of the fluorescent probe prepared in example 1 was weighed, dissolved in DMSO, and the volume was adjusted to the scale line with a 10mL volumetric flask to prepare a 1mM fluorescent probe stock solution, which was stored in a refrigerator at 4 ℃.

Preparing a metal ion solution to be detected: preparing each metal chloride salt into 1mM solution to be tested by using deionized water, and storing the solution in a refrigerator at 4 ℃. The metal ions include: zn²⁺、Ca²⁺、Ni²⁺、Co²⁺、Cu²⁺、Fe³⁺Mg²⁺、Na⁺、K⁺、Mn²⁺、Pb²⁺、Ag⁺、Hg^{2 +}。

All tests were performed at room temperature.

100 mu L of the prepared 1mM p-RSP-TPA solution is put into a 10mL volumetric flask, 9mL, 8mL, 7mL, 6mL, 5mL, 4mL, 3mL, 2mL and 1mL of deionized water are respectively measured into 9 10mL volumetric flasks by a pipette, after the solutions are uniformly mixed, the volume is determined to a scale mark by a THF solution, and a 10 mu M p-RSP-TPA fluorescent probe solution with the water content (water/tetrahydrofuran, v/v) of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% and 10% is obtained. Then, fluorescence spectrum measurement was carried out with an excitation wavelength of 355nm and a slit width of 5 nm. As a result, as shown in FIG. 1, the fluorescence intensity was weak at a water content of 0% to 60% in the solution, and the emission peak was gradually red-shifted from 485nm to 515nm as the water content increased. At water contents above 60%, the fluorescence intensity increased and reached a maximum at 475nm at water contents of 70%, with the spectrum blue shifted from 515nm to 475 nm. Then as the water content increased to 80%, the fluorescence intensity gradually decreased and the spectrum continued to blue shift. When the water content increased to 90%, there were two emission peaks at 460nm and 595nm, respectively. The test conditions were selected to be 60% moisture.

Example 11:

diluting 1mM p-RSP-TPA solution to 20 μ M with tetrahydrofuran, taking 1mL of the diluted 20 μ M p-RSP-TPA solution in a 2mL volumetric flask, respectively adding hydrochloric acid solution with pH of 1-6 and deionized water,

and NaOH solution with the pH of 8-14 is added to a constant volume to a scale mark, the concentration of the mixed fluorescent probe solution is 10 mu M, and the volume ratio of tetrahydrofuran to water in the mixed solution is 1: 1. the solution was subjected to fluorescence spectroscopy with an excitation wavelength of 355nm and a slit width of 5 nm. The results are shown in FIG. 2, where pH ranges from 2 to 10 have substantially no effect on fluorescence. When the pH is greater than 10, the fluorescence gradually quenches. When pH 12, there was essentially no fluorescence. The fluorescent probe has fluorescence emission under the conditions from acidity to alkalescence, and the fluorescence is quenched when the alkalinity is strong. Water and an organic solvent which are neutral in pH are selected as a common reaction solution.

Example 12:

diluting 1mM p-RSP-TPA solution to 25 μ M with tetrahydrofuran, collecting 800 μ L diluted p-RSP-TPA solution in 2mL volumetric flask, adding Hg with different concentrations²⁺The solution is subjected to constant volume to scale marks to obtain the fluorescent probe with the concentration of 10 mu M and Hg^{2 +}Mixed solutions at concentrations of 12. mu.M, 10. mu.M, 9. mu.M, 8. mu.M, 7. mu.M, 6. mu.M, 5. mu.M, 4. mu.M, 3. mu.M, 2. mu.M, and 1. mu.M, respectively. Fluorescence spectrum test was carried out with an excitation wavelength of 355nm and a slit width of 5 nm. The results are shown in FIG. 3, as Hg is plotted²⁺The increase in concentration, the intensity of fluorescence at 515nm gradually decreased and gradually disappeared, gradually blue-shifted. Meanwhile, a fluorescence peak appears at 580nm, gradually red-shifted to 595nm when Hg is present²⁺At a concentration of 12. mu.M, the fluorescence intensity at 595nm reached a maximum.

Example 13:

diluting 1mM p-RSP-TPA solution to 25 μ M with tetrahydrofuran, placing 800 μ L diluted p-RSP-TPA solution in 2mL volumetric flask, adding different concentrationsDegree of Hg²⁺The solution is subjected to constant volume to scale marks to obtain the fluorescent probe with the concentration of 10 mu M and Hg^{2 +}Mixed solutions at concentrations of 12. mu.M, 10. mu.M, 9. mu.M, 8. mu.M, 7. mu.M, 6. mu.M, 5. mu.M, 4. mu.M, 3. mu.M, 2. mu.M, and 1. mu.M, respectively. And (5) carrying out ultraviolet absorption spectrum test. The results are shown in FIG. 4, without the addition of Hg²⁺When the solution had almost no absorption peak at 570nm, Hg was added²⁺Thereafter, the absorption peak dissolved at 570nm gradually increased, and Hg²⁺The maximum value is reached at a concentration of 12. mu.M. The ratio of the absorption intensity at 570nm to that at 276nm, i.e. A, is calculated₅₇₀/A₂₇₆. The results are shown in FIG. 5, HgCl₂The concentration has a good linear relationship in the range of 0-5. mu.M.

Example 14:

diluting 1mM of p-RSP-TPA solution to 25 mu M by using tetrahydrofuran, diluting 1mM of each metal cation solution to 167 mu M by using deionized water, putting 800 mu L of diluted 25 mu M of fluorescent probe solution into a 2mL volumetric flask, adding the diluted 167 mu M of each metal cation solution into a 2mL volumetric flask, and reaching a scale mark to obtain a mixed solution to be tested, wherein the concentration of the fluorescent probe is 10 mu M, the concentration of the metal cations is 100 mu M, and the volume ratio of the tetrahydrofuran to the water in the solution to be tested is 2/3. Fluorescence spectrum test was carried out with an excitation wavelength of 355nm and a slit width of 5 nm. The metal cation solution is respectively magnesium chloride, sodium chloride, potassium chloride, aluminum chloride, ferric chloride, manganese chloride, mercuric chloride, zinc chloride, nickel chloride, mercuric chloride, cobalt chloride and silver nitrate solution. The results are shown in FIG. 6, only after addition of Hg²⁺After that, there was an emission peak at 595 nm. Shows that the fluorescent probe p-RSP-TPA can selectively identify metal Hg²⁺。

Example 15:

cell bioimaging tests. At 5% CO₂And HeLa cells were cultured in high-glucose DMEM minimal essential medium containing 10% fetal bovine serum and 1% double antibody (mixed solution of streptomycin) at 37 ℃ for 30 min. HeLa cells were ratiometrically imaged using a laser scanning confocal microscope (LeicaDMI 8). The blue channel is 450-490 nm, the red channel is 570-610 nm, and the excitation wavelength is 405 nm. Cells were washed three times with HBSS buffer solution prior to imaging. The scaled image was processed using the image analysis program ImageJ. Proportional Hg can be performed in HeLa cells using confocal laser scanning microscope²⁺Imaging of (2). In the absence of Hg²⁺In the case of (2 h) HeLa cells stained with p-RSP-TPA (10. mu.M) showed fluorescence emission in the blue channel (450 to 490nm) and only weak fluorescence in the red channel (570 to 610nm), indicating no Hg in the cells²⁺. Further 10. mu.M and 50. mu.M of Hg²⁺After the materials are respectively added into the culture medium to react for 30min, the fluorescence intensity in a blue channel is reduced, the fluorescence in a red channel is obviously enhanced, and a proportion image is changed from green to orange, which shows that the proportion changes with Hg²⁺With increasing concentration, the fluorescence emission will change accordingly, with a red-shift in emission, as shown in FIG. 7. Specific sample compositions and fluorescence intensity vs. ratio table 1:

table 1: sample composition and fluorescence intensity control

The result proves that the p-RSP-TPA can be used as a ratio metering type imaging agent with good effect to be applied to biological Hg²⁺And (6) detecting.

Example 16:

1.0g of polyvinyl alcohol PVA (molecular weight of 75000), 1.0g of polyethylene glycol PEG (molecular weight of 20000), and 5g of a 10% polyacrylamide (PAM, molecular weight of 400000-800000) aqueous solution were dissolved in 20mL of water, and the solution was heated at 95 ℃ for 8 hours to obtain a mixed solution of 3.7% PVA, 3.7% PEG and 18.5% PAM in terms of mass fraction. Coating the mixture on filter paper, and drying the filter paper at 60 ℃. The filter paper was then coated with 10. mu.M p-RSP-TPA (ethanol as solvent) and dried at 60 ℃. Obtaining the detection paper containing the p-RSP-TPA fluorescent probe reagent. The structural schematic diagram of the detection paper is shown in figure 8, and the detection paper comprises a fluorescent probe dye layer, a polyethylene glycol/polyvinyl alcohol/polyacrylamide layer and a filter paper substrate layer from top to bottom.

Dipping various metal cation solutions (C) by using a sponge₂H₅OH/H₂O-3/2, 100 μ M) is dipped and coated on the prepared detection paper material to realize the detection of the fluorescent probe material on the metal ions; the fluorescence change of the contact area of the detection paper with the metal cation is observed under the excitation of a UV lamp at 365 nm. The material meets Hg²⁺After ionization, red fluorescence can be emitted under the irradiation of a 365nm ultraviolet lamp. When meeting other metal cations, the fluorescence is not changed under the irradiation of an ultraviolet lamp. The results are shown in FIG. 9, which is a graph of the effect of the first row, from left to right, after dipping in a solution containing 3/2 metal cations in a volume ratio of ethanol to water, under an ultraviolet lamp: zn²⁺、Ca²⁺、Ni²⁺、Co²⁺、Cu²⁺、Fe³⁺The second row sequentially dipping Mg²⁺、Na⁺、K⁺、Mn²⁺、Pb²⁺、Ag⁺The third row is the dipping of Hg containing 3/2 ethanol/water volume ratio²⁺The latter effect diagram. The first and second rows were dipped under an ultraviolet lamp and contained Hg as compared to the third row²⁺The test paper of the solution fluoresces red. Whereas the first and second rows did not change significantly in fluorescence. The result proves that the p-RSP-TPA detection paper material can be applied to rapidly detecting whether the solution contains Hg²⁺。