A dual-channel detection of Hg 2+ and Ag + Near-infrared fluorescent probe and preparation method and application thereof

Abstract

The invention discloses a method for dual-channel detection of Hg 2+ and Ag + The invention discloses a near-infrared fluorescent probe and a preparation method and application thereof, wherein the chemical structure of the probe is as shown in formula (I). The preparation method comprises the steps of mixing POCl3 and N,N-dihydroxyethylaniline to obtain 4-(bis(2-chloroethyl)amino)benzaldehyde; reacting sodium ethoxide with mercaptoethanol, and then adding 4-(bis(2-chloroethyl)amino)benzaldehyde to obtain 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde; and finally mixing 2-(1-(9-ethyl-9-hydro-carbazole-3-ethyl)malononitrile with 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde to obtain a near-infrared fluorescent probe for dual-channel detection of Hg. 2+ and Ag + The steps of the near-infrared fluorescent probe have the advantages of strong stability, fast detection speed, good selectivity, high sensitivity and accuracy.

Description

Technical Field

[0001] The present invention relates to a near-infrared fluorescent probe, in particular to a probe for dual-channel detection of Hg 2+ and Ag + Near-infrared fluorescent probe and its preparation method and application. Background Art

[0002] Among heavy metal pollution, mercury is the most common pollutant and the most harmful pollutant. Trace amounts of mercury can cause harm to the environment and even people's lives and health. Mercury and its compounds have been widely used in industrial production, electrical instruments, pharmaceutical manufacturing, metal refining and military industry. With the large-scale use of mercury and its compounds and the unreasonable treatment of mercury-containing wastes, the water, air and soil environments have been seriously harmed, and at the same time, it has also posed a great threat to human safety. At the same time, monitoring the health of workers who have been exposed to high-concentration mercury environments for a long time has found that the neurobehavioral functions of workers who have been exposed to high-concentration mercury for a long time have declined significantly, mainly in terms of visual perception, psychological stability, hand reaction ability and attention.

[0003] As one of the metal pollutants, silver is not only biologically toxic, but also can pollute soil and water. + It can enter the hydra through skin loading, feeding, etc., causing acute poisoning and strong stress response. + It also affects the activities of fungi, bacteria, algae, viruses and some small protists in the microbial world. + When it occurs, it can cause a depolarization effect on the cell membrane, inducing Na + and Ca 2+ Influx eventually leads to cell necrosis. + It easily combines with proteins in the body, affecting people's metabolism and the normal functioning of various organs.

[0004] At present, the detection of heavy metal ions mainly includes ultraviolet visible spectrophotometry, atomic fluorescence spectroscopy, and inductively coupled plasma mass spectrometry. The above methods have cumbersome detection steps, high costs, and require professional operators. Therefore, it is of great significance to develop a heavy metal detection probe with low cost, fast detection, high sensitivity, good stability, and strong specificity.

[0005] Fluorescent probes are widely used in the detection of metal ions due to their ease of operation, good selectivity, high spatial and temporal resolution, and high sensitivity. These probes have important application prospects in the fields of fluorescence sensing, quantitative analysis, and biomedicine. Near-infrared (NIR) fluorescent probes are widely used due to their less background interference, stronger tissue penetration, and less tissue damage. At present, there are many studies on dual-channel detection of Hg2+ and Ag + There are no reports on the research of small molecule near-infrared fluorescent probes. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting Hg in a dual channel with strong stability, fast detection speed, good selectivity, high sensitivity and accuracy. 2+ and Ag + Near-infrared fluorescent probe and its preparation method and application.

[0007] The technical solution adopted by the present invention to solve the above technical problems is: a method for dual-channel detection of Hg 2+ and Ag + A near-infrared fluorescent probe, wherein the chemical structure of the near-infrared fluorescent probe is shown in the following formula (I):

[0008]

[0009] The present invention also provides the above-mentioned dual-channel detection of Hg 2+ and Ag + The preparation method of the near-infrared fluorescent probe comprises the following steps:

[0010] Step 1, mixing a dimethylformamide solution containing POCl3 and N,N-dihydroxyethylaniline to obtain 4-(bis(2-chloroethyl)amino)benzaldehyde;

[0011] Step 2, reacting the ethanol solution containing sodium ethoxide with mercaptoethanol, adding 4-(bis(2-chloroethyl)amino)benzaldehyde to react completely, extracting with dichloromethane, washing with saturated brine, drying with a desiccant, and then performing silica gel column chromatography to obtain 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde;

[0012] Step 3: Mix 2-(1-(9-ethyl-9-hydrogen-carbazole-3-ethylidene)malononitrile and 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde, use piperidine as a catalyst, and perform silica gel column chromatography after the reaction is complete to obtain a dual-channel detection method for Hg 2+ and Ag + A near-infrared fluorescent probe, whose chemical structure is shown in the following formula (I):

[0013]

[0014] Further, the specific steps are as follows:

[0015] Step 1, 5.5-6.5 mL of POCl3 is slowly added to 15.5-17.50 mL of dimethylformamide, stirred at 0°C for 0.5 h, then 6-7.5 g of N, N-dihydroxyethylaniline is added, and the reaction is continued at 90°C for 2.5-3.5 h. After the reaction is completed, the reaction product is cooled to room temperature, poured into pure water to terminate the reaction, and the pH is adjusted to 7.0-7.5 with Na2CO3, filtered, and the white solid precipitate is washed, dried and recrystallized to obtain yellow-brown needle-shaped crystals, i.e. 4-(bis(2-chloroethyl)amino)benzaldehyde;

[0016] Step 2, add 0.6-0.8g sodium ethoxide to 20mL ethanol, then add 3-4mL mercaptoethanol, react at room temperature for 1h, then add 3.5-4.5g 4-(bis(2-chloroethyl)amino)benzaldehyde, continue to react at room temperature for 1h, heat to 60°C for 1h, pour the reaction product into pure water to terminate the reaction after the reaction is completed, add 50-100mL dichloromethane for extraction, add 20mL saturated brine to the organic layer for washing, take the upper organic solution, add 10-15g desiccant to the organic solution, remove the desiccant after shaking and filtering, and then use a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:1 as a chromatographic solution, perform silica gel column chromatography, collect the solution with yellow fluorescence in the silica gel column under 365nm ultraviolet light, and rotary evaporate the collected solution at 50°C to obtain a yellow solid, i.e. 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde;

[0017] Step 3: 300-330 mg of 2-(1-(9-ethyl-9-hydrogen-carbazole-3)ethylidene)malononitrile and 300-350 mg of 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde were mixed, 20 mL of ethanol was added, and 300-500 μL of piperidine was added dropwise as a reaction catalyst. The mixture was heated to reflux at 60° C. overnight. After the reaction was completed, the reaction product was filtered through an organic filter membrane with a pore size of 0.22 μm. The filtered solution was subjected to silica gel column chromatography. A mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1 was used as a chromatographic solution. Under irradiation with a 365 nm ultraviolet lamp, an oily liquid with red fluorescence in the silica gel column was taken, which was the product for dual-channel detection of Hg. 2+ and Ag + A near-infrared fluorescent probe, whose chemical structure is shown in the above formula (I).

[0018] Further, the desiccant described in step 2 is any one of magnesium oxide, calcium oxide, calcium sulfate, copper sulfate, sodium sulfate and magnesium sulfate.

[0019] The present invention also provides the above-mentioned near-infrared fluorescent probe to simultaneously detect Ag + and Hg2+ The method comprises the following steps:

[0020] Step 1, instrument parameter setting: set the excitation wavelength of the fluorescence spectrophotometer to 420nm, the emission band to 500-800nm, the photomultiplier voltage to 600V, the slit width to 5nm, and use a quartz cuvette with four sides transparent to light;

[0021] Step 2, dissolving the near-infrared fluorescent probe in a mixed solvent of methanol and HEPES buffer in a volume ratio of (4-5): (6-5) to obtain a near-infrared fluorescent probe solution with a concentration of 10 μmol / L, adjusting the pH of the near-infrared fluorescent probe solution to 5.5-6.5, mixing the near-infrared fluorescent probe solution with the test solution in a volume ratio of 9:1, reacting for 30 seconds, and then using a fluorescence spectrophotometer to measure the fluorescence intensity at 610 nm. + and Hg 2+ The linear relationship between the total concentrations is calculated to obtain the Ag in the test solution. + and Hg 2+ Total concentration;

[0022] Step 3: Ag + Concentration determination: After adjusting the pH of the near-infrared fluorescent probe solution prepared in step 2 to 7-11, the near-infrared fluorescent probe solution and the test solution were mixed in a volume ratio of 9:1, reacted for 30 seconds, and then the fluorescence intensity at 610 nm was measured by a fluorescence spectrophotometer. + The linear relationship between the concentrations is calculated to obtain the Ag in the test solution. + concentration;

[0023] Step 4: Hg 2+ Concentration determination: With the same volume of the test liquid, + and Hg 2+ The total concentration minus Ag + The concentration of Hg in the test solution 2+ concentration.

[0024] Preferably, the volume ratio of methanol to HEPES buffer in the mixed solvent described in step 2 is 4.5:5.5.

[0025] Compared with the prior art, the advantages of the present invention are: 2+ and Ag +The invention discloses a near-infrared fluorescent probe and a preparation method and application thereof. The near-infrared fluorescent probe prepared by the cyclization reaction of the 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde recognition group is basically not affected by the environmental pH and other interfering ions and has strong stability. At the same time, the desulfurization effect produced by the recognition group combined with the 2-(1-(9-ethyl-9H-carbazole)ethylidene)malononitrile fluorophore can realize the detection of Ag under different pH conditions. + and Hg 2+ In addition, the near-infrared fluorescent probe can complete the determination of Ag within 30 seconds. + and Hg 2+ The detection speed is fast. 2+ and Ag + It has high selectivity and can produce visible staining changes. Cytotoxicity experiments show that the half-lethal concentration of the probe for Hela cells is 30μmol / L, the cytotoxicity is low, and it can penetrate the cell membrane to detect Hg in the cell. 2+ Perform imaging tests.

[0026] In summary, the present invention provides a method for dual-channel detection of Hg 2+ and Ag + The near-infrared fluorescent probe and its preparation method and application are used to detect Hg 2+ It can coordinate with groups containing thio substituents (such as thioamide, thiourea, thioketone, etc.) to undergo desulfurization reaction, while utilizing Ag + Cyclization reaction with sulfur-containing substituents is carried out to achieve Hg 2+ and Ag + It has the advantages of strong stability, fast detection speed, good selectivity, high sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a flow chart of the synthesis steps of the near-infrared fluorescent probe in the first specific embodiment;

[0028] Figure 2 is the nuclear magnetic resonance hydrogen spectrum of compound 2 (a) and near-infrared fluorescent probe (b) in specific example 1;

[0029] Figure 3 The results of the effect of the ratio of methanol to HEPES buffer solution in the mixed solvent on the fluorescence intensity of the near-infrared fluorescent probe in the specific embodiment 3;

[0030] Figure 4 The detection effect of the near-infrared fluorescent probe on mercury ions (a) and silver ions (b) at different pH values ​​in the specific embodiment 3;

[0031] Figure 5is the selective ability measurement result of the near-infrared fluorescent probe in the specific embodiment 4, wherein a is the selective ultraviolet absorption intensity value of the near-infrared fluorescent probe for various ions, b is the selective fluorescence intensity value of the near-infrared fluorescent probe for various ions, and c is the color change diagram of the near-infrared fluorescent probe solution after adding various ions;

[0032] Figure 6 The near-infrared fluorescent probe for Hg 2+ The sensitivity measurement results of a are as follows: 2+ The fluorescence intensity value of the near-infrared fluorescence probe changes with the concentration of Hg 2+ The UV absorption intensity value of the concentration change, c is the near-infrared fluorescence probe with Hg 2+ Linear curve of concentration change;

[0033] Figure 7 The near-infrared fluorescent probe for Ag in the specific embodiment 5 + Sensitivity measurement results, a is the near-infrared fluorescent probe with Ag + The fluorescence intensity value of the near-infrared fluorescent probe changes with the concentration of Ag. + The UV absorption intensity value of the concentration change, c is the near-infrared fluorescence probe with Ag + Linear curve of concentration change;

[0034] Figure 8 The near-infrared fluorescent probe in the specific embodiment 6 and the addition of Hg 2+ Imaging of near-infrared fluorescent probes in cells;

[0035] Fig. 9 The results of the near-infrared fluorescent probe cytotoxicity experiment in the specific embodiment 6, wherein a is the relationship between different concentrations of near-infrared fluorescent probe and cell half inhibition rate, and b is the LC of the near-infrared fluorescent probe on the cell 50 Half lethal concentration curve. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below with reference to the accompanying drawings.

[0037] The structure of the compound is determined by nuclear magnetic resonance (abbreviated as NMR) and / or mass spectrometry (abbreviated as MS). NMR is measured using a nuclear magnetic spectrometer (brand: Bruker; model: ADVANCE III 500MHz), the measurement solvent is deuterated chloroform (CDCl3), deuterated dimethyl sulfoxide (DMSO-d6), and the internal standard is tetramethylsilane. The thin layer chromatography silica gel plate used for MS measurement uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, and the silica gel plate used in thin layer chromatography uses a specification of 0.20mm-0.25mm. Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as a carrier.

[0038] Specific embodiment 1: A method for dual-channel detection of Hg 2+ and Ag + A near-infrared fluorescent probe, whose chemical structure is shown in the following formula (I):

[0039]

[0040] The preparation method of the above-mentioned near-infrared fluorescent probe is as follows Figure 1 As shown, the following steps are included:

[0041] Step 1, 5.5-6.5 mL of POCl3 is slowly added to 15.5-17.50 mL of dimethylformamide, stirred at 0°C for 0.5 h, then 6-7.5 g of N, N-dihydroxyethylaniline is added, and the reaction is continued at 90°C for 2.5-3.5 h. The reaction progress is tracked by thin layer chromatography. When the silica gel plate shows only one band under 365 nm ultraviolet light, the reaction is completed. The reaction product is cooled to room temperature and poured into pure water to terminate the reaction. The pH is adjusted to 7.0-7.5 with Na2CO3, filtered, and the white solid precipitate is washed, dried and recrystallized to obtain yellow-brown needle-shaped crystals, i.e., 4-(bis(2-chloroethyl)amino)benzaldehyde, abbreviated as compound 1, with a yield of 73-87%.

[0042] Step 2, add 0.6-0.8g sodium ethoxide to 20mL ethanol, then add 3-4mL mercaptoethanol, react at room temperature for 1h, add 3.5-4.5g compound 1, continue to react at room temperature for 1h, heat to 60℃ for 1h, track the reaction progress by thin layer chromatography, when the silica gel plate shows only one band under 365nm ultraviolet light, the reaction is complete, pour the reaction product into pure water to terminate the reaction, add 50-100mL dichloromethane for extraction, add 20mL saturated brine to the organic layer for washing, then take the upper organic solution, add to the organic 10-15g of desiccant (the desiccant is any one of magnesium oxide, calcium oxide, calcium sulfate, copper sulfate, sodium sulfate and magnesium sulfate) is added to the solution, and the desiccant is removed after shaking and filtering. Then, a mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1 is used as a chromatographic liquid for silica gel column chromatography. Under irradiation with a 365nm ultraviolet lamp, the solution with yellow fluorescence in the silica gel column is collected, and the collected solution is rotary evaporated at 50°C to obtain a yellow solid, namely 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde, abbreviated as compound 2, with a yield of 65-76%. The nuclear magnetic resonance spectrum is as follows Figure 2 As shown in a, 1H NMR (600 MHz, Chloroform-d) δ9.72 (s, 1H), 7.73 (d, J = 8.9 Hz, 2H), 6.70 (d, J = 8.9 Hz, 2H), 3.79 (t, J = 5.9 Hz, 4H), 3.70-3.63 (m, 4H), 2.85-2.74 (m, 8H), indicating that the target substance 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde was successfully synthesized.

[0043] Step 3, weigh 300-330 mg of 2-(1-(9-ethyl-9-hydrogen-carbazole-3)ethylidene)malononitrile (abbreviated as compound 3) and 300-350 mg of compound 2 and place them in a flask, add 20 mL of ethanol to the beaker, and drop 300-500 μL of piperidine as a reaction catalyst, heat and reflux at 60° C. overnight, and track the reaction progress by thin layer chromatography. When the silica gel plate shows only one band under 365 nm ultraviolet light, the reaction is completed. Then, an organic filter membrane with a pore size of 0.22 μm is used to filter the reaction product, and the filtered solution is subjected to silica gel column chromatography, using a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:1 as a chromatographic solution. Under 365 nm ultraviolet light, an oily liquid with red fluorescence in the silica gel column is taken, which is used for dual-channel detection of Hg 2+ and Ag + The yield of near-infrared fluorescent probe is 43%-50%. Figure 2 As shown in b, 1H NMR(500MHz,Chloroform-d)δ8.23-7.83(m,2H),7.59-7.11(m,8H),6.88(d,J=15.2Hz,1H),6.60(d,J=8.5Hz,2H),4.37(q, J=6.6Hz,2H),3.67(dt,J=74.6,6.3Hz,8H),2.73(q,J=6.0,5.2Hz,10H),1.46(t,J=7.0Hz,3H).HRESIMS(m / z):calcd.forC 34 H 37 O2N4S2,597.23, indicating that the target substance was successfully synthesized, and its chemical structure is shown in the above formula (I).

[0044] Specific Example 2: Detection of Ag by using the near-infrared fluorescent probe prepared by the method of Specific Example 1 + and Hg 2+ The method comprises the following steps:

[0045] Step 1, instrument parameter setting: set the excitation wavelength of the F-4600 fluorescence spectrophotometer to 420nm, the emission band to 500-800nm, the photomultiplier voltage to 600V, the slit width to 5nm, and use a quartz cuvette with four sides transparent to light;

[0046] Step 2: Ag + and Hg 2+ Total concentration determination: The near-infrared fluorescent probe was dissolved in a mixed solvent of methanol and HEPES buffer at a volume ratio of 4.5:5.5 to obtain a near-infrared fluorescent probe solution with a concentration of 10 μmol / L. The pH of the near-infrared fluorescent probe solution was adjusted to 5.5-6.5 with hydrochloric acid, and 900 μL of the near-infrared fluorescent probe solution was mixed with 100 μL of the test solution. The reaction was continued for 30 seconds, and then the fluorescence intensity at 610 nm was measured by a fluorescence spectrophotometer. The fluorescence intensity value was compared with the Ag + and Hg 2+ The linear relationship between the total concentrations is calculated to obtain the Ag in the test solution. + and Hg 2+ Total concentration;

[0047] Step 3: Ag + Concentration determination: adjust the pH of the near-infrared fluorescent probe solution prepared in step 2 to 7-11 with sodium hydroxide, take 900 μL of the near-infrared fluorescent probe solution and mix it with 100 μL of the test solution, react for 30 seconds, and then use a fluorescence spectrophotometer to measure the fluorescence intensity at 610 nm. + The linear relationship between the concentrations is calculated to obtain the Ag in the test solution. + concentration;

[0048] Step 4: Hg 2+ Concentration determination: With the same volume of the test liquid, add + and Hg 2+ The total concentration minus Ag + The concentration of Hg in the test solution 2+ concentration.

[0049] Specific embodiment 3: Using the method of specific embodiment 2 to carry out Hg 2+ and Ag + Optimization of detection conditions.

[0050] 1. Screening of solvent system: The volume ratios of methanol and HEPES buffer in the mixed solvent of step 2 of specific embodiment 2 were set to 0:10, 0.5:9.5, 1:9, 1.5:8.5, 2:8, 2.5:7.5, 3:7, 3.5:6.5, 4:6, 4.5:5.5, and 5:5, respectively. The near-infrared fluorescent probe was dissolved in the mixed solvents in different proportions to make the final concentration of the near-infrared fluorescent probe be 10 μmol / L. The fluorescence intensity of the near-infrared fluorescent probe at 610 nm was measured by a fluorescence spectrophotometer, and the optimal solvent ratio was screened according to the fluorescence intensity.

[0051] The results are as follows Figure 3 As shown in the figure, the fluorescence intensity of the near-infrared fluorescent probe gradually increases with the increasing proportion of the organic phase in the mixed solvent. When the volume ratio of methanol to HEPES buffer is 4.5:5.5, the near-infrared fluorescent probe obtains the maximum fluorescence intensity. When the organic phase proportion further increases, the fluorescence intensity begins to weaken. Therefore, the mixed volume ratio of methanol to HEPES buffer is 4.5:5.5 as the optimal mixed solvent ratio.

[0052] 2. Optimization of pH setting: The pH values ​​of the near-infrared fluorescent probe solution prepared in step 2 of the specific embodiment 2 were set to 3, 4, 5, 6, 7, 8, 9, 10, and 11, and then the fluorescence intensity of the near-infrared fluorescent probe at 610 nm was measured by a fluorescence spectrophotometer. Then, 900 μL of the near-infrared fluorescent probe solution and 100 μL of Hg-containing 2+ and Ag + Aqueous solutions with a total concentration of 50 μmol / L were mixed and reacted for 30 seconds. The fluorescence intensity of the near-infrared fluorescent probe at 610 nm was measured by a fluorescence spectrophotometer, and the fluorescence intensity values ​​at 610 nm were used to draw a dot-line graph.

[0053] The results are as follows Figure 4 As shown, the fluorescence intensity of the near-infrared fluorescent probe has basically no significant change in the pH range of 3 to 11, and is relatively stable; Figure 4The near-infrared fluorescent probe and Ag-containing + and Hg 2+ After mixing with Hg aqueous solution, the fluorescence intensity decreased significantly in the pH range of 5.5-6.5. When the pH was 7-11, the fluorescence intensity of the near-infrared fluorescent probe was not affected by 2+ This indicates that the near-infrared fluorescent probe can only detect Hg in the pH range of 5.5 to 6.5. 2+ Response, at pH 7-11 to Hg 2+ No response. Figure 4 Middle b shows that the near-infrared fluorescent probe is sensitive to Ag + The detection effect is less affected by pH, and the detection effect is stable for Ag in the concentration range of pH 5.5 to 11.0. + There is an obvious fluorescence response, and with the change of pH value, the near-infrared fluorescent probe is sensitive to Ag. + The responses were basically the same.

[0054] Specific embodiment 4: The method of specific embodiment 2 is used to measure the selectivity of the near-infrared fluorescent probe.

[0055] Experimental method: Deionized water was used to prepare 21 common cation and anion solutions, including Na + , K + 、Al 3+ , Ca 2 + Mg 2+ , Fe 2+ , Fe 3+ , Cu 2+ 、Cd 2+ Cr 3+ Cr 6+ 、Zn 2+ 、Ni 2+ 、Co 2+ , Ba 2+ , Mn 2+ , Pb 2+ NH 4+ 、HCO3 - 、CO3 2- and HPO4 2- The concentration of each solution was 100 μmol / L. At the same time, Hg 2+ solution and Ag with a concentration of 50 μmol / L +Solution. A fluorescence spectrophotometer was used to measure the ultraviolet absorption intensity at 510nm and the fluorescence intensity at 610nm of a near-infrared fluorescent probe solution with a pH of 6 and a concentration of 10μmol / L. Then, 100μL of each pre-prepared ion solution was added to 900μL of the near-infrared fluorescent probe solution. The reaction lasted for 30s. The ultraviolet absorption intensity at 510nm and the fluorescence intensity at 610nm of the near-infrared fluorescent probe with different ions added were measured respectively. At the same time, pictures of the near-infrared fluorescent probe solution with different ions added were taken under a fluorescent lamp. The results are shown in the figure. Figure 5 shown.

[0056] Depend on Figure 5 As shown in Figure a, the ultraviolet absorption peak of the near-infrared fluorescent probe appears at 510 nm. 2+ After that, the ultraviolet absorption peak of the near-infrared fluorescent probe was significantly blue-shifted from 510nm to 360nm, and the ultraviolet absorption peak of the near-infrared fluorescent probe was + After adding ions, the blue shift from 510nm to 450nm, but after adding other ion solutions, the ultraviolet absorption peak of the near-infrared fluorescent probe did not change significantly. Figure 5 As shown in Figure b, the fluorescence emission peak of the near-infrared fluorescent probe appears at 610 nm. 2+ After that, the fluorescence intensity of the near-infrared fluorescent probe decreased significantly and slightly blue-shifted from 610nm to 598nm. + After adding Ag + It had dropped 3 times before, but after adding other ion solutions, the fluorescence emission peak of the near-infrared fluorescent probe did not change significantly. Figure 5 From c, we can see that when Hg is added 2+ or Ag + After addition of ions, the color of the near-infrared fluorescent probe solution changed significantly from light red to light yellow, but after adding other ion solutions, the color of the near-infrared fluorescent probe solution did not change. The above conclusions prove that the near-infrared fluorescent probe can detect Hg 2+ and Ag + Has good selectivity and can be used for Hg 2+ and Ag + Dual channel detection.

[0057] Specific Example 5: Using the method of Specific Example 2 to carry out Hg 2+ and Ag + Sensitivity determination.

[0058] Hg 2+ Sensitivity test: the concentrations were 0, 0.5μmol / L, 1μmol / L, 5μmol / L,

[0059] 10μmol / L, 15μmol / L, 30μmol / L, 50μmol / L Hg 2+ 100 μL of Hg 2+ The standard solution was added to 900 μL of a near-infrared fluorescent probe solution with a pH of 5.5-6.5 and a concentration of 10 μmol / L, and the reaction was continued for 30 seconds. 2+ The ultraviolet absorption intensity of the near-infrared fluorescent probe at 510nm and the fluorescence intensity at 610nm.

[0060] result Figure 6 As shown in a, with the increase of Hg 2+ With the increase of Hg concentration, the fluorescence intensity of the near-infrared fluorescent probe at 610 nm gradually decreased. 2+ When the concentration is 50 μmol / L, the fluorescence intensity of the near-infrared fluorescent probe tends to be stable. Figure 6 As shown in b, the ultraviolet absorption of the near-infrared fluorescent probe at 510 nm is obviously blue-shifted. 2+ When the concentration is 50 μmol / L, the ultraviolet absorption position of the near-infrared fluorescent probe basically does not change. Figure 6 As shown in c, when Hg 2+ The fluorescence intensity of the near-infrared fluorescent probe is similar to that of Hg 2+ There is a good linear relationship between the concentrations y = -57.30x + 1622.55, and the detection limit is 0.52μmol / L, which can be used for Hg in the test solution of unknown concentration. 2+ Concentration detection.

[0061] Ag + Sensitivity test: the concentrations were 0, 0.5μmol / L, 1μmol / L, 5μmol / L,

[0062] 10μmol / L, 15μmol / L, 30μmol / L, 50μmol / L Ag + Standard solution, 100 μL of different concentrations of Ag + The standard solution was added to 900 μL of a near-infrared fluorescent probe solution with a pH of 7-11 and a concentration of 10 μmol / L, and the reaction was continued for 30 seconds. + The ultraviolet absorption intensity of the near-infrared fluorescent probe at 510nm and the fluorescence intensity at 610nm.

[0063] The results are as follows Figure 7 As shown in a, with Ag +With the increase of concentration, the fluorescence intensity of the near-infrared fluorescent probe at 610 nm gradually decreased. + When the concentration is 50 μmol / L, the fluorescence intensity of the near-infrared fluorescent probe tends to be stable. Figure 7 As shown in b, with the Ag + With the increase of concentration, the ultraviolet absorption of the near-infrared fluorescent probe at 510nm gradually shifts to the blue. + When the concentration is 50 μmol / L, the ultraviolet absorption of the near-infrared fluorescent probe remains basically unchanged. Figure 7 As shown in c, when Ag + The fluorescence intensity of the near-infrared fluorescent probe is similar to that of Ag in the concentration range of 0-15 μmol / L. + The concentration of Ag is linearly related to the concentration of Ag in the test solution. + Concentration detection.

[0064] Specific embodiment 6: The method of specific embodiment 2 is used to carry out cell imaging and toxicity experiments of near-infrared fluorescent probes.

[0065] Cell imaging experiment: 20 μL of near-infrared fluorescent probe solution with a pH of 6 and a concentration of 10 μmol / L was mixed with 100 μL of 5×10 4 Cells / mL of Hela cell solution was added to each well of a 96-well culture plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. Cell fluorescence images were collected at an excitation wavelength of 420 nm, and the cell status was observed under a laser confocal microscope and the fluorescence intensity was recorded. Subsequently, 20 μL of 10 μmol / L Hg 2+ The solution was incubated for 1 h, and the state of the cells was observed under a laser confocal microscope and the fluorescence intensity was recorded.

[0066] The results are as follows Figure 8 As shown in the figure, the cell structure is intact under bright field. Under the excitation wavelength of 420nm, the cell emits red fluorescence, proving that the near-infrared fluorescent probe can penetrate the cell membrane and enter the cell. 2+ After 1 h of incubation, the red fluorescence of the cells weakened, indicating that the near-infrared fluorescent probe can be used to detect Hg in cells. 2+ Detection.

[0067] Cytotoxicity experiment: 20 μL of 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 25 μmol / L, 50 μmol / L and 100 μmol / L near-infrared fluorescent probe solution (pH 6) were mixed with 100 μL of 5×10 4Cells / mL of Hela cell solution were cultured. After incubation for 24 h, cell fluorescence images were collected at an excitation wavelength of 420 nm to observe and record the survival status of the cells, and the half-inhibition rate was calculated using the prism drawing software.

[0068] like Fig. 9 As shown in (a), as the concentration of the near-infrared fluorescent probe increases, the cell survival rate gradually decreases. Fig. 9 Middle b shows the LC of the near-infrared fluorescent probe calculated by logarithmic inversion. 50 , the half lethal concentration of the near-infrared fluorescent probe is 30μmol / L. When the concentration of the near-infrared fluorescent probe is 10μmol / L, the inhibition rate of the near-infrared fluorescent probe on cells is less than 20%, and the cytotoxicity is relatively small. + and Hg 2+ It is feasible to select a near-infrared fluorescent probe concentration of 10 μmol / L in the process. At the same time, this concentration has low cytotoxicity and can be used for intracellular Ag. + and Hg 2+ Detection.

[0069] Specific Example 7: Using the method of Specific Example 2 to measure Hg in actual water samples 2+ and Ag + Carry out determination.

[0070] Deionized water, tap water and reservoir water were selected as samples to explore the use of near-infrared fluorescent probes for Hg in actual water samples. 2+ and Ag + The feasibility of the test,results are shown in Table 1.

[0071] Table 1 Near-infrared fluorescence probe for Hg in water samples 2+ and Ag + The spike recovery

[0072]

[0073] As shown in Table 1, the near-infrared fluorescent probe is 2+ The recovery rates of pure water were between 78.04% and 105.09%, tap water was between 77.02% and 113.8%, and reservoir water was between 95.37% and 109.65%. The recovery rates of Ag+ were between 72.14% and 109.01% for pure water, between 81.21% and 118.52% for tap water, and between 85.47% and 120% for reservoir water, all within the appropriate range, indicating that the detection method has high accuracy and the near-infrared fluorescent probe has the ability to detect Hg in actual samples. 2+ and Ag + potential application value.

[0074] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A dual-channel detection method for Hg 2+ and Ag + A near-infrared fluorescent probe characterized by The chemical structure of the near-infrared fluorescent probe is shown in the following formula (I):

2. A dual-channel detection method for Hg according to claim 1 2+ and Ag + A method for preparing a near-infrared fluorescent probe, characterized in that The following steps are involved: Step 1, mixing a dimethylformamide solution containing POCl3 and N,N-dihydroxyethylaniline to obtain 4-(bis(2-chloroethyl)amino)benzaldehyde; Step 2, reacting the ethanol solution containing sodium ethoxide with mercaptoethanol, adding 4-(bis(2-chloroethyl)amino)benzaldehyde to react completely, extracting with dichloromethane, washing with saturated brine, drying with a desiccant, and then performing silica gel column chromatography to obtain 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde; Step 3: Mix 2-(1-(9-ethyl-9-hydrogen-carbazole-3-ethylidene)malononitrile and 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde, use piperidine as a catalyst, and perform silica gel column chromatography after the reaction is complete to obtain a dual-channel detection method for Hg 2+ and Ag + A near-infrared fluorescent probe, whose chemical structure is shown in the following formula (I):

3. A method for dual-channel detection of Hg as claimed in claim 2 2+ and Ag + A method for preparing a near-infrared fluorescent probe, characterized in that The specific steps are as follows: Step 1, 5.5-6.5 mL of POCl3 is slowly added to 15.5-17.50 mL of dimethylformamide, stirred at 0°C for 0.5 h, then 6-7.5 g of N, N-dihydroxyethylaniline is added, and the reaction is continued at 90°C for 2.5-3.5 h. After the reaction is completed, the reaction product is cooled to room temperature, poured into pure water to terminate the reaction, and the pH is adjusted to 7.0-7.5 with Na2CO3, filtered, and the white solid precipitate is washed, dried and recrystallized to obtain yellow-brown needle-shaped crystals, i.e. 4-(bis(2-chloroethyl)amino)benzaldehyde; Step 2, add 0.6-0.8g sodium ethoxide to 20mL ethanol, then add 3-4mL mercaptoethanol, react at room temperature for 1h, then add 3.5-4.5g 4-(bis(2-chloroethyl)amino)benzaldehyde, continue to react at room temperature for 1h, heat to 60°C for 1h, pour the reaction product into pure water to terminate the reaction after the reaction is completed, add 50-100mL dichloromethane for extraction, add 20mL saturated brine to the organic layer for washing, take the upper organic solution, add 10-15g desiccant to the organic solution, remove the desiccant after shaking and filtering, and then use a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 1:1 as a chromatographic solution, perform silica gel column chromatography, collect the solution with yellow fluorescence in the silica gel column under 365nm ultraviolet light, and rotary evaporate the collected solution at 50°C to obtain a yellow solid, i.e. 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde; Step 3: 300-330 mg of 2-(1-(9-ethyl-9-hydrogen-carbazole-3)ethylidene)malononitrile and 300-350 mg of 4-(bis(2-((2-hydroxyethyl)thio)ethyl)amino)benzaldehyde were mixed, 20 mL of ethanol was added, and 300-500 μL of piperidine was added dropwise as a reaction catalyst. The mixture was heated to reflux at 60° C. overnight. After the reaction was completed, the reaction product was filtered through an organic filter membrane with a pore size of 0.22 μm. The filtered solution was subjected to silica gel column chromatography. A mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1 was used as a chromatographic solution. Under irradiation with a 365 nm ultraviolet lamp, an oily liquid with red fluorescence in the silica gel column was taken, which was the product for dual-channel detection of Hg. 2+ and Ag + A near-infrared fluorescent probe, whose chemical structure is shown in the above formula (I).

4. A method for dual-channel detection of Hg according to claim 3. 2+ and Ag + The method for preparing a near-infrared fluorescent probe is characterized by: The desiccant described in step 2 is any one of magnesium oxide, calcium oxide, calcium sulfate, copper sulfate, sodium sulfate and magnesium sulfate.

5. A method for simultaneously detecting Ag using the near-infrared fluorescent probe described in claim 1 + and Hg 2+ The method is characterized in that The following steps are involved: Step 1, instrument parameter setting: set the excitation wavelength of the fluorescence spectrophotometer to 420nm, the emission band to 500-800nm, the photomultiplier voltage to 600V, the slit width to 5nm, and use a quartz cuvette with four sides transparent to light; Step 2: Ag + and Hg 2+ Total concentration determination: The near-infrared fluorescent probe was dissolved in a mixed solvent of methanol and HEPES buffer in a volume ratio of (4-5): (6-5) to obtain a near-infrared fluorescent probe solution with a concentration of 10 μmol / L. After the pH of the near-infrared fluorescent probe solution was adjusted to 5.5-6.5, the near-infrared fluorescent probe solution was mixed with the test solution in a volume ratio of 9:1, and the reaction was carried out for 30 seconds. Then, the fluorescence intensity at 610 nm was measured by a fluorescence spectrophotometer. The fluorescence intensity value was compared with the Ag value. + and Hg 2+ The linear relationship between the total concentrations is calculated to obtain the Ag in the test solution. + and Hg 2+ Total concentration; Step 3: Ag + Concentration determination: After adjusting the pH of the near-infrared fluorescent probe solution prepared in step 2 to 7-11, the near-infrared fluorescent probe solution and the test solution were mixed in a volume ratio of 9:1, reacted for 30 seconds, and then the fluorescence intensity at 610 nm was measured by a fluorescence spectrophotometer. + The linear relationship between the concentrations is calculated to obtain the Ag in the test solution. + concentration; Step 4: Hg 2+ Concentration determination: With the same volume of the test liquid, add + and Hg 2+ The total concentration minus Ag + The concentration of Hg in the test solution 2+ concentration.

6. The near-infrared fluorescent probe according to claim 5 simultaneously detects Ag + and Hg 2+ The method is characterized in that: The volume ratio of methanol to HEPES buffer in the mixed solvent described in step 2 is 4.5:5.5.

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