A ratiometric fluorescent probe for detecting heavy metal palladium and its preparation method and application

Abstract

A ratio-type fluorescent probe for detecting heavy metal palladium, and its preparation method and application. The structure of the fluorescent probe is shown in Formula I, wherein R is a different halogen element or an alkoxy substituted group. After the fluorescent probe provided by the present invention reacts with palladium, it exhibits an obvious spectral red shift, so palladium can be subjected to high-sensitivity and high-selectivity ratio-type recognition detection at two independent analysis wavelengths to eliminate the fluorescence intensity measurement error caused by fluctuations in factors such as probe concentration, environmental conditions, and excitation light source intensity, thereby obtaining more accurate detection results. Furthermore, after the fluorescent probe provided by the present invention reacts with palladium, an obvious color change occurs, which can be applied to the colorimetric analysis of palladium. In addition, the bifunctional fluorescent probe provided by the present invention has stable properties, a simple preparation method, and can be mass-produced, and has great application prospects in the fields of environmental testing, biological analysis, and the like.

Description

Technical Field

[0001] The invention relates to a ratio-type fluorescent probe for detecting heavy metal palladium and a preparation method and application thereof, belonging to the technical field of environmental analysis and biochemical analysis. Background Art

[0002] Palladium is a heavy metal with excellent chemical properties and plays a very important role in modern organic synthesis. However, in recent decades, with the widespread use of palladium-containing catalysts such as Pd(PPh3)4, PdCl2, Pd(CH3COO)2, (CH3CN)2PdCl2, etc., the toxicity of heavy metal palladium has also attracted great attention. A large number of studies have shown that palladium can bind to biological molecules containing thiol groups, such as proteins, DNA and RNA, thereby disrupting various basic cellular processes and causing serious harm to human health. In order to avoid the above hazards, the palladium content in industrial products or environmental samples must be strictly limited. For example, the residual level of palladium in drugs must be less than 5-10ppm; and the daily palladium intake per person should be less than 1.5-15μg. Therefore, it is particularly important to develop efficient palladium detection methods. Inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectroscopy (AAS) are the main methods for detecting palladium in food, drug and environmental analysis. However, these methods rely on expensive large instruments, have disadvantages such as difficult sample preparation, high instrument maintenance costs, and high requirements for experimental technicians, and are not suitable for real-time in situ analysis in biological systems. In contrast, organic small molecule fluorescent probes can achieve rapid and simple detection of palladium with the help of small analytical instruments such as fluorescence spectrophotometers, greatly improving the efficiency and feasibility of analytical detection and reducing detection costs. More importantly, combined with imaging equipment such as fluorescence microscopes, fluorescent probes can achieve imaging analysis and detection of palladium at the cell, tissue and even living level. Ratio response is a special fluorescent probe response mode. The detection of analytes is achieved by monitoring the change in the ratio of fluorescence intensity at two different wavelengths before and after the interaction of the fluorescent probe and the analyte. It can eliminate the fluorescence intensity measurement error caused by fluctuations in probe concentration, environmental conditions and excitation light source intensity, thereby obtaining more accurate detection results. Therefore, the development of ratio-response fluorescent probes is of great practical significance for efficient and accurate detection of heavy metal palladium. Summary of the invention

[0003] The present invention aims to solve the technical problem of how to develop a ratio-type response fluorescent probe for efficient and accurate detection of palladium ratio-type fluorescent probes.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] In a first aspect, the present invention provides a ratiometric fluorescent probe for detecting palladium, the structure of which is shown in Formula I:

[0006]

[0007] Wherein, R is a different halogen element or an alkoxy substituent group.

[0008] Preferably, the ratiometric fluorescent probe for detecting palladium has a structure as shown in Formula II to Formula V,

[0009]

[0010] In a second aspect, the present invention provides a method for preparing a ratiometric fluorescent probe for detecting palladium of formula I, comprising the following steps: step 1: compound 1 and compound 2 are condensed in an organic solvent by base catalysis to generate intermediate 3; step 2: intermediate 3 and compound 4 are condensed in an organic solvent by base catalysis to generate the probe of formula I; see the following reaction scheme:

[0011]

[0012] Wherein, R is a different halogen element or an alkoxy substituent group.

[0013] Further, compound 2 can be specifically compound 2a-2d, and intermediate 3 can be specifically intermediate 3a-3d, to prepare fluorescent probes represented by formula II to formula V, as shown in the following reaction scheme:

[0014]

[0015] Furthermore, in the preparation method, the organic solvent in step 1 is at least one of acetic anhydride, methanol, ethanol and toluene, the base is at least one of sodium acetate, potassium carbonate and pyridine, the reaction temperature of the condensation reaction is 20-150°C, and the reaction time is 1-10h; the organic solvent in step 2 is at least one of dichloromethane, N,N-dimethylformamide, acetonitrile and toluene, the base is at least one of potassium carbonate, triethylamine and pyridine, the reaction temperature of the substitution reaction is 20-150°C, and the reaction time is 1-10h.

[0016] In a third aspect, the present invention provides application of the ratiometric fluorescent probe in palladium detection.

[0017] The ratiometric fluorescent probe for detecting palladium provided by the present invention has the following characteristics and advantages:

[0018] 1. Detecting palladium through ratiometric response can eliminate the fluorescence intensity measurement error caused by fluctuations in probe concentration, environmental conditions, and excitation light source intensity, thereby obtaining more accurate detection results.

[0019] 2. After reacting with palladium, it exhibits a large range of spectral red shift, thus avoiding mutual interference between the two signal detection channels.

[0020] 3. After reacting with palladium, it shows a significant color change and can be used in the colorimetric analysis of palladium.

[0021] 4. The detection selectivity of palladium is good and is not interfered by other common metal ions and physiological substances.

[0022] 5. The detection sensitivity of palladium is high, and the detection limit can reach the nanomolar level.

[0023] 6. The preparation method is simple, can be produced in large quantities, and is easy to store. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the fluorescence response diagram after the fluorescent probes shown in Formula II-Formula V react with Pd(PPh3)4.

[0025] Figure 2 (A) Fluorescence spectrum response diagram and (B) linear relationship diagram after the fluorescent probe shown in Formula II reacts with different concentrations of Pd(PPh3)4.

[0026] Figure 3 This is an investigation of the detection selectivity of the fluorescent probe represented by formula II for palladium.

[0027] Figure 4 The colorimetric analysis application of the fluorescent probe test paper shown in Formula II to palladium: (A) Colorimetric analysis results after the test paper responds to different concentrations of Pd(PPh3)4; (B) Colorimetric analysis comparison of the test paper with 1mM Pd(PPh3)4 and 1mM other common metals.

[0028] Figure 5 The fluorescence imaging diagram of the fluorescent probe shown in formula II on 4T1 cells under incubation conditions of different concentrations of Pd(PPh3)4. DETAILED DESCRIPTION

[0029] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0030] Example 1 Preparation of fluorescent probes represented by formula II to formula V:

[0031] This embodiment provides a method for preparing a fluorescent probe represented by Formula II to Formula V, and the synthetic route is as follows:

[0032]

[0033] The specific steps are as follows:

[0034] Take 1g of compound 1 (5.37mmol) and 5.37mmol of the corresponding benzaldehyde derivative 2a-2d, dissolve in 40mL of acetonitrile, then drop 0.15mL of piperidine, and reflux the resulting solution at 80°C for 6h. After the reaction is completed, evaporate under reduced pressure to remove the solvent, redissolve the solid in 50mL of ethyl acetate, and wash it three times with HCl (1M) and deionized water (50mL each time), add anhydrous Na2SO4 to dry the organic phase, and then evaporate under reduced pressure to remove ethyl acetate. The crude product is separated and purified by silica gel column chromatography (DCM: petroleum ether = 7:3, v / v) to obtain orange solid 3a-3d. 3a: 1 H NMR (400 MHz, DMSO-d6) δ10.43(s,1H),7.61–7.54(m,1H),7.33–7.22(m,2H),7.17(d,J=16.1 Hz,1H),6.93(t,J=8.7 Hz,1H),6.80(s,1H),2.57(s,2H),2.48(s,2H),0.98(s,6H). 13 C NMR(101 MHz, DMSO-d6)δ170.8,156.7,152.8,150.4,147.1,137.4,128.2,126.1,122.5,118.3,115.1,114.5,113.6,75.9,42.7,38.5,32.1,27.9.HR-ES I-MS m / z calcd.for C 19H16FN2O[MH] - 307.1252, found 307.1252.

[0035] 3b: 1 H NMR (400 MHz, DMSO-d6) δ10.74(s,1H),7.79(d,J=2.0 Hz,1H),7.47(s,1H),7.25(d,J=32.0 Hz,2H),6.98(d,J=8.5 Hz,1H),6.84(s,1H),2.60(s,2H),2.51(s,2H),1.01(s,6H). 13C NMR(101 MHz,DMSO-d6)δ170.7,156.7,154.9,137.0,129.5,128.9,128.9,128.2,122.5,120.9,117.2,114.5,113.6,76.0,42.7,38.5,32.1,27.9.HR-ES I-MSm / z cal cd.for C 19H16ClN2O[M-H] - 323.0957,found 323.0956.

[0036] 3c: 1 H NMR(400 MHz,DMSO-d6)δ10.82(s,1H),7.93(d,J=2.0 Hz,1H),7.52(dd,J=8.5,2.0 Hz,1H),7.34–7.14(m,2H),6.96(d,J=8.4 Hz,1H),6.84(s,1H),2.59(s,2H),2.50(s,2H),1.00(s,6H). 13 C NMR(101 MHz,DMSO-d6)δ170.72,156.66,155.92,136.91,132.58,129.48,129.35,128.11,122.50,116.92,114.46,113.63,110.57,75.93,42.67,40.53,40.32,40.11,39.90,39.69,39.48,39.27,38.50,32.10,27.88.HR-ES I-MS m / zcalcd.for C 19H16BrN2O[M-H] - 367.0451,found367.0450.

[0037] 3d: 1 H NMR(400 MHz,DMSO-d6)δ9.63(s,1H),7.35(d,J=1.4Hz,1H),7.31–7.17(m,2H),7.10(dd,J=8.2,1.5 Hz,1H),6.84–6.76(m,2H),3.83(s,3H),2.60(s,2H),2.53(s,2H),1.01(s,6H). 13C NMR(101 MHz, DMSO-d6)δ170.7,157.3,149.3,148.4,139.2,128.1,126.9,123.5,121. 7,116.0,114.6,113.9,110.8,75.0,56.0,42.7,38.6,32.1,27.9.HR-ESI-MS m / z cal cd.for C20H19N2O2[MH] - 319.1452, found 319.1452.

[0038] 1 mmol of each compound 3a-3d was taken respectively and dissolved together with 1.5 eq of compound 4 (1.5 mmol, 180 mg) in 15 mL of dichloromethane, and 0.15 mL of triethylamine was dropped. The resulting solution was reacted at room temperature for 4 h. After the reaction was completed, the solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20: 1, v / v) to obtain a yellow solid, i.e., the fluorescent probes represented by Formula II to Formula V.

[0039] Fluorescent probe represented by formula II: 1 H NMR (400MHz, CDCl3) δ7.36–7.31(m,1H),7.29–7.25(m,2H),6.95(d,J=3.9Hz,2H),6.86(s,1H),6.08–5.92(m,1H ),5.45(dd,J=17.2,1.3Hz,1H),5.39–5.34(m,1H),4.78(d,J=5.8Hz,2H),2.61(s,2H),2.46(s,2H),1.09(s,6H). 13 C NMR (101MHz, CDCl3) δ169.01,155.46,152.80,152.32,139.09,135.41,134.38,130.63,124.42,123. 83,123.72,119.89,115.28,115.09,113.25,112.47,79.69,69.86,42.95,39.17,32.04,28.01.HR-ES I-MS m / z calcd.for C23H22FN2O3[M+H] + 393.1609, found 393.1610. Fluorescent probe represented by formula III: 1H NMR (400MHz, CDCl3) δ7.59(d,J=2.0Hz,1H),7.42(dd,J=8.5,2.0Hz,1H),7.27(d,J=7.3Hz,1H),6.95(s,2H),6.87(s,1H),6.06–5.96( m,1H),5.46(dd,J=17.2,1.3Hz,1H),5.36(dd,J=10.4,1.1Hz,1H),4.78(dt,J=5.8,1.2Hz,2H),2.61(s,2H),2.45(s,2H),1.09(s,6H). 13 C NMR (101MHz, CDCl3) δ169.0,152.8,152.3,147.6,135.2,134.1,130.7,129.1,127.6 ,126.7,124.4,123.7,119.8,113.2,112.4,79.7,69.8,42.9,39.1,32.0,28.0.HR-ES I-MS m / z cal cd.for C23H22ClN2O3[M+H] + 409.1313, found 409.1317.

[0040] Fluorescent probe represented by formula IV: 1 H NMR (400MHz, CDCl3) δ7.76(d,J=1.9Hz,1H),7.47(dd,J=8.5,2.0Hz,1H),7.27(d,J=2.8Hz,1H),6.95(s,2H),6.86(s,1H) ,6.06–5.96(m,1H),5.50–5.41(m,1H),5.38–5.31(m,1H),4.79(d,J=5.8Hz,2H),2.61(s,2H),2.45(s,2H),1.09(s,6H). 13 C NMR (101MHz, CDCl3) δ169.0,152.8,152.3,148.8,135.5,133.9,132.2,130.7,127.4,1 24.5,123.7,119.9,116.7,113.2,112.5,79.7,69.8,42.9,39.1,32.0,28.0.HR-ESI-MS m / z cal cd.for C23H22BrN2O3[M+H] + 453.0808, found 453.0813.

[0041] Fluorescent probe represented by formula V: 1H NMR (400MHz, CDCl3) δ7.17–7.11(m,2H),7.07(dd,J=8.2,1.8Hz,1H),7.02(d,J=16.1Hz,1H),6.94(d,J=16.1Hz,1H),6.86(s,1H),6.05–5.95( m,1H),5.44(dq,J=17.2,1.4Hz,1H),5.33(dd,J=10.4,1.2Hz,1H),4.75(d,J=5.8Hz,2H),3.92(s,3H),2.61(s,2H),2.47(s,2H),1.09(s,6H). 13 C NMR (101MHz, CDCl3) δ169.3,153.6,152.8,151.5,141.1,136.0,134.9,131.0,129.5,123.8,122 .8,120.8,119.3,113.4,112.7,110.4,78.8,69.4,56.0,50.7,42.9,39.1,32.0,28.0.HR-ESI-MS m / z calcd.forC24H25N2O4[M+H] + 405.1809, found 405.1817.

[0042] Example 2 Determination of metallic palladium by fluorescent probes represented by formulas II to V:

[0043] 3 mL of 20 mM phosphate buffer (containing 20% ​​DMSO) was added to a 5 mL test tube, followed by an appropriate volume of a fluorescent probe solution of Formula II-Formula V (1 mM, dissolved in DMSO) to make the final probe concentration 10 μM, and finally 5 μM of Pd(PPh3)4 was added. After reacting for 30 min, the fluorescence intensity of the reaction solution was measured on a fluorescence spectrophotometer, with an excitation wavelength of 440 nm.

[0044] Figure 1 The fluorescence response diagram of the fluorescent probes shown in formula II to formula V after reacting with 5 μM Pd(PPh3)4 for 30 minutes. Figure 1 It can be seen that after the fluorescent probes shown in Formula II to Formula V react with Pd(PPh3)4, they all show a significant spectral red shift. The fluorescence intensity ratio at two wavelengths before and after the reaction (I after / I before ) were enhanced by 72.8 times, 53.7 times, 43.5 times and 8.1 times, respectively, indicating that the fluorescent probes shown in Formula II to Formula V can be used for ratiometric detection of palladium, and the fluorescent probe shown in Formula II has the highest sensitivity. Therefore, the fluorescent probe shown in Formula II was used in Examples 3 to 6.

[0045] Example 3: Determination of different concentrations of metal palladium by the fluorescent probe shown in formula II:

[0046] 3 mL of 20 mM phosphate buffer (containing 20% ​​DMSO) was added to a 5 mL test tube, followed by an appropriate volume of the fluorescent probe solution of Formula II (1 mM, dissolved in DMSO) to make the final probe concentration 10 μM, and finally different concentrations of Pd(PPh3)4 were added. After 30 min of reaction, the fluorescence intensity of the reaction solution was measured on a fluorescence spectrophotometer, with an excitation wavelength of 440 nm.

[0047] Figure 2 The fluorescence response diagram of the fluorescent probe shown in formula II after reacting with different concentrations of Pd(PPh3)4 for 30min. Figure 2 It can be seen from A in Formula II that after the fluorescent probe reacts with different concentrations of Pd(PPh3)4, the fluorescence intensity at 517nm gradually decreases, while the fluorescence intensity at 665nm gradually increases; Figure 2 From B in the figure, we can see that the fluorescence intensity ratio at the above two wavelengths (I 665 / I 517 ) increases with the increase of Pd(PPh3)4 concentration, and shows a good linear relationship in the concentration range of 0.5-3.0μM. The detection limit is calculated and shown as LOD=3σ / k (σ is the fluorescence intensity ratio of 11 blank probe solutions I 665 / I 517 The standard deviation of the linear equation (k is the slope of the linear equation) was calculated to be 2.11 nM (equivalent to 0.22 μg / L of Pd), which is much lower than the residual limit of palladium in drugs of 5-10 ppm.

[0048] Example 4 Investigation of the detection selectivity of the fluorescent probe represented by formula II for palladium:

[0049] 3 mL of 20 mM phosphate buffer (containing 20% ​​DMSO) was added to a 5 mL test tube, followed by an appropriate volume of the fluorescent probe solution of Formula II (1 mM, dissolved in DMSO) to make the final probe concentration 10 μM, and finally 3 μM Pd(PPh3)4 and 150 mM K + , 2.5 mM Ca 2+ , 2.5 mM Mg 2+ , 100 μM other common metal ions, 5 mM glutathione, 1 mM common amino acids. After 30 min of reaction, the fluorescence intensity of the reaction solution was measured on a fluorescence spectrophotometer with an excitation wavelength of 440 nm.

[0050] Figure 3is the fluorescence ratio (I) of the fluorescent probe shown in formula II after reacting with Pd(PPh3)4 and the above-mentioned investigation substance for 30min respectively 665 / I 517 ) response graph. Figure 3 It can be seen that after the fluorescent probe shown in Formula II reacts with Pd(PPh3)4, the fluorescence ratio (I 665 / I 517 ) was significantly enhanced, while other investigated substances did not cause changes in the fluorescence ratio, including 100 μM Pd 2+ , indicating that the fluorescent probe shown in Formula II has good detection selectivity for palladium and can be used for Pd 0 Selective detection.

[0051] Example 5 Preparation of fluorescent probe test paper shown in formula II and colorimetric analysis of palladium:

[0052] Cut the filter paper into 1cm×2cm strips and soak them in the ethanol solution (10mM) of the fluorescent probe shown in Formula II. After soaking for 10 minutes, take out the strips, evaporate the solvent ethanol, and prepare the fluorescent probe detection test paper shown in Formula II. Then, take 100μL of Pd(PPh3)4 aqueous solution of different concentrations and drop them on the test paper respectively, let it stand for 10min to observe the color change of the test paper. Further, take 100μL of Pd(PPh3)4 aqueous solution (1mM) and 100μL of other common metal ion aqueous solutions (1mM) and drop them on the test paper respectively, let it stand for 10min to observe the color change of the test paper. Figure 4 The colorimetric analysis result of the fluorescent probe test paper shown in formula II for metal palladium. Figure 4 It can be seen from A in Formula II that after the fluorescent probe test paper reacts with Pd(PPh3)4, the color changes from yellow to brown, and the brown color deepens as the concentration of Pd(PPh3)4 increases, indicating that it can be used in the colorimetric analysis of palladium; Figure 4 It can be seen from B in Formula II that the fluorescent probe test paper shown in Formula II does not undergo obvious color changes after reacting with other common metal ions, indicating that it has good palladium selectivity.

[0053] Example 6 The fluorescent probe represented by Formula II is applied to palladium fluorescence imaging analysis in 4T1 cells: 1) 4T1 cells are cultured in a glass-bottomed culture dish dedicated to a laser confocal fluorescence microscope for 24 hours. The culture solution is a DMEM culture medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture conditions are 37°C and 5% (v / v) CO2 atmosphere conditions, so that the cells are fully attached to the bottom of the culture dish.

[0054] 2) Add 10 μL of the fluorescent probe represented by Formula II into the above cell culture dish and incubate with the cells for 30 min.

[0055] 3) After the cell incubation in step 2 is completed, the culture medium is aspirated, the cells are washed with PBS buffer, and then 1 mL of DMEM culture medium is added, and then different concentrations of Pd(PPh3)4 are added, and the culture is continued for 30 min.

[0056] 4) After the cell culture in step 3 is completed, the culture dish is placed on a confocal fluorescence microscope for fluorescence imaging, wherein the green channel excitation light source is a 405nm laser, and the fluorescence signal collection range is 480-550nm; the red channel excitation light source is a 405nm laser, and the fluorescence signal collection range is 620-680nm.

[0057] Figure 5 The fluorescence imaging diagram of the fluorescent probe shown in formula II on 4T1 cells under incubation conditions of different concentrations of Pd(PPh3)4. Figure 5 It can be seen that with the increase of Pd(PPh3)4 concentration, the ratio of the red channel and green through-fluorescence signals of the fluorescent probe shown in Formula II gradually increases, indicating that the fluorescent probe shown in Formula II can be used for ratiometric fluorescence imaging analysis of metallic palladium in living cells.

[0058] Finally, it should be noted that the above embodiments only illustrate the preparation and application of the fluorescent probes represented by Formula II to Formula V. The preparation methods, test conditions, etc. of the remaining fluorescent probes are not listed one by one because of their similar structures and properties.

[0059] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A ratiometric fluorescent probe for detecting heavy metal palladium, characterized in that: The structure of the fluorescent probe is shown in Formula II: 。

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