CN117777115B - Mercury ion near infrared fluorescent probe and preparation method and application thereof - Google Patents
Mercury ion near infrared fluorescent probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN117777115B CN117777115B CN202410211259.4A CN202410211259A CN117777115B CN 117777115 B CN117777115 B CN 117777115B CN 202410211259 A CN202410211259 A CN 202410211259A CN 117777115 B CN117777115 B CN 117777115B
- Authority
- CN
- China
- Prior art keywords
- fluorescent probe
- formula
- near infrared
- mercury
- mercury ions
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 85
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 229910052753 mercury Inorganic materials 0.000 claims abstract description 59
- -1 mercury ions Chemical class 0.000 claims abstract description 57
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 241000252212 Danio rerio Species 0.000 claims abstract description 17
- BRNULMACUQOKMR-UHFFFAOYSA-N thiomorpholine Chemical compound C1CSCCN1 BRNULMACUQOKMR-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 37
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- 239000000975 dye Substances 0.000 claims description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 21
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 18
- VGCXGMAHQTYDJK-UHFFFAOYSA-N Chloroacetyl chloride Chemical compound ClCC(Cl)=O VGCXGMAHQTYDJK-UHFFFAOYSA-N 0.000 claims description 14
- 238000004440 column chromatography Methods 0.000 claims description 14
- 239000013067 intermediate product Substances 0.000 claims description 14
- 239000000178 monomer Substances 0.000 claims description 12
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 8
- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 229910052786 argon Inorganic materials 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 2
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 2
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 abstract description 12
- 125000004433 nitrogen atom Chemical group N* 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000003786 synthesis reaction Methods 0.000 abstract description 6
- 125000004430 oxygen atom Chemical group O* 0.000 abstract description 3
- 230000004044 response Effects 0.000 abstract description 3
- 125000004434 sulfur atom Chemical group 0.000 abstract description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 36
- 238000003384 imaging method Methods 0.000 description 13
- 238000011534 incubation Methods 0.000 description 13
- 229910021642 ultra pure water Inorganic materials 0.000 description 12
- 239000012498 ultrapure water Substances 0.000 description 12
- 239000007853 buffer solution Substances 0.000 description 10
- 230000005284 excitation Effects 0.000 description 9
- 239000012467 final product Substances 0.000 description 9
- 238000012544 monitoring process Methods 0.000 description 7
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 6
- 238000004809 thin layer chromatography Methods 0.000 description 6
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 238000004624 confocal microscopy Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 101100394734 Aspergillus oryzae (strain ATCC 42149 / RIB 40) hepG gene Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000028389 Nerve injury Diseases 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000003968 anodic stripping voltammetry Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 1
- 238000001636 atomic emission spectroscopy Methods 0.000 description 1
- 231100000693 bioaccumulation Toxicity 0.000 description 1
- 238000012984 biological imaging Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 238000010249 in-situ analysis Methods 0.000 description 1
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000008764 nerve damage Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a mercury ion near infrared fluorescent probe and a preparation method and application thereof, and belongs to the technical field of organic synthesis. The fluorescent probe takes hemicyanine as a fluorescent signal reporting group, thiomorpholine as a recognition group, acetamide as a connector, oxygen atoms and nitrogen atoms on acetamide and nitrogen atoms and sulfur atoms on thiomorpholine can quickly coordinate with mercury ions, so that the fluorescent intensity is quickly and obviously reduced, the specificity is high, the sensitivity is high (the detection limit is 1.41 mu M), the detection speed is quick (the response can be completed within 30 seconds, and the balance is achieved within 1 minute), and the fluorescent probe can be applied to the detection of mercury ions in aqueous solutions, living cells, living bodies (such as zebra fish) and plants (such as pea sprouts).
Description
Technical Field
The invention relates to a fluorescent probe and a preparation method and application thereof, in particular to a mercury ion near infrared fluorescent probe and a preparation method and application thereof, and belongs to the technical field of organic synthesis.
Background
Mercury is a globally recognized heavy metal contaminant with strong toxicity and bioaccumulation. The mercury ions have extremely strong affinity with sulfur-containing substances in organisms, can be combined with proteins and enzymes, cause dysfunction and cause central nerve injury, and further cause cognitive and motor impairment. Mercury accumulation can cause serious environmental and human hazards. Therefore, detection of mercury ions is of great importance in environmental pollution monitoring and clinical disease diagnosis. Traditional techniques for qualitative and quantitative analytical determination of mercury ions include: atomic absorption spectrometry, atomic emission spectrometry, inductively coupled plasma mass spectrometry, anodic stripping voltammetry and the like, and the problems that instruments and equipment are expensive, a sample pretreatment process is complex and time-consuming and the like exist in the technologies, and particularly, real-time in-situ analysis cannot be realized. Therefore, it is very interesting to develop a method that can detect mercury ions in situ in real time.
Fluorescent analysis detection technology has tended to be mature in the detection field because of the advantages of high sensitivity, high selectivity, high efficiency, high speed, biological damage free, real-time in-situ visualization and the like. At present, a lot of reports on fluorescent probes for detecting mercury ions exist, but the existing fluorescent probes have shorter wavelength, are easily interfered by background signals and strong light scattering of biological samples, have reduced signal-to-noise ratio and sensitivity, and are limited to a certain extent in the field of biological analysis. To avoid the above drawbacks, near infrared fluorescent probes have been the focus of attention. By adjusting the absorption and emission wavelengths of the fluorescent probe, self-absorption and background signal interference can be avoided to a certain extent, and more accurate response signals are provided for detection and imaging of biological samples. The cyanine dye is easy to modify, has longer absorption and emission wavelength, is easy to dissolve in water, has small cytotoxicity, and is suitable for cell and biological imaging experiments. However, through structural modification, designing a near infrared fluorescent probe based on hemicyanine dye and using the probe for specifically detecting the mercury ion content in organisms still has a certain challenge.
Disclosure of Invention
The first object of the present invention is to provide a mercury ion near infrared fluorescent probe based on hemicyanine dye, the second object of the present invention is to provide a method for obtaining the mercury ion near infrared fluorescent probe based on hemicyanine dye in a large quantity with a simple synthetic route, and the third object of the present invention is to provide the application of the mercury ion near infrared fluorescent probe based on hemicyanine dye in detecting mercury ions in aqueous solution, living cells, living bodies (such as zebra fish) and plants (such as pea sprouts).
In order to achieve the above object, the present invention adopts the following technical scheme:
a mercury ion near infrared fluorescent probe having the structure shown below:
Formula I.
The preparation method of the mercury ion near infrared fluorescent probe comprises the following synthetic route:
formula II formula III formula I.
The preparation method of the mercury ion near infrared fluorescent probe comprises the following steps:
step 1: dissolving a hemicyanine dye molecular monomer shown in a formula II in dichloromethane, dropwise adding chloroacetyl chloride, dropwise adding triethylamine, stirring at room temperature to fully react the hemicyanine dye molecular monomer with the chloroacetyl chloride, and separating by column chromatography after the reaction is finished to obtain an intermediate shown in a formula III;
Step 2: adding the intermediate product shown in the formula III into acetonitrile, dropwise adding N, N-diisopropylethylamine and potassium iodide, reacting for 30min at 40 ℃ under the protection of argon, dropwise adding acetonitrile containing thiomorpholine, refluxing for 10h, and separating by column chromatography after the reaction is finished to obtain the mercury ion near infrared fluorescent probe based on the hemicyanine dye shown in the formula I.
Preferably, in step 1, the molar ratio of the hemicyanine dye molecular monomer, chloroacetyl chloride and triethylamine shown in formula II is 1: 2-3: 3 to 5; the column chromatography adopts a silica gel column, and the eluent is dichloromethane and methanol with a weight ratio of 20-40: mixing at a volume ratio of 1.
Preferably, in step 2, the molar ratio of the intermediate of formula III, N-diisopropylethylamine, potassium iodide and thiomorpholine is 1: 5-8: 0.100 to 0.125:2 to 3; the column chromatography adopts a silica gel column, and the eluent is dichloromethane and methanol with a ratio of 40-50: mixing at a volume ratio of 1.
The application of the mercury ion near infrared fluorescent probe in detecting mercury ions in aqueous solution, living cells, living bodies and plants is the application aiming at diagnosis and treatment of non-diseases. Preferably, the living body is zebra fish; the plant is bean sprout.
The invention has the advantages that:
(1) The near infrared fluorescent probe for mercury ions, which is shown in the formula I, provided by the invention, takes half cyanine as a fluorescent signal reporting group, thiomorpholine as an identification group, acetamide as a connector, and oxygen atoms and nitrogen atoms on acetamide and nitrogen atoms and sulfur atoms on thiomorpholine can quickly coordinate with mercury ions, so that the fluorescent intensity is quickly and obviously reduced, the specificity is high, the sensitivity is high (the detection limit is 1.41 mu M), the detection speed is quick (the response can be completed within 30 seconds, and the balance is achieved within 1 min);
(2) When the near infrared fluorescent probe for mercury ions shown in the formula I provided by the invention is used for detecting mercury ions in aqueous solution, the amount of an analysis sample is small, the detection sensitivity is high, real-time in-situ detection can be realized, and the near infrared fluorescent probe for mercury ions has great advantages in the aspect of detecting mercury ions in aqueous solution;
(3) The mercury ion near-infrared fluorescent probe shown in the formula I has good cell permeability and biocompatibility, can detect mercury ions in living cells, living bodies (such as zebra fish) and plants (such as pea sprouts) in a near-infrared wavelength range, and has a wide application field;
(4) The preparation method of the mercury ion near infrared fluorescent probe shown in the formula I provided by the invention has a simple synthetic route, and can obtain the mercury ion near infrared fluorescent probe shown in the formula I in a large quantity.
Drawings
FIG. 1 is a graph showing the change in fluorescence intensity of a fluorescent probe of formula I for detecting mercury ions in PBS buffer solution;
FIG. 2 is a graph showing the change in fluorescence intensity of the fluorescent probe of formula I after reacting with different metal ions in PBS buffer solution;
FIG. 3 is a graph showing the time-dependent fluorescence intensity change of the fluorescent probe of formula I in PBS buffer solution for detecting mercury ions at different concentrations;
FIG. 4 is a confocal microscopy imaging of the fluorescent probes of formula I for detecting the bright field and fluorescent field of mercury ions in HepG 2 cells;
FIG. 5 is a confocal microscope imaging of the bright field and fluorescence field of the fluorescent probe of formula I for detecting mercury ions in zebra fish;
Fig. 6 is a confocal microscope imaging chart of the fluorescent probe shown in formula I for detecting the bright field and the fluorescent field of mercury ions in pea sprouts.
Detailed Description
The invention is described in detail below with reference to the drawings and the specific embodiments.
1. Structure of mercury ion near infrared fluorescent probe based on hemicyanine dye
The structure of the mercury ion near infrared fluorescent probe based on the hemicyanine dye is shown as a formula I:
I is a kind of
The mercury ion near infrared fluorescent probe based on the hemicyanine dye takes hemicyanine as a fluorescent signal reporting group, thiomorpholine as a recognition group, acetamide as a connector (connecting the fluorescent signal reporting group and the recognition group), and oxygen atoms and nitrogen atoms on the acetamide and nitrogen atoms and sulfur atoms on the thiomorpholine can quickly coordinate with mercury ions, so that the fluorescence intensity is quickly and obviously reduced, and the rapid detection of the mercury ions is realized.
2. Preparation method of mercury ion near infrared fluorescent probe based on hemicyanine dye
The synthetic route of the mercury ion near infrared fluorescent probe based on the hemicyanine dye provided by the invention is as follows:
formula II and III formula I
Firstly, dissolving a hemicyanine dye molecular monomer shown in a formula II in dichloromethane, dropwise adding chloroacetyl chloride, dropwise adding triethylamine (used for providing alkaline conditions), stirring at room temperature to enable the hemicyanine dye molecular monomer and the chloroacetyl chloride to fully react, monitoring the reaction progress by using thin-layer chromatography, and separating by using column chromatography after the reaction is finished to obtain an intermediate product shown in a formula III.
Then, the intermediate product shown in the formula III is added into acetonitrile, N-diisopropylethylamine (alkali) and potassium iodide (catalyst) are dropwise added, the mixture is reacted for 30min at 40 ℃ under the protection of argon, then acetonitrile containing thiomorpholine is dropwise added, the mixture is refluxed for 10h, the reaction progress is monitored by thin layer chromatography, and after the reaction is finished, the mercury ion near infrared fluorescent probe based on the hemicyanine dye shown in the formula I is obtained through column chromatography separation.
Example 1
Step 1: synthesis of intermediate product by reacting hemicyanine dye with chloroacetyl chloride
Dissolving 0.23mmol of hemicyanine dye molecular monomer shown in formula II in 20mL of dichloromethane, firstly dripping 0.46mmol of chloroacetyl chloride, then dripping 0.69mmol of triethylamine, stirring at room temperature for 12h to enable the hemicyanine dye molecular monomer and the chloroacetyl chloride to fully react, monitoring the reaction progress by thin layer chromatography, separating by column chromatography (silica gel column, eluent is dichloromethane and methanol mixed in a volume ratio of 40:1) after the reaction is finished to obtain an intermediate product, and drying for later use.
The results of the characterization of 1H NMR、13 C NMR and ESI-MS of this intermediate are as follows:
1H NMR（500MHz,CDCl3）δ11.42（s,1H）,8.63（d,J=14.0Hz,1H）,8.18（s,1H）,7.76（d,J=8.2Hz,1H）,7.49（dd,J=9.8,7.5Hz,2H）,7.40（t,J=7.4Hz,1H）,7.33-7.24（m,2H）,7.18（s,1H）,6.30（d,J=13.6Hz,1H）,4.48（s,2H）,4.33（q,J=6.9Hz,2H）,2.78-2.69（m,2H）,2.66（t,J=5.8Hz,2H）,2.02-1.87（m,2H）,1.79（s,6H）,1.53（t,J=7.2Hz,3H）.
13C NMR（126MHz,CDCl3）δ176.73,166.40,162.26,153.54,145.97,143.18,141.89,140.92,134.49,129.15,127.89,127.82,127.25,122.80,118.31,117.61,114.65,111.83,106.36,102.36,50.69,44.16,40.52,29.70,29.09,28.22,20.28,12.67.
ESI-MS [ C 29H30N2O2Cl]+: calculated 473.35, theoretical 473.20.
From the above characterization results, it can be determined that: the intermediate product synthesized by the method has a structure shown in a formula III.
The yield of this intermediate was calculated to be 47%.
Step 2: synthesis of fluorescent probe by reaction of intermediate product and thiomorpholine
0.17Mmol of intermediate product shown in formula III is added into 20mL of acetonitrile, 0.85mmol of N, N-diisopropylethylamine and 0.02mmol of potassium iodide are added dropwise, the mixture is reacted for 30min at 40 ℃ under the protection of argon, 5mL of acetonitrile containing 0.34mmol of thiomorpholine is added dropwise, the reflux is carried out for 10h at 80 ℃, the reaction progress is monitored by thin layer chromatography, after the reaction is finished, the final product is obtained by separation through column chromatography (silica gel column, eluent is methylene dichloride and methanol in a volume ratio of 50:1), and the final product is dried for standby.
The results of characterization of 1H NMR、13 C NMR and ESI-MS of the final product were as follows:
1H NMR（500MHz,DMSO-d6）δ10.50（s,1H）,8.58（d,J=15.0Hz,1H）,8.05（s,1H）,7.83（d,J=7.4Hz,1H）,7.71（d,J=8.0Hz,1H）,7.60-7.51（m,3H）,7.51-7.39（m,2H）,6.60（d,J=15.0Hz,1H）,4.46（q,J=7.1Hz,2H）,3.27（s,2H）,2.85-2.57（m,12H）,1.87-1.80（m,2H）,1.76（s,6H）,1.38（t,J=7.2Hz,3H）.
13C NMR（126MHz,DMSO-d6）δ177.53,169.91,160.72,153.38,145.49,142.64,142.49,141.49,137.60,133.07,129.43,128.52,127.65,123.44,120.51,117.72,117.33,114.48,113.61,105.72,104.97,62.69,54.80,50.92,27.75,27.45,24.09,22.56,20.34,14.43,13.20.
ESI-MS [ C 33H38N3O2S]+: calculated 540.35, theoretical 540.27.
From the above characterization results, it can be determined that: the structure of the final product synthesized by the method is shown in the formula I.
The yield of the final product was calculated to be 32%.
Example 2
Step 1: synthesis of intermediate product by reacting hemicyanine dye with chloroacetyl chloride
Dissolving 0.23mmol of hemicyanine dye molecular monomer shown in formula II in 25mL of dichloromethane, firstly dripping 0.69mmol of chloracetyl chloride, then dripping 1.15mmol of triethylamine, stirring at room temperature for 12h to enable the hemicyanine dye molecular monomer and chloracetyl chloride to fully react, monitoring the reaction progress by thin layer chromatography, separating by column chromatography (silica gel column, eluent is dichloromethane and methanol mixed in a volume ratio of 20:1) after the reaction is finished to obtain an intermediate product, and drying for later use.
The intermediate product was characterized by 1H NMR、13 C NMR and ESI-MS and the structure was determined to be that shown in formula III. The yield of this intermediate was calculated to be 45%.
Step 2: synthesis of fluorescent probe by reaction of intermediate product and thiomorpholine
0.17Mmol of intermediate product shown in formula III is added into 30mL of acetonitrile, 1.36mmol of N, N-diisopropylethylamine and 0.017mmol of potassium iodide are added dropwise, the mixture is reacted for 30min at 40 ℃ under the protection of argon, 5mL of acetonitrile containing 0.51mmol of thiomorpholine is added dropwise, the reflux is carried out for 10h at 85 ℃, the reaction progress is monitored by thin layer chromatography, and after the reaction is finished, the final product is obtained by separation through column chromatography (silica gel column, eluent is methylene dichloride and methanol in a volume ratio of 40:1), and the final product is dried for standby.
The final product was characterized by 1H NMR、13 C NMR and ESI-MS to confirm that the structure was that shown in formula I. The yield of the final product was calculated to be 30%.
As can be seen from examples 1 and 2, the preparation method provided by the invention has a simple synthetic route, and can be used for obtaining a large amount of mercury ion near infrared fluorescent probes based on the hemicyanine dye.
3. Application of mercury ion near infrared fluorescent probe based on hemicyanine dye
1. Detection of mercury ions in aqueous solutions
The fluorescent probe of formula I prepared in example 1 (dissolved in DMSO at a concentration of 1 mM) was added to a PBS buffer solution (10 mM, ph=7.40), and the final concentration of the fluorescent probe of formula I was 10 μm, and the fluorescence intensity was detected at an excitation wavelength of 615 nm. Then, mercury ions (dissolved in ultrapure water at a concentration of 1 mM) were continuously added to the PBS buffer solution, the final concentration of mercury ions was 100. Mu.M, and the fluorescence intensity was measured at an excitation wavelength of 615 nm.
The result of the fluorescence intensity detection is shown in FIG. 1. As can be seen from fig. 1: when only the fluorescent probe shown in the formula I exists in the PBS buffer solution, a strong fluorescence emission peak is generated at the position of 674nm under the excitation wavelength of 615 nm; after continued addition of mercury ions to the PBS buffer, a significant drop in fluorescence emission peak at 674nm occurred. Therefore, the fluorescent probe shown in the formula I can achieve the purpose of near infrared detection of mercury ions in the aqueous solution.
And (3) according to the fluorescence change curve, the detection limit of the fluorescent probe shown in the formula I on mercury ions can be calculated through linear fitting. Specifically, a fluorescent probe (dissolved in DMSO) and a mercury ion (dissolved in ultrapure water) represented by formula I were added to a PBS buffer solution (10 mm, ph=7.40), wherein the final concentration of the fluorescent probe was 10 μm, and the final concentrations of the mercury ion were 0 μm, 5 μm, 10 μm, 15 μm, 20 μm, and 25 μm, respectively, and the fluorescence intensity was detected at 615nm excitation wavelength and at 674nm emission wavelength, to obtain a curve (fluorescence change curve) in which the fluorescence intensity was changed with the concentration of mercury ion. The resulting fluorescence change curve was fitted linearly and then the limit of detection of mercury ions by the fluorescent probe of formula I was calculated from the limit of detection = 3 sigma/k. The detection limit of the fluorescent probe shown in the formula I on mercury ions is 1.41 mu M.
The fluorescent probe (dissolved in DMSO) of formula I and the ions （Cu2+、Ag+、Mg2+、Fe3+、Fe2+、Cd2+、K+、Cr3+、Ba2+、Na+、Mn2+、Co2+、Ca2+、Ni2+、Zn2+ and Hg 2+ to be measured, dissolved in ultrapure water or methanol) were added to a PBS buffer solution (10 mm, ph=7.40), wherein the final concentration of the fluorescent probe was 10μM,Cu2+、Ag+、Mg2+、Fe3+、Fe2+、Cd2+、K+、Cr3+、Ba2+、Na+、Mn2+、Co2+、Ca2+、Ni2+ and the final concentration of Zn 2+ were 100 μm, the corresponding solutions were respectively noted as 1 to 15, the final concentration of Hg 2+ was 50 μm and 100 μm, the corresponding solutions were respectively noted as 16 and 17, and the fluorescence intensity was detected at 615nm excitation wavelength and at 674nm emission wavelength.
The result of the fluorescence intensity detection is shown in FIG. 2. As can be seen from fig. 2: when Hg 2+ is added into the PBS buffer solution, the fluorescence intensity of the fluorescent probe at 674nm is obviously reduced; while when other metal ions （Cu2+、Ag+、Mg2+、Fe3+、Fe2+、Cd2+、K+、Cr3+、Ba2+、Na+、Mn2+、Co2+、Ca2+、Ni2+、Zn2+） were added to the PBS buffer, the fluorescence intensity of the fluorescent probe at 674nm was hardly changed. The following is explained: the fluorescent probe shown in the formula I can realize high-selectivity detection of mercury ions in aqueous solution.
The fluorescent probe (dissolved in DMSO) and mercury ions (dissolved in ultrapure water) of formula I were added to a PBS buffer solution (10 mm, ph=7.40), wherein the final concentration of the fluorescent probe was 10 μm, and the final concentrations of mercury ions were 0 μm, 5 μm, 10 μm, 15 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, respectively, and the fluorescence intensities were detected at 615nm excitation wavelength, 674nm emission wavelength at different time points (30 s, 1.5min, 2.5min, 3.5min, 4.5min, 5.5min, 6.5min, 7.5min, 8.5min, 9.5min, 10.5min, 11.5 min).
The result of the fluorescence intensity detection is shown in FIG. 3. As can be seen from fig. 3: the fluorescent probe and mercury ions react for 30s, and stable balance is achieved after 1 min; the greater the mercury ion concentration, the weaker the fluorescence intensity. The following is explained: the fluorescent probe shown in the formula I can realize rapid and sensitive detection of mercury ions.
The fluorescent probe shown in the formula I has high selectivity and high sensitivity, and can rapidly detect mercury ions (within 1 min).
2. Detection of mercury ions in living cells
HepG 2 cells are taken as an example.
Group a: hepG 2 cells were incubated at 37℃for 20min.
Group b: to HepG 2 cells, the fluorescent probe of formula I (dissolved in DMSO at a concentration of 10 mM) was added to a final concentration of 10. Mu.M, followed by incubation at 37℃for 20min.
Group c: the fluorescent probe of formula I (dissolved in DMSO at a concentration of 10 mM) was added to HepG 2 cells, the final concentration of the fluorescent probe of formula I was 10 μm, then incubated at 37 ℃ for 20min, washed 3 times with PBS buffer (10 mM, ph=7.40) after the incubation was completed (for the purpose of washing off excess fluorescent probe), then mercury ions (dissolved in ultrapure water at a concentration of 10 mM) were added, the final concentration of mercury ions was 10 μm, and incubated at 37 ℃ for 10min.
D group: fluorescent probe of formula I (dissolved in DMSO at a concentration of 10 mM) was added to HepG 2 cells, the final concentration of fluorescent probe of formula I was 10 μm, then incubated at 37 ℃ for 20min, washed 3 times with PBS buffer (10 mM, ph=7.40) after the incubation was completed, and then mercury ion (dissolved in ultrapure water at a concentration of 10 mM) was added, the final concentration of mercury ion was 10 μm, and incubated at 37 ℃ for 20min.
And detecting confocal microscope imaging conditions of each group of HepG 2 cells in a bright field and a fluorescence field, wherein the excitation wavelength of the fluorescence field is 635nm, and the collection wavelength is 650-750 nm.
The confocal microscopy imaging results are shown in fig. 4. Imaging results show that: when the HepG 2 cells are only incubated with the fluorescent probe, the HepG 2 cells after the incubation are subjected to strong red fluorescent signals, and after the incubation with mercury ions is continued, the fluorescent signals of the HepG 2 cells after the incubation are subjected to the second incubation are obviously weakened along with the increase of the time of interaction with the mercury ions. The following is explained: the fluorescent probe shown in the formula I can realize the monitoring effect of mercury ions in living cells, and has good biological applicability.
3. Detection of mercury ions in animals
Zebra fish is taken as an example.
Group a: zebra fish of 3 days old is not treated.
Group b: zebra fish of 3 days old were placed in a fluorescent probe (dissolved in DMSO at a concentration of 20. Mu.M) of formula I and incubated at room temperature for 10min.
Group c: zebra fish of 3 days old is placed in a fluorescent probe (dissolved in DMSO at a concentration of 20. Mu.M) of formula I, incubated at room temperature for 10min, washed 3 times with ultrapure water after the incubation is completed (the aim of washing off the excess fluorescent probe), then placed in mercury ions (dissolved in ultrapure water at a concentration of 10. Mu.M), and incubated at room temperature for 5min.
D group: zebra fish of 3 days old is placed in a fluorescent probe (dissolved in DMSO at a concentration of 20. Mu.M) shown in formula I, incubated at normal temperature for 10min, washed 3 times with ultrapure water after the incubation is completed, then placed in mercury ions (dissolved in ultrapure water at a concentration of 10. Mu.M), and incubated at normal temperature for 10min.
And detecting confocal microscopic imaging conditions of the zebra fish in the bright field and the fluorescence field, wherein the excitation wavelength of the fluorescence field is 635nm, and the collection wavelength is 660-760 nm.
The confocal microscopy imaging results are shown in fig. 5. Imaging results show that: when the zebra fish is only incubated with the fluorescent probe, the zebra fish after the incubation shows a stronger red fluorescent signal, and after the incubation with mercury ions is continuously added, the fluorescent signal of the zebra fish after the incubation is again ended is obviously weakened along with the increase of the time of acting with the mercury ions. The following is explained: the fluorescent probe shown in the formula I can realize the monitoring effect of mercury ions in zebra fish and has good biocompatibility.
4. Detection of mercury ions in plants
Taking pea sprouts as an example.
Group a: 4-day-old pea buds are placed in water and incubated for 12h at normal temperature.
Group b: 4-day-old pea buds were placed in a fluorescent probe (dissolved in DMSO at a concentration of 10. Mu.M) of formula I and incubated at room temperature for 12h.
Group c: 4-day-old pea buds were placed in a fluorescent probe (dissolved in DMSO at a concentration of 10. Mu.M) of formula I, incubated at room temperature for 12 hours, washed 3 times with ultrapure water after the incubation was completed (the aim of washing off the excess fluorescent probe), then placed in mercury ions (dissolved in ultrapure water at a concentration of 10. Mu.M), and incubated at room temperature for 12 hours.
And (3) longitudinally cutting each group of pea buds, and detecting confocal microscope imaging conditions of root parts of each group of pea buds in a bright field and a fluorescence field, wherein the excitation wavelength of the fluorescence field is 635nm, and the collection wavelength is 660-760 nm.
The confocal microscopy imaging results are shown in fig. 6. Imaging results show that: the fluorescent probe shown in the formula I can enter the root of the pea sprout, and presents an obvious red fluorescent signal, and the fluorescent signal of the root of the pea sprout is obviously weakened when mercury ions exist. The following is explained: the fluorescent probe shown in the formula I can realize the monitoring effect of mercury ions in pea sprouts, and has wider application field.
Therefore, the mercury ion near-infrared fluorescent probe based on the hemicyanine dye shown in the formula I can be applied to rapid detection of low-concentration mercury ions in complete aqueous solution, living cells, living bodies (such as zebra fish) and plants (such as pea sprouts). Has important significance for rapid detection of mercury ions in environment, industrial wastewater and life bodies.
It should be noted that the above examples are only examples for clearly illustrating the present invention, and are not limiting to the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. All obvious changes or modifications which are obvious from the technical proposal of the invention are still within the protection scope of the invention.
Claims (10)
1. The mercury ion near infrared fluorescent probe is characterized by comprising the following structure:
。
2. the method for preparing the mercury ion near infrared fluorescent probe as set forth in claim 1, wherein the synthetic route is as follows:
。
3. The preparation method according to claim 2, characterized by comprising the steps of:
step 1: dissolving a hemicyanine dye molecular monomer shown in a formula II in dichloromethane, dropwise adding chloroacetyl chloride, dropwise adding triethylamine, stirring at room temperature to fully react the hemicyanine dye molecular monomer with the chloroacetyl chloride, and separating by column chromatography after the reaction is finished to obtain an intermediate shown in a formula III;
Step 2: adding the intermediate product shown in the formula III into acetonitrile, dropwise adding N, N-diisopropylethylamine and potassium iodide, reacting for 30min at 40 ℃ under the protection of argon, dropwise adding an acetonitrile solution containing thiomorpholine, refluxing for 10h, and separating by column chromatography after the reaction is finished to obtain the mercury ion near infrared fluorescent probe based on the hemicyanine dye shown in the formula I.
4. A process according to claim 3, characterized in that in step 1, the molar ratio of the hemicyanine dye molecular monomer of formula II, chloroacetyl chloride and triethylamine is 1: 2-3: 3 to 5.
5. The method according to claim 3, wherein in step 1, the column chromatography is performed by using a silica gel column, and the eluent is dichloromethane and methanol in an amount of 20 to 40: mixing at a volume ratio of 1.
6. A process according to claim 3, wherein in step 2, the molar ratio of the intermediate of formula III, N-diisopropylethylamine, potassium iodide and thiomorpholine is 1: 5-8: 0.100 to 0.125:2 to 3.
7. A method according to claim 3, wherein in step 2, the column chromatography is performed using a silica gel column with 40 to 50% of eluent of dichloromethane and methanol: mixing at a volume ratio of 1.
8. Use of the near infrared fluorescent probe for mercury ions according to claim 1 for the detection of mercury ions in aqueous solutions, living cells, living bodies and plants, said use being for the diagnosis and treatment of non-diseases.
9. The use according to claim 8, wherein the living organism is zebra fish.
10. The use according to claim 8, wherein the plants are bean sprouts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410211259.4A CN117777115B (en) | 2024-02-27 | 2024-02-27 | Mercury ion near infrared fluorescent probe and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410211259.4A CN117777115B (en) | 2024-02-27 | 2024-02-27 | Mercury ion near infrared fluorescent probe and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117777115A CN117777115A (en) | 2024-03-29 |
| CN117777115B true CN117777115B (en) | 2024-04-30 |
Family
ID=90387473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410211259.4A Active CN117777115B (en) | 2024-02-27 | 2024-02-27 | Mercury ion near infrared fluorescent probe and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117777115B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109081836A (en) * | 2018-08-23 | 2018-12-25 | 许昌学院 | A kind of mercury ion near infrared fluorescent probe and its preparation method and application based on half flower cyanines structure |
| CN116751210A (en) * | 2023-06-10 | 2023-09-15 | 郑州大学 | Water-soluble probe for detecting mercury ions and preparation method and application thereof |
-
2024
- 2024-02-27 CN CN202410211259.4A patent/CN117777115B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109081836A (en) * | 2018-08-23 | 2018-12-25 | 许昌学院 | A kind of mercury ion near infrared fluorescent probe and its preparation method and application based on half flower cyanines structure |
| CN116751210A (en) * | 2023-06-10 | 2023-09-15 | 郑州大学 | Water-soluble probe for detecting mercury ions and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117777115A (en) | 2024-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110540837B (en) | Preparation and application of hydrogen peroxide near-infrared fluorescent probe | |
| CN111423423B (en) | Application of ratiometric fluorescent probe in detecting peroxynitrite anion | |
| CN108003869B (en) | Fluorescent probe for detecting hypochlorite with high sensitivity and synthesis method and application thereof | |
| CN113121513B (en) | Carbazole-coumarin hydrazone compound as well as preparation method and application thereof | |
| Bhatti et al. | New water soluble p-sulphonatocalix [4] arene chemosensor appended with rhodamine for selective detection of Hg2+ ion | |
| CN107556305B (en) | Fluorescent probe for detecting aluminum ions, preparation method and application | |
| CN111807993A (en) | Near-infrared fluorescent compound for specifically detecting hydrazine and preparation method thereof | |
| CN106749034A (en) | Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker | |
| CN111892552A (en) | Triphenylamine derivative, preparation method thereof and application thereof in double-channel fluorescence detection of hydrogen sulfide | |
| CN109021000B (en) | Fluorescent probe for detecting hydrogen peroxide, synthetic method and application | |
| CN114105927B (en) | Construction of benzopyran nitrile fluorescent molecular probe and in-vitro diagnosis application thereof | |
| CN117777115B (en) | Mercury ion near infrared fluorescent probe and preparation method and application thereof | |
| CN110878085B (en) | Rapid high-selectivity hypobromous acid fluorescent probe, preparation method and application | |
| CN107365289A (en) | A kind of preparation method of hypochlorous acid response type fluorescent sensing material and application | |
| CN114773305B (en) | Preparation method and application of 2-cycloarone pH fluorescence ratio probe | |
| CN113582985B (en) | Mitochondrion targeted pH and viscosity dual-channel detection fluorescent probe and preparation method and application thereof | |
| CN109970730B (en) | Difunctional fluorescent probe and preparation method and application thereof | |
| CN110156858B (en) | Water-soluble hydrogen sulfide fluorescent probe, preparation method thereof and application thereof in detection of water quality sulfide and cell hydrogen sulfide | |
| CN109134483B (en) | Hydrogen sulfide fluorescent probe and preparation method and application thereof | |
| CN110818702A (en) | Pyridine-coumarin derivative fluorescent probe and preparation method and application thereof | |
| CN115073434B (en) | Near-infrared fluorescent probe for detecting hydrazine and preparation method and application thereof | |
| CN114507204B (en) | Golgi apparatus targeted superoxide anion fluorescent probe, preparation method and application thereof | |
| CN103436254B (en) | Functional multi-nitrogen heterocyclic ring fluorescent probe, preparation method and application thereof | |
| CN117430804B (en) | Preparation method of fluorescent probe for rapidly detecting hypochlorite in pure water environment | |
| CN116003427B (en) | Be used for detecting Hg 2+ RBLS of fluorescent probe (R) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |