CN110642771B - Molecular probe and preparation method and application thereof - Google Patents
Molecular probe and preparation method and application thereof Download PDFInfo
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- CN110642771B CN110642771B CN201910809213.1A CN201910809213A CN110642771B CN 110642771 B CN110642771 B CN 110642771B CN 201910809213 A CN201910809213 A CN 201910809213A CN 110642771 B CN110642771 B CN 110642771B
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- 239000003068 molecular probe Substances 0.000 title claims abstract description 133
- 238000002360 preparation method Methods 0.000 title abstract description 24
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims abstract description 91
- GDIYMWAMJKRXRE-UHFFFAOYSA-N (2z)-2-[(2e)-2-[2-chloro-3-[(z)-2-(1,3,3-trimethylindol-1-ium-2-yl)ethenyl]cyclohex-2-en-1-ylidene]ethylidene]-1,3,3-trimethylindole Chemical compound CC1(C)C2=CC=CC=C2N(C)C1=CC=C1C(Cl)=C(C=CC=2C(C3=CC=CC=C3[N+]=2C)(C)C)CCC1 GDIYMWAMJKRXRE-UHFFFAOYSA-N 0.000 claims abstract description 39
- 239000003960 organic solvent Substances 0.000 claims abstract description 34
- HFBMWMNUJJDEQZ-UHFFFAOYSA-N acryloyl chloride Chemical compound ClC(=O)C=C HFBMWMNUJJDEQZ-UHFFFAOYSA-N 0.000 claims abstract description 31
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 24
- 239000011261 inert gas Substances 0.000 claims abstract description 24
- 239000001632 sodium acetate Substances 0.000 claims abstract description 24
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 24
- 238000003756 stirring Methods 0.000 claims abstract description 24
- 238000010438 heat treatment Methods 0.000 claims abstract description 10
- 239000003513 alkali Substances 0.000 claims abstract description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 42
- 238000000034 method Methods 0.000 claims description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 claims description 8
- 238000003384 imaging method Methods 0.000 claims description 5
- 150000007530 organic bases Chemical class 0.000 claims description 5
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- 238000003786 synthesis reaction Methods 0.000 abstract description 4
- 239000000975 dye Substances 0.000 description 40
- 239000000543 intermediate Substances 0.000 description 31
- 239000000243 solution Substances 0.000 description 24
- 238000001514 detection method Methods 0.000 description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical group CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 21
- 238000010521 absorption reaction Methods 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 238000001917 fluorescence detection Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- 230000005284 excitation Effects 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 239000000460 chlorine Substances 0.000 description 4
- 238000004821 distillation Methods 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000010898 silica gel chromatography Methods 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 238000004737 colorimetric analysis Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000035515 penetration Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 238000003918 potentiometric titration Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 1
- 101710134784 Agnoprotein Proteins 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000013064 chemical raw material Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 238000005536 corrosion prevention Methods 0.000 description 1
- 238000003869 coulometry Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000006575 electron-withdrawing group Chemical group 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 1
- 150000002429 hydrazines Chemical class 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- ILWRPSCZWQJDMK-UHFFFAOYSA-N triethylazanium;chloride Chemical compound Cl.CCN(CC)CC ILWRPSCZWQJDMK-UHFFFAOYSA-N 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
- C07D209/12—Radicals substituted by oxygen atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/56—Ring systems containing three or more rings
- C07D209/58—[b]- or [c]-condensed
- C07D209/60—Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/0066—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
- C09B23/086—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines more than five >CH- groups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/1702—Systems in which incident light is modified in accordance with the properties of the material investigated with opto-acoustic detection, e.g. for gases or analysing solids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a molecular probe and a preparation method and application thereof, wherein the preparation method comprises the steps of dissolving heptamethine cyanine dye and sodium acetate in a first organic solvent, heating and stirring in inert gas to obtain a molecular probe intermediate; and dissolving the molecular probe intermediate and organic alkali in a second organic solvent, adding an acryloyl chloride solution under the protection of inert gas, and stirring at room temperature in sequence in an ice bath to obtain the molecular probe. According to the invention, through modifying and improving the structure of the heptamethine cyanine dye, a molecular probe with an ester bond and a strong conjugated structure is obtained, and the prepared molecular probe has good selectivity and high sensitivity when detecting hydrazine; and the preparation method is simple, the synthesis condition is not harsh, and the operation is convenient.
Description
Technical Field
The invention relates to the technical field of molecular probes, in particular to a molecular probe and a preparation method and application thereof.
Background
Hydrazine (N) 2 H 4 ) Is a common chemical raw material, has strong reducibility and strong corrosiveness, and has wide application in the chemical industry, including corrosion prevention of metals, important precursors of synthetic polymers, textile dyes and medical intermediates, and the like. Meanwhile, because of the explosiveness and combustibility of hydrazine, the hydrazine is widely applied to rocket propulsion systems. But at the same time, hydrazine is a potential carcinogen, has strong erosion ability to skin, and has serious injury effect to eyes, livers and other organs. Therefore, the effective detection of the residual hydrazine in the environment has very important significance for human health and environmental protection.
Among the existing detection methods, the methods for detecting hydrazine mainly include a fluorescence detection method, a coulometric method and a potentiometric titration method. Among these techniques, the fluorescence detection method is widely used because of its advantages of high sensitivity, short acquisition time and high time resolution. However, the fluorescence imaging has an excitation light range of 400-700nm, the penetration depth is limited, deep tissues still cannot be accurately detected, and the body autofluorescence background seriously interferes with the in-vivo application. And other detection methods have complex operation and long detection time. In addition, these detection methods require treatment of ex vivo tissues or cell lysates, and do not have the function of detecting living biological tissues, and thus are not suitable for detection of in vivo hydrazines.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
The invention aims to solve the technical problems that aiming at the defects in the prior art, a molecular probe and a preparation method and application thereof are provided, and aims to solve the problems that the existing hydrazine detection method is complex in operation and long in detection time, needs to treat isolated tissues or cell lysates, has no function of detecting living biological tissues and is not suitable for detecting in-vivo hydrazine.
The technical scheme adopted by the invention for solving the technical problems is as follows: a molecular probe, wherein the molecular probe has the structural formula:
wherein R is-CH 3 、-CH 2 CH 3 、
The invention also provides a preparation method of the molecular probe, wherein the method comprises the following steps:
dissolving heptamethine cyanine dye and sodium acetate in a first organic solvent, heating and stirring in inert gas to obtain a molecular probe intermediate;
and dissolving the molecular probe intermediate and organic alkali in a second organic solvent, adding an acryloyl chloride solution under the protection of inert gas, and stirring at room temperature in sequence in an ice bath to obtain the molecular probe.
The preparation method of the molecular probe comprises the following steps of:
wherein R is-CH 3 、-CH 2 CH 3 、
The preparation method of the molecular probe comprises the step of preparing a molecular probe, wherein the molar ratio of the heptamethine cyanine dye to the sodium acetate is 1:1-1:4.
The preparation method of the molecular probe comprises the steps of dissolving heptamethine cyanine dye and sodium acetate in a first organic solvent, heating and stirring in inert gas, and obtaining a molecular probe intermediate, wherein the steps comprise:
the heptamethine cyanine dye and sodium acetate are dissolved in a first organic solvent, and stirred for 3 to 5 hours at the temperature of 80 to 100 ℃ under the protection of inert gas, so as to obtain a molecular probe intermediate.
The preparation method of the molecular probe comprises the step of preparing a first organic solvent, wherein the first organic solvent is one of dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP) or N, N-Dimethylformamide (DMF).
The preparation method of the molecular probe comprises the steps of dissolving the molecular probe intermediate and organic alkali in a second organic solvent, adding an acryloyl chloride solution under the protection of inert gas, and stirring at ice bath and room temperature in sequence to obtain the molecular probe, wherein the step of obtaining the molecular probe comprises the following steps of:
dissolving the molecular probe intermediate and the organic alkali in a second organic solvent, stirring for 15min at 0-4 ℃, then dropwise adding an acryloyl chloride solution under the protection of inert gas, continuously stirring for 20min at 0-4 ℃, and continuously stirring for 3-5 h at room temperature to obtain the molecular probe.
The preparation method of the molecular probe comprises the step of preparing a second organic solvent, wherein the second organic solvent is one of dichloromethane or chloroform.
The molecular probe is prepared by the method, wherein the molar ratio of the molecular probe intermediate to the acryloyl chloride is 1:1-1:4.
The molecular probe is applied to the field of hydrazine detection, wherein the detection method is an ultraviolet colorimetric method or a photoacoustic imaging method.
The beneficial effects are that: according to the invention, through modifying and improving the structure of the heptamethine cyanine dye, a molecular probe with an ester bond and a strong conjugated structure is obtained, and the prepared molecular probe can realize accurate detection of hydrazine by an ultraviolet colorimetric method and photoacoustic imaging, and has strong selectivity and high sensitivity; has good application prospect in the aspect of hydrazine detection. Meanwhile, the synthesis method of the molecular probe is simple, the synthesis conditions are not harsh, and the operation is convenient.
Drawings
FIG. 1 is a synthetic route diagram of a molecular probe in example 1 of the present invention;
FIG. 2 is an ultraviolet absorption spectrum of the molecular probe prepared in example 1 of the present invention;
FIG. 3 is a graph showing the ultraviolet absorption spectrum of the molecular probe in example 2 of the present invention in response to hydrazine at various concentrations;
FIG. 4 is a graph showing the change in the response of a molecular probe to different ions in example 3 of the present invention;
FIG. 5 is a graph of photoacoustic signals of the response of a molecular probe to different ions at 808nm in example 4 of the present invention;
FIG. 6 is a graph of photoacoustic signals of molecular probes at 808nm responding to different concentrations of hydrazine in example 4 of the present invention;
FIG. 7 is a graph showing the linear relationship between the response of the molecular probe at 808nm to hydrazine at various concentrations in example 4 of the present invention.
Detailed Description
The invention provides a molecular probe, a preparation method and application thereof, and the invention is further described in detail below in order to make the purposes, technical schemes and advantages of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Specifically, the molecular probe has the structural formula:
wherein R is-CH 3 、-CH 2 CH 3 、/>
The molecular probe has a very strong conjugated structure, and ester bonds of the molecular probe are hydrolyzed under the action of hydrazine, so that the conjugated structure of the molecular probe is damaged, an ultraviolet absorption peak blue is moved out to form a new absorption peak, and the original absorption peak is gradually reduced and the newly generated absorption peak is gradually enhanced along with the increase of the concentration of the hydrazine.
Specifically, the preparation method of the molecular probe comprises the following steps:
s1, dissolving heptamethine cyanine dye and sodium acetate in a first organic solvent, heating and stirring in inert gas to obtain a molecular probe intermediate;
the existing hydrazine detection methods comprise a fluorescence detection method, a coulomb method and a potentiometric titration method, the other methods have the defects of long detection time, high sensitivity, short acquisition time and high time resolution, but the excitation light range of the fluorescence detection method is 400-700nm, the penetration depth is limited, deep tissues still cannot be accurately detected, and the application of the fluorescence detection method in body detection is seriously interfered by an autofluorescence background of an organism. To solve this problem, a heptamethine cyanine dye was selected as a parent in this example to prepare a molecular probe for hydrazine detection. The heptamethine cyanine dye is one of methine cyanine dyes, and the absorption and emission spectrum of the methine cyanine dye is between 530 and 1200nm (near infrared fluorescence lambda) em > 600 nm), relative to conventional fluorescence(λ em < 600 nm), in the near infrared fluorescence region, the organism light absorption or fluorescence intensity of the biological sample is small, so that the background interference is greatly reduced, and the raman scattering is rapidly reduced with the increase of the wavelength, so that the scattering interference is also greatly reduced, because the scattered light intensity is inversely proportional to the fourth power of the wavelength. The heptamethine cyanine dye is used as one of methine cyanine dyes, has longer absorption wavelength and smaller background interference, so that the problem that the existing fluorescence detection method is easily influenced by the autofluorescence background of an organism and has limited penetration depth during hydrazine detection can be solved. Preferably, the structural formula of the heptamethine cyanine dye in the embodiment is
Wherein R is-CH 3 、-CH 2 CH 3 、/>
Although the existing cyanine dye has absorption and emission spectra in the near infrared region, and can avoid the autofluorescence of biological tissues in biological application, the cyanine dye has the defects of small stokes shift, which causes the excitation light wavelength to be too close to the fluorescence detection wavelength, and the scattered light of the excitation light can interfere with analysis, thereby influencing the detection sensitivity. Therefore, in this embodiment, the heptamethine cyanine dye and sodium acetate are dissolved in the first organic solvent, and heated and stirred in inert gas, so that the heptamethine cyanine dye reacts with sodium acetate to modify and improve the structure of the heptamethine cyanine dye, and the heptamethine cyanine dye has larger stokes shift. Specifically, after the heptamethine cyanine dye and sodium acetate are dissolved in a first organic solvent, chlorine atoms in the heptamethine cyanine dye leave, electron-deficient centers are formed on cyclohexane and are combined with oxygen atoms with negative charges in sodium acetate to form C=O bonds, so that the electron cloud density in a heptamethine cyanine dye conjugated system is increased, the excitation energy of a molecular ground state is reduced, and the fluorescence of heptamethine cyanine dye molecules can be enhanced.
Specifically, in this embodiment, the heptamethine cyanine dye and sodium acetate are dissolved in a first organic solvent, and under the protection of inert gas, the mixture is heated and stirred for several hours, then cooled to room temperature, and the solvent is removed to obtain the molecular probe intermediate. Wherein the inert gas is nitrogen or argon. If the heating temperature is too high, the heptamethine cyanine dye is easy to decompose, and if the temperature is too low, the reaction rate is too slow, preferably, the heating temperature is 80-100 ℃ and the heating time is 3-5 h in the embodiment. After the completion of the reaction, the mixed solution after the reaction was cooled to room temperature, and the solvent was removed by distillation under reduced pressure to obtain a crude product, which was then purified using petroleum ether: ethyl acetate = 3: and 1 is an eluent, and the molecular probe intermediate is obtained by silica gel column chromatography purification.
Further, the first organic solvent in this embodiment is a high boiling point solvent such as Dimethylsulfoxide (DMSO), N-methylpyrrolidone (NMP), N-Dimethylformamide (DMF), or the like, and the heptamethine cyanine dye and sodium acetate can be well dissolved in the first organic solvent during the reaction. For example: when heptamethine cyanine dye is
When the heptamethine cyanine dye and sodium acetate are dissolved in N-methyl pyrrolidone (NMP), and stirred under the protection of nitrogen to obtain a molecular probe intermediate, the reaction formula is shown as follows:
in a specific embodiment, in this embodiment, the molar ratio of the heptamethine cyanine dye to the sodium acetate is 1:1-1:4, the amount of sodium acetate relative to the heptamethine cyanine dye is too small, the heptamethine cyanine dye does not react completely, the amount of sodium acetate relative to the heptamethine cyanine dye is too large, resulting in sodium acetate waste, and preferably, the molar ratio of the heptamethine cyanine dye to the sodium acetate is 1:3, and under this ratio, the heptamethine cyanine dye can be guaranteed to react sufficiently.
Specifically, the preparation method of the molecular probe provided by the invention further comprises the following steps:
s2, dissolving the molecular probe intermediate and the organic alkali into a second organic solvent, adding an acryloyl chloride solution under the protection of inert gas, and stirring at room temperature in sequence to obtain the molecular probe.
In specific implementation, after the molecular probe intermediate is obtained in this embodiment, the molecular probe intermediate and the organic base are further dissolved in a second organic solvent, an acrylic chloride solution is added under the protection of inert gas, and the mixture is stirred at ice bath and room temperature in sequence, so as to obtain the final molecular probe. In the previous step, the oxygen atom on the molecular probe intermediate is an electron donating group, after the acryloyl chloride solution is added, the acryloyl chloride with the electron withdrawing group is combined with the molecular probe intermediate with the electron donating group to form a molecular probe with an ester bond, and the molecular probe with the ester bond is hydrolyzed under the action of hydrazine, so that the conjugated structure of the molecular probe is damaged, and the ultraviolet absorption peak blue is removed from a new absorption peak. And the chloride ion separated from the acrylic chloride is combined with the hydrogen ion to form hydrochloric acid, and the organic base is used as an acid applying agent to promote the reaction of the molecular probe intermediate and the acrylic chloride. When the organic base is triethylamine, the lone pair electrons on the nitrogen atom of the triethylamine are easy to combine with chloride ions discharged by the reaction of the acryloyl chloride to form triethylamine hydrochloride, so that the charge movement promotes the reaction of the molecular probe intermediate and the acryloyl chloride.
Specifically, in the implementation, the molecular probe intermediate and the organic alkali are dissolved in a second organic solvent, stirred in an ice bath at 0-4 ℃ for 15min, then the acryloyl chloride solution is added dropwise under the protection of inert gas, and the mixture is continuously stirred in the ice bath at 0-4 ℃ for 20min, and then the mixture is stirred at room temperature for 3-5 h to obtain the molecular probe. The method is characterized in that the anhydrous dichloromethane solution of the acryloyl chloride is dropwise added, the reaction temperature is controlled to be 0-4 ℃ in the dropwise adding process, the purpose of avoiding self-polymerization of the acryloyl chloride is to continuously stir the acryloyl chloride solution at the temperature of 0-4 ℃ for 20min after the completion of dropwise adding of the acryloyl chloride solution, and then the mixture is gradually warmed to room temperature and continuously stirred for 3-5 h. The solvent was then removed by distillation under reduced pressure using methylene chloride: methanol=30:1 is used as an eluent, and the molecular probe is obtained by silica gel column chromatography purification.
As a specific embodiment, in this example, dichloromethane or chloroform which has a good solubility for the molecular probe intermediate, the organic base and the acryl chloride is selected as the second organic solvent, and the second organic solvent is an anhydrous organic solvent such as anhydrous dichloromethane or anhydrous chloroform because the acryl chloride is easily hydrolyzed. The molecular probe intermediates obtained still as mentioned in the previous step
An illustration is given. The molecular probe intermediate and Triethylamine (TEA) obtained were further dissolved in anhydrous Dichloromethane (DCM), and an acryloyl chloride solution was added under the protection of inert gas, followed by stirring at room temperature in an ice bath, to obtain a molecular probe having the following reaction formula:
in a specific embodiment, the molar ratio of the molecular probe intermediate to the acryloyl chloride is 1:1-1:4, the quantity of the acryloyl chloride is too small, and the molecular probe intermediate does not react completely; the molar ratio of the molecular probe intermediate to the acryloyl chloride in this embodiment is preferably 1:3, and the complete reaction of the molecular probe intermediate can be ensured without excessive acryloyl chloride.
The invention also provides application of the molecular probe in the field of hydrazine detection, wherein the molecular probe is prepared by adopting the preparation method, and the detection method is an ultraviolet colorimetric method or a photoacoustic imaging method.
According to the invention, the prepared molecular probe is modified and improved in structure of the heptamethine cyanine dye to obtain the molecular probe with an ester bond and a strong conjugated structure, the ester bond of the molecular probe is hydrolyzed under the action of hydrazine, so that the conjugated structure of the molecular probe is damaged, an ultraviolet absorption peak blue is removed to form a new absorption peak, the original absorption peak is gradually reduced along with the increase of the concentration of the hydrazine, the newly generated absorption peak is gradually enhanced, and the prepared molecular probe has good selectivity and high sensitivity when detecting the hydrazine; and the preparation method is simple, the synthesis condition is not harsh, and the operation is convenient.
The invention is further illustrated by the following examples.
Example 1
Preparation of a molecular probe: the synthetic scheme of the molecular probe is shown in figure 1, wherein DMF is N, N-dimethylformamide, TEA is triethylamine, and DCM is dichloromethane. The preparation steps of the molecular probe are as follows:
(1) Seven methine cyanine dye IR-813 (305 mg) and sodium acetate (123 mg) were dissolved in DMF (10 mL) to obtain a mixture solution; the mixture was then heated to 90℃under nitrogen and stirred for 3 hours before cooling to room temperature. The solvent was removed by distillation under reduced pressure to give the crude product, using petroleum ether: ethyl acetate = 3:1 is eluent, silica gel column chromatography purification is carried out to obtain 151mg of red solid powder molecular probe intermediate, and the yield is 65%;
(2) Molecular probe intermediate (780 mg) and triethylamine (170 mg) were dissolved in anhydrous Dichloromethane (DCM) (20 mL). The solution was then stirred in an ice bath for 15min. Then, a methylene chloride solution of acryloyl chloride (456 mg) was added dropwise under nitrogen protection, followed by stirring at 0℃for 20min. After 20min, the reaction was allowed to stir at room temperature for a further 3h. The solvent was then removed by distillation under reduced pressure using methylene chloride: methanol=30: 1 is eluent, and the molecular probe 392mg of green solid powder is obtained by silica gel column chromatography purification, and the yield is 42%. The ultraviolet absorption spectrum of the prepared molecular probe is shown in figure 2, and the maximum absorption wavelength is 808nm, so that the self-background fluorescence of biological tissues can be avoided in biological application.
Example 2
Measuring the response of the molecular probe to hydrazine: the molecular probe obtained in example 1 was prepared as 3mL of a methanol solution of the molecular probe having a concentration of 10. Mu.M. Dripping hydrazine solutions with different concentrations into the molecular probeIn methanol solution, probe A was measured simultaneously 808nm /A 550nm Is characterized by means of an ultraviolet-visible spectrophotometer. As shown in fig. 3, after hydrazine is added into the methanol solution of the molecular probe, the absorbance of the molecular probe at 808nm gradually decreases with the increase of the hydrazine concentration; at the same time, a new absorption peak appears at 550nm, and the absorbance at this position gradually increases with the increase of the hydrazine concentration. The Stokes shift of the prepared molecular probe is 258nm, and compared with the Stokes shift of the traditional cyanine dye, the Stokes shift of the prepared molecular probe is 20nm, and the Stokes shift of the prepared molecular probe is obviously improved.
Example 3
Determination of selectivity of molecular probes for hydrazine: the molecular probe obtained in example 1 was prepared as 3mL of a methanol solution of the molecular probe having a concentration of 10. Mu.M. Hydrazine hydrate and corresponding salts of various ions (respectively Na 2 SO 4 ，NaNO 3 ，NaCl，KBr，CH 3 COONa，NH 4 Cl，NaCl，KCl，AgNO 3 ，Zn(NO 3 ) 2 ，HgCl 2 ，MgCl 2 ，CuCl 2 ，MnCl 2 And (CH) 3 COO) 2 Pb) was dissolved in deionized water to prepare various solutions (SO 4 2- 、NO 3- 、Cl - 、Br - 、CH 3 COO - 、NH 4 + 、Na + 、K + 、Ag + 、Zn 2+ 、Hg 2+ 、Mg 2+ 、Cu 2+ 、Mn 2+ 、Pb 2+ And hydrazine, 10 μm). Subsequently, 10. Mu.M of the anion and cation and hydrazine solution were added to the methanol solution of the molecular probe, respectively, and detection was performed by ultraviolet absorption. As shown in fig. 4, only the molecular probe added with hydrazine has significantly reduced absorption at 808nm and significantly enhanced absorption at 550 nm; and a significant color change of the probe solution was observed with naked eyes: the color changes from green to red, indicating that the molecular probe has high selectivity for hydrazine detection.
Example 4
Application of molecular probe in detecting hydrazine by photoacoustic imaging method: under the same conditions, a control group was established: molecular probes plus hydrazine groups and molecular probes plus other ions (SO 4 2- 、NO 3- 、Cl - 、Br - 、CH 3 COO - 、NH 4 + 、Na + 、K + 、Ag + 、Zn 2+ 、Hg 2+ 、Mg 2+ 、Cu 2+ 、Mn 2+ 、Pb 2+ ) A group; the photoacoustic signal was detected at 808nm and probe photoacoustic signal variation at 808nm was detected by varying hydrazine concentration (0-10. Mu.M). As shown in FIG. 5 (1 to 17 represent SO in this order) 4 2- 、NO 3- 、Cl - 、Br - 、CH 3 COO - 、NH 4 + 、Na + 、K + 、Ag + 、Zn 2+ 、Hg 2+ 、Mg 2+ 、Cu 2+ 、Mn 2+ 、Pb 2+ And hydrazine), only the signal of the molecular probe added with hydrazine at 808nm is greatly reduced, and the molecular probes added with other ions do not cause the change of the photoacoustic signal; as shown in fig. 6, the photoacoustic signal decreases with increasing hydrazine concentration; as shown in fig. 7, PA 808nm The concentration of hydrazine is linearly related to the range of 0-10 mu M, and the detection limit is as low as 0.50 mu mol/L.
In summary, the invention discloses a molecular probe and a preparation method and application thereof, wherein the preparation method comprises the steps of dissolving heptamethine cyanine dye and sodium acetate in a first organic solvent, heating and stirring in inert gas to obtain a molecular probe intermediate; and dissolving the molecular probe intermediate and organic alkali in a second organic solvent, adding an acryloyl chloride solution under the protection of inert gas, and stirring at room temperature in sequence in an ice bath to obtain the molecular probe. According to the invention, through modifying and improving the structure of the heptamethine cyanine dye, a molecular probe with an ester bond and a strong conjugated structure is obtained, the ester bond of the molecular probe is hydrolyzed under the action of hydrazine, so that the conjugated structure of the molecular probe is damaged, an ultraviolet absorption peak blue is removed to form a new absorption peak, the original absorption peak is gradually reduced along with the increase of the concentration of the hydrazine, the newly generated absorption peak is gradually enhanced, and the prepared molecular probe has good selectivity and high sensitivity when detecting the hydrazine; and the preparation method is simple, the synthesis condition is not harsh, and the operation is convenient.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (8)
1. An application of a molecular probe in detecting hydrazine by a photoacoustic imaging method, wherein the molecular probe has a structural formula as follows:
wherein R is-CH 3 、-CH 2 CH 3 、
2. The use according to claim 1, wherein the method for preparing the molecular probe comprises:
dissolving heptamethine cyanine dye and sodium acetate in a first organic solvent, heating and stirring in inert gas to obtain a molecular probe intermediate;
dissolving the molecular probe intermediate and organic alkali in a second organic solvent, adding an acryloyl chloride solution under the protection of inert gas, and stirring sequentially at room temperature in an ice bath to obtain a molecular probe;
the structural formula of the heptamethine cyanine dye is as follows:
wherein R is-CH 3 、-CH 2 CH 3 、
3. The use according to claim 2, characterized in that the molar ratio of the heptamethine cyanine dye to the sodium acetate is between 1:1 and 1:4.
4. The use according to claim 2, wherein the step of dissolving the heptamethine cyanine dye and sodium acetate in the first organic solvent, heating and stirring in an inert gas, obtaining a molecular probe intermediate comprises:
the heptamethine cyanine dye and sodium acetate are dissolved in a first organic solvent, and stirred for 3 to 5 hours at the temperature of 80 to 100 ℃ under the protection of inert gas, so as to obtain a molecular probe intermediate.
5. The use according to claim 4, wherein the first organic solvent is one of Dimethylsulfoxide (DMSO), N-methylpyrrolidone (NMP) or N, N-Dimethylformamide (DMF).
6. The use according to claim 2, wherein the step of dissolving the molecular probe intermediate and the organic base in a second organic solvent, adding an acrylic chloride solution under the protection of an inert gas, and stirring sequentially in an ice bath at room temperature to obtain the molecular probe comprises:
dissolving the molecular probe intermediate and the organic alkali in a second organic solvent, stirring for 15min at 0-4 ℃, then dropwise adding an acryloyl chloride solution under the protection of inert gas, continuously stirring for 20min at 0-4 ℃, and continuously stirring for 3-5 h at room temperature to obtain the molecular probe.
7. The use according to claim 6, wherein the second organic solvent is one of dichloromethane or chloroform.
8. The use according to claim 6, wherein the molar ratio of the molecular probe intermediate to the acryloyl chloride is from 1:1 to 1:4.
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| CN108982447A (en) * | 2018-07-19 | 2018-12-11 | 曲阜师范大学 | It is a kind of for detecting the preparation method and application of the ratio formula fluorescence probe of hydrazine |
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| CN106432041A (en) * | 2016-09-26 | 2017-02-22 | 济南大学 | Preparation and application of ratio type near infrared hydrazine fluorescent probe compound |
| CN108982447A (en) * | 2018-07-19 | 2018-12-11 | 曲阜师范大学 | It is a kind of for detecting the preparation method and application of the ratio formula fluorescence probe of hydrazine |
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| A near-infrared fluorescent probe for rapid and selective detection of hydrosulfide and imaging in live cells;Yichen Jiang et al.;《Res Chem Intermed》;20161115;Scheme 1、Scheme 2、2948-2949页 * |
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