WO2022242147A1 - 一种近红外花菁类比色荧光探针及其制备方法和应用 - Google Patents
一种近红外花菁类比色荧光探针及其制备方法和应用 Download PDFInfo
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- WO2022242147A1 WO2022242147A1 PCT/CN2021/138482 CN2021138482W WO2022242147A1 WO 2022242147 A1 WO2022242147 A1 WO 2022242147A1 CN 2021138482 W CN2021138482 W CN 2021138482W WO 2022242147 A1 WO2022242147 A1 WO 2022242147A1
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- fluorescent probe
- infrared cyanine
- colorimetric
- infrared
- colorimetric fluorescent
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 114
- 238000002360 preparation method Methods 0.000 title claims abstract 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 title claims abstract 3
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 claims abstract description 106
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- 238000001228 spectrum Methods 0.000 claims description 11
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 10
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- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 10
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- CVBGNAKQQUWBQV-QFFDRWTDSA-N n'-acetyl-2,3,5,6-tetradeuteriopyridine-4-carbohydrazide Chemical compound [2H]C1=NC([2H])=C([2H])C(C(=O)NNC(C)=O)=C1[2H] CVBGNAKQQUWBQV-QFFDRWTDSA-N 0.000 description 3
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- KQDJZZWCYTXUDE-UHFFFAOYSA-N hydron;thiophene;chloride Chemical compound Cl.C=1C=CSC=1 KQDJZZWCYTXUDE-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
- C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1092—Heterocyclic compounds characterised by ligands containing sulfur as the only heteroatom
Definitions
- the invention relates to the technical field of detection and identification of toxic substances, in particular to a near-infrared cyanine-like colorimetric fluorescent probe and its preparation method and application.
- Anti-tuberculosis drugs are the most common drug agents, which can cause a series of liver damage in patients.
- Isoniazid is the first choice for effective treatment of tuberculosis agent, which is widely used to inhibit the activity of mycobacterial tuberculosis.
- Rifampicin and isoniazid are widely used in the treatment of tuberculosis by enzymatically inducing an increase in hydrazine levels.
- isoniazid and its major metabolite hydrazine may be associated with mitochondrial dysfunction.
- hydrazine The effect of hydrazine (generally referred to as hydrazine) on mitochondrial metabolic activity is mainly divided into two types. The first one needs to prevent the conversion of amine neurotransmitters by inhibiting monoamine oxidase in mitochondria. The second is associated morphological changes, including mitochondrial swelling and hypodensity, leading to disruption of homeostasis.
- hydrazine is an important chemical organic material with strong reducibility and nucleophilicity, and is widely used in the production of pesticides, pharmaceuticals, emulsifiers, corrosion inhibitors, related materials, and photographic materials.
- Fluorescent molecular probes are widely used in industry, agriculture, medicine, environmental detection, etc. It has a wide range of applications and has attracted extensive attention from researchers in the field of chemistry. In recent years, designing and synthesizing highly selective fluorescent probes to detect highly toxic pollutant hydrazine is one of the important research directions in the field of chemical research.
- near-infrared fluorescent probes can reduce the absorption and scattering of biomolecules, improve the signal-to-noise ratio of fluorescence imaging, and non-invasively realize the visualization of physiological processes in cells and living organisms. It has been widely studied in the field of fluorescence imaging. . At present, a large number of organic small molecule fluorescent probes for the detection of hydrazine have been reported, but the near-infrared fluorescent probes for the detection of hydrazine are relatively rare, and the existing near-infrared hydrazine fluorescent probes can only be used in vivo The detection of non-targeted exogenous hydrazine has low selectivity and sensitivity, poor anti-interference ability, and low detection limit.
- the present invention proposes a near-infrared cyanine-like colorimetric fluorescent probe and its preparation method and application.
- the structural formula of the probe is shown in formula (I).
- the probe can recognize N 2 H 4 in liquid phase and gas phase through fluorescence and ultraviolet-visible absorption channels, and has good sensitivity and strong anti-interference ability, and can be applied to exogenous hydrazine and drug metabolites in living cells fluorescence imaging detection.
- the present invention provides a near-infrared cyanine-like colorimetric fluorescent probe, the structural formula of which is shown in formula (I):
- R is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl, benzene ring;
- R is any one of methyl, ethyl, propyl , butyl, propanesulfonic acid
- n 0 or 1.
- the present invention also provides a method for preparing a near-infrared cyanine-like colorimetric fluorescent probe, comprising the following steps:
- R is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl, and benzene ring;
- R is any one of methyl, ethyl, propyl , butyl, propanesulfonic acid
- n 0 or 1.
- the second compound is dissolved in N,N-dimethylformamide, then anhydrous sodium acetate is added, and the first compound is generated under an argon atmosphere at 75-90°C;
- the structural formula of the second compound is shown in formula (III);
- R is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl, benzene ring;
- R is any one of methyl, ethyl, propyl , butyl, propanesulfonic acid
- n 0 or 1.
- the first compound is dissolved in anhydrous dichloromethane, followed by adding triethylamine and thienyl chloride for reaction, and after the reaction is completed, it is separated and purified to obtain the near-infrared cyanine-like colorimetric fluorescence probe.
- the present invention also provides a method for identifying N2H4 by using a near - infrared cyanine-like colorimetric fluorescent probe.
- the near-infrared cyanine-like colorimetric fluorescent probe is used to detect Spectral identification of N 2 H 4 ; the structural formula of the near-infrared cyanine-like colorimetric fluorescent probe is shown in formula (I):
- R is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl, benzene ring;
- R is any one of methyl, ethyl, propyl , butyl, propanesulfonic acid
- n 0 or 1.
- the mixed solution is a mixed solution of absolute ethanol and the PBS buffer of 10 ⁇ 50 mmol/L, and the volume ratio of the absolute ethanol and the PBS buffer of 10 ⁇ 50 mmol/L is 99: 1 ⁇ 1:99, the pH value of the PBS buffer solution is 4 ⁇ 10.
- the purpose of the present invention can be better achieved: that is, the probe has an obvious detection and recognition effect on hydrazine. However, under test conditions outside the parameter range, the probe has no obvious detection and recognition effect on hydrazine.
- the detection spectrum is a fluorescence spectrum.
- the detection spectrum is an ultraviolet-visible absorption spectrum.
- the present invention also provides the application of the near-infrared cyanine-like colorimetric fluorescent probe in the in - situ rapid detection of N2H4 in the environment.
- the present invention also provides the application of the near-infrared cyanine-like colorimetric fluorescent probe in the mitochondrial targeted tracer imaging detection of anti - tuberculosis drug metabolite N2H4 in vitro and in vivo.
- the fluorescent probe of the present invention has excellent selectivity for the recognition of N 2 H 4 , basically does not change the fluorescence signal or absorption signal when it interacts with other common amine substances and cations, and has strong anti-interference ability.
- the fluorescent probe of the present invention has good sensing properties and high sensitivity.
- the fluorescent probe of the present invention has an ultra-low detection limit of 0.5 ppb.
- the probe has the advantages of near-infrared fluorescence and can realize the portable on-site analysis and visual detection of hydrazine.
- the near-infrared fluorescent probe can not only realize the imaging detection of exogenous hydrazine, but more importantly, it can realize the tracer imaging detection of the anti-tuberculosis drug isoniazid metabolite (hydrazine).
- Fig. 1 is a schematic diagram of the fluorescent probe of the present invention for recognizing and detecting N 2 H 4 .
- Fig. 2 is a high-resolution mass spectrum characterization diagram of the near-infrared cyanine-like colorimetric fluorescent probe VI.
- Fig. 3 is a high-resolution mass spectrum characterization diagram of the near-infrared cyanine-like colorimetric fluorescent probe IX.
- Fig. 4 is a high-resolution mass spectrum characterization diagram of the near-infrared cyanine-like colorimetric fluorescent probe XII.
- Fig. 5 is a high-resolution mass spectrum characterization diagram of the near-infrared cyanine-like colorimetric fluorescent probe XV.
- Fig. 6 is a high-resolution mass spectrum characterization diagram of the near-infrared cyanine-like colorimetric fluorescent probe XVII.
- Fig. 7 is a graph of ultraviolet-visible light absorption spectra of different analytes.
- Figure 8 is a graph of fluorescence emission spectra of different analytes.
- Fig. 9 is an ultraviolet-visible light absorption spectrum diagram of different concentrations of hydrazine.
- Fig. 10 is a graph of fluorescence emission spectra of different concentrations of hydrazine.
- Fig. 11 is a titration linear curve diagram of different concentrations of hydrazine.
- Fig. 12 is a competitive relationship diagram.
- Figure 13 is a linear correlation diagram for calculating detection limits.
- Figure 14 is an RGB linearity plot for different hydrazine concentrations.
- Fig. 15 is a co-staining diagram of fluorescent probe VI and mitochondrial dye Mito-tracker FM488 in Hela cells.
- Fig. 16 is a confocal fluorescence image of the metabolic decomposition of fluorescent probe VI in Hela cells.
- Fig. 17 is a confocal fluorescence image of fluorescent probe VI in Hela cells after adding different concentrations of hydrazine.
- the present invention provides a near-infrared cyanine-like colorimetric fluorescent probe, the structural formula of which is shown in formula (I):
- R is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl, benzene ring;
- R is any one of methyl, ethyl, propyl , butyl, propanesulfonic acid
- n 0 or 1.
- R 1 and R 2 represent different benzene ring substituents and side chain groups, which are only modifications to the main structure. It does not affect its structural and functional properties, so those skilled in the art can infer that the different choices of R 1 and R 2 above can all achieve the technical effect of identifying and detecting N 2 H 4 , and the same reasoning applies when n is 0 or 1.
- the present invention also provides the application of a near-infrared cyanine colorimetric fluorescent probe, (1) the probe can identify N 2 H 4 (such as fluorescent Spectrum and UV-visible absorption spectrum), the principle is shown in Figure 1.
- the probe solution itself has a weak background fluorescence, and the color is cyanine green. After adding hydrazine, it reacts with the thiophene ester recognition group of the probe, and the thiophene ester protecting group is detached.
- Fluorescent parent the PET process is inhibited, the solution fluorescence is significantly enhanced and at the same time it changes from cyanine green to red, so the probe can perform colorimetric and fluorescent dual-channel recognition detection of hydrazine; (2) The probe can be used in vivo to target external Tracer imaging detection of source and drug metabolite hydrazine.
- the mixed solution is a mixed solution obtained by mixing absolute ethanol and 10-50 mmol/L PBS solution at a volume ratio of 99:1-1:99, wherein the pH value of the PBS buffer solution is 4-10.
- the purpose of the present invention can be better achieved: that is, the probe has an obvious detection and recognition effect on hydrazine. However, under test conditions outside the parameter range, the probe has no obvious detection and recognition effect on hydrazine.
- the detection spectrum for identifying N 2 H 4 is fluorescence spectrum and ultraviolet-visible absorption spectrum.
- the fluorescent probe After the fluorescent probe is co-incubated with cervical cancer cells, after adding different concentrations of hydrazine and isoniazid respectively, the hydrazine in the cell mitochondria is visually monitored by a fluorescent confocal microscope.
- the fluorescent probe VI of this embodiment corresponds to the compound of formula (I) in which R 1 is hydrogen, R 2 is propyl, and n is 1.
- the fluorescent probe IX of this embodiment corresponds to the compound of formula (I) in which R 1 is a benzene ring, R 2 is a propyl group, and n is 0.
- the fluorescent probe XII of this embodiment corresponds to the compound of formula (I) in which R 1 is chlorine, R 2 is methyl, and n is 1.
- the fluorescent probe XV of this embodiment corresponds to the compound of formula (I) in which R 1 is hydrogen, R 2 is propanesulfonic acid group, and n is 1.
- the fluorescent probe XVII of this embodiment corresponds to the compound of formula (I) in which R 1 is hydrogen, R 2 is ethyl, and n is 0.
- the detection results are shown in Figure 7 and Figure 8, and the near-infrared
- the cyanine-like colorimetric fluorescent probe itself has almost no emission peak at 627 nm, and almost no absorption peak at 537 nm; when N 2 H 4 is added, the near-infrared cyanine-like colorimetric fluorescent probe solution has a strong Emission peak; however other metal ions such as Mg 2+ , Al 3+ , Ca 2+ , Cr 3+ , Mn 2+ , Fe 2+ , Fe 3+ , Co 2+ , Ni 2+ , Zn 2+ , Ag + , Cd 2+ , Sn 2+ , Ba 2+ , Hg 2+ , Pb 2+ , La 3+ , Ce 3+ , Pr 3+ , Nd 3+ , Sm 3+ , Eu 3+ , Dy 3+ , After Er 3+ and Th 4+ were added, the emission peak at 627 nm of the near-infrared cyanine color
- the probes are loaded on ordinary filter paper to prepare a portable paper-based film.
- the volume fraction of N 2 H 4 is 0%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50% aqueous solution, and the gaseous hydrazine is detected by the probe film.
- the solution is naturally volatilized
- use a smartphone to scan the color of the paper-based film after the action of gaseous hydrazine, and obtain the RGB value of the corresponding concentration.
- the fluorescent probe has good targeting (Pearson colocalization coefficient is 0.93).
- fluorescence confocal microscopy imaging was performed. The fluorescent signal is gradually enhanced; then, after the probe is incubated with cervical cancer cells, isoniazid, isoniazid, rifampicin, and rifampicin are added respectively.
- Fig. 15 is a co-staining diagram of fluorescent probe VI and mitochondrial dye Mito-tracker FM488 in Hela cells.
- A is the bright field image
- B is the confocal fluorescence image of the fluorescent probe VI in the cell
- C is the confocal fluorescence image of the mitochondrial dye Mito-tracker FM488 in the cell
- D is the overlay image of A, B, and C.
- Figure 17 is the confocal fluorescence image of fluorescent probe VI in HeLa cells after adding different concentrations of hydrazine; wherein, A is the confocal fluorescence image of only fluorescent probe VI in HeLa cells; B is the confocal fluorescence image of fluorescent probe VI and Co-staining image of 10 ⁇ M hydrazine; C is the co-staining image of fluorescent probe VI and 20 ⁇ M hydrazine; D is the co-staining image of fluorescent probe VI and 50 ⁇ M hydrazine.
- the present invention provides a near-infrared cyanine-based colorimetric fluorescent probe and its preparation method and application.
- the structural formula of the probe is shown in formula (I).
- the fluorescent probe can identify and detect N 2 H 4 by using fluorescence and ultraviolet-visible absorption spectrum.
- the fluorescent probe of the present invention has excellent selectivity for the recognition of N2H4 , basically no change in fluorescence signal or absorption signal when interacting with other common amine substances and cations, strong anti - interference ability, and good sensing properties and high sensitivity with an ultra-low detection limit of 0.5 ppb.
- the probe has the advantages of near-infrared fluorescence and can realize the portable on-site analysis and visual detection of hydrazine. It can not only realize the imaging detection of exogenous hydrazine, but more importantly, it can realize the display of the anti-tuberculosis drug isoniazid metabolite (hydrazine). Tracking imaging detection.
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Abstract
本发明公开了一种近红外花菁类比色荧光探针及其制备方法和应用，该探针的结构式如式（I）所示。该荧光探针在无水乙醇和PBS缓冲液的混合溶液中，利用荧光和紫外可见吸收光谱可识别检测N 2H 4。该探针对N 2H 4的识别具有高选择性和灵敏度，抗干扰能力强，而且具有非常低的检测限；所述荧光探针可以应用于环境污染或生物体外以及生物体内抗结核药物代谢物N 2H 4的检测。
Description
本发明涉及毒性物质检测识别技术领域，特别涉及一种近红外花菁类比色荧光探针及其制备方法和应用。
抗结核药是最普遍的一种药试剂，可导致病人一系列肝损伤。异烟肼则是首选的有效治疗结核病试剂，被广泛用于抑制分枝杆菌结核病的活性。研究表明，异烟肼的肝毒性取决于多种酶产生的代谢物（肼）。利福平和异烟肼被广泛用于治疗结核病，通过酶诱导提高联氨水平。有实验证据表明异烟肼及其主要代谢物肼可能与线粒体功能障碍有关。联氨（一般指肼）对线粒体代谢活性的影响主要分为两种，第一种需要通过抑制线粒体中的单胺氧化酶来阻止胺类神经递质转化的过程。第二种是相关的形态学改变，包括线粒体肿胀和低密度，导致体内平衡的破坏。此外，联氨是一种重要的化学有机材料，具有很强的还原性和亲核性，在农药、药品、乳化剂、缓蚀剂、相关材料和照相材料的生产中有着广泛的应用。然而，伴随着它的重要性和巨大的应用范围，含肼工业废物大量排放到环境中会不可避免地破坏生态平衡，严重污染土地和水资源，严重损害人的肝、肺和肾等重要器官，并可通过皮肤和肺部的吸收诱发高毒性和致癌性。正因为联氨的高毒性和致癌性，美国环保局规定的饮用水中联氨的最大阈限值为10 ppb。因此，需要一种可靠有效的方法来分析检测肼（N
2H
4）。
荧光分子探针因其具有好的选择性、高的灵敏度、价格低廉和操作简便，并且能够快速、实时、原位定量检测和分析等优点，在工业、农业、医药学、环境检测等各个方面都有广泛的应用，引起了化学领域研究者的广泛关注。近年来，设计和合成高选择性荧光探针检测高毒污染物肼是化学研究领域重要研究方向之一。
近红外荧光探针的使用可以降低生物分子的吸收与散射，能够提高荧光成像的信噪比，可以非侵入性地实现细胞及生命体中的生理过程的可视化，在荧光成像领域得到了广泛研究。目前，已报道过大量用于检测肼的有机小分子荧光探针，但其中用于检测肼的近红外类荧光探针却较稀少，而且已有的近红外肼荧光探针仅可用于生物体内非靶向性外源肼的检测，且选择性和灵敏度低、抗干扰能力差、检测限也不够低，目前亟需一种具有高选择性和灵敏度、抗干扰能力强、检测限足够低、且能用于生物体内靶向性内外源肼的可视化检测的近红外荧光探针。
针对现有技术中的缺陷，本发明提出了一种近红外花菁类比色荧光探针及其制备方法和应用。该探针的结构式如式（I）所示。该探针可以通过荧光和紫外可见吸收两种通道识别液相和气相的N
2H
4，并且具有良好的灵敏度、较强的抗干扰能力，可以应用于活细胞中外源性肼以及药物代谢产物的荧光成像检测。
本发明提供一种近红外花菁类比色荧光探针，所述荧光探针的结构式如式（I）所示：
其中，R
1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；
R
2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；
n为0或1。
本发明还提供一种近红外花菁类比色荧光探针的制备方法，包括如下步骤：
S1：合成第一化合物；第一化合物的结构式如式（II）所示；
S2：由所述第一化合物合成近红外花菁类比色荧光探针，所述近红外花菁类比色荧光探针的结构式如式（I）所示：
其中，所述第一化合物和所述近红外花菁类比色荧光探针的结构式中R
1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；
R
2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；
n为0或1。
进一步的，所述步骤S1中将第二化合物溶解在N,N-二甲基甲酰胺中，随后加入无水醋酸钠，在75~90℃氩气气氛下，生成所述第一化合物；所述第二化合物的结构式如式（III）所示；
其中，R
1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；
R
2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；
n为0或1。
进一步的，所述步骤S2中将所述第一化合物溶于无水二氯甲烷中，依次加入三乙胺、噻吩酰氯进行反应，反应结束后分离纯化，得到所述近红外花菁类比色荧光探针。
本发明还提供一种利用近红外花菁类比色荧光探针识别N
2H
4的方法，所述近红外花菁类比色荧光探针在无水乙醇和PBS缓冲液的混合溶液中，利用检测光谱识别N
2H
4；所述近红外花菁类比色荧光探针的结构式如式（I）所示：
其中，R
1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；
R
2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；
n为0或1。
进一步的，所述混合溶液为无水乙醇与10~50 mmol/L的PBS缓冲液的混合溶液，所述无水乙醇与所述10~50 mmol/L的PBS缓冲液的体积比为99:1~1:99，所述PBS缓冲液的pH值为4~10。在上述测试条件的参数范围内，能较好地实现本发明目的：即所述探针对肼有明显的检测识别效果。而在所述参数范围之外的测试条件下，所述探针对肼则无明显检测识别效果。
进一步的，所述检测光谱是荧光光谱。
进一步的，所述检测光谱是紫外-可见吸收光谱。
本发明还提供所述的近红外花菁类比色荧光探针在环境中现场原位快速检测N
2H
4中的应用。
本发明还提供所述的近红外花菁类比色荧光探针在生物体外以及生物体内抗结核药物代谢物N
2H
4的线粒体靶向示踪成像检测中的应用。
综上，与现有技术相比，本发明达到了以下技术效果：
（1）本发明的荧光探针对N
2H
4的识别有优良的选择性，与其它常见胺类物质和阳离子作用时荧光信号或吸收信号基本没有变化，抗干扰能力强。
（2）本发明的荧光探针具有较好的传感性质和高灵敏度。
（3）本发明的荧光探针具有0.5 ppb的超低检测限。
（4）该探针具有近红外荧光优点同时可实现肼的可便携式现场分析可视化检测。
（5）该近红外荧光探针不仅可实现对外源性肼的成像检测，更重要的是可实现抗结核药物异烟肼代谢产物（肼）的示踪成像检测。
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1是本发明的荧光探针识别检测N
2H
4的原理图。
图2是近红外花菁类比色荧光探针VI的高分辨质谱表征图。
图3是近红外花菁类比色荧光探针IX的高分辨质谱表征图。
图4是近红外花菁类比色荧光探针XII的高分辨质谱表征图。
图5是近红外花菁类比色荧光探针XV的高分辨质谱表征图。
图6是近红外花菁类比色荧光探针XVII的高分辨质谱表征图。
图7是不同分析物的紫外可见光吸收光谱图。
图8是不同分析物的荧光发射光谱图。
图9是不同浓度肼的紫外可见光吸收光谱图。
图10是不同浓度肼的荧光发射光谱图。
图11是不同浓度肼的滴定线性曲线图。
图12是竞争性关系图。
图13是计算检出限的线性相关图。
图14是不同肼浓度的RGB线性图。
图15是荧光探针VI在Hela细胞中，与线粒体染料Mito-tracker FM488的共染图。
图16是荧光探针VI在Hela细胞中，药物代谢分解的共聚焦荧光图。
图17是荧光探针VI在Hela细胞中，加入不同浓度肼之后的共聚焦荧光图。
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
本发明提供了一种近红外花菁类比色荧光探针，该荧光探针的结构式如式（I）所示：
其中R
1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；
R
2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；
n为0或1。
所述近红外花菁类比色荧光探针的主体结构如式（I）所示，因为R
1、R
2分别代表不同的苯环取代基以及侧链基团，仅是对主体结构的修饰，而不影响其结构功能特性，因此本领域技术人员可以推断上述R
1、R
2的不同选择均能够实现识别检测N
2H
4的技术效果，n为0或1同理。
上述近红外花菁类比色荧光探针的反应式为：
具体步骤如下：
（1）将式III溶解在N,N-二甲基甲酰胺中，随后加入无水醋酸钠，按照上述反应式进行反应，生成式II，产率为58~85%；
（2）将式II溶于无水二氯甲烷中，最后依次加入三乙胺、噻吩酰氯进行反应，反应结束后，萃取、柱层析分离纯化后得到近红外花菁类比色荧光探针式I，产率为52~80%。
本发明还提供了一种近红外花菁类比色荧光探针的应用，（1）该探针在乙醇和PBS缓冲液的混合溶液中，可利用不同的检测光谱识别N
2H
4（如荧光光谱和紫外-可见吸收光谱），其原理见图1，探针溶液本身具有较弱的背景荧光，颜色为菁绿色，加入肼之后与探针的噻吩酯识别基发生反应，噻吩酯保护基脱离荧光母体，PET过程被抑制使溶液荧光显著增强同时由菁绿色变为红色，由此探针可以对肼进行比色和荧光双通道识别检测；（2）该探针可用于生物体内靶向外源性以及药物代谢产物肼的示踪成像检测。
其中，所述混合溶液为无水乙醇与10~50 mmol/L的PBS溶液按体积比99:1~1:99混合得到的混合液，其中PBS缓冲液的pH值为4~10。在上述测试条件的参数范围内，能较好地实现本发明目的：即所述探针对肼有明显的检测识别效果。而在所述参数范围之外的测试条件下，所述探针对肼则无明显检测识别效果。
所述的识别N
2H
4的检测光谱是荧光光谱和紫外-可见吸收光谱。
所述荧光探针与宫颈癌细胞共同孵育后，分别加入不同浓度肼和异烟肼后，通过荧光共聚焦显微镜对细胞线粒体内的肼进行可视化监测。
实施例 1 合成近红外花菁类比色荧光探针VI
本实施例的荧光探针VI对应式（I）化合物的R
1为氢，R
2为丙基，n为1。
合成近红外花菁类比色荧光探针VI的反应式如下式（A）：
合成近红外花菁类比色荧光探针VI的具体步骤：
（1）中间体V的合成：IV和乙酸钠按照1:5~1:2的摩尔比溶在无水N,N-二甲基甲酰胺（15 mL）中，75~90℃氩气气氛下加热2~6 h。混合物冷却至室温后，过滤，将得到的溶液经减压浓缩得到油状粗产品。最后，硅胶柱层析分离纯化（二氯甲烷作为洗脱液），得到中间体V纯品，收率50~70%。
（2）近红外花菁类比色荧光探针VI的合成：中间体V和噻吩酰氯按照1:6~1:2摩尔比溶于N,N-二甲基甲酰胺中；置于恒压滴液漏斗中，缓慢滴加至上述溶液中，于室温下搅拌反应30 min，最后，硅胶柱层析分离纯化（二氯甲烷和甲醇作为洗脱液，其体积比1:0~100:1)。得到所述近红外花菁类比色荧光探针VI，并用质谱、核磁共振氢谱/碳谱表征了其结构。
高分辨质谱（电喷雾，正模式）表征：C
41H
47N
2O
2S的分子质量M理论计算值为631.34；实测值[M
+H]
+为631.2（如图2）。
实施例 2 合成近红外花菁类比色荧光探针IX
本实施例的荧光探针IX对应式（I）化合物的R
1为苯环，R
2为丙基，n为0。
合成近红外花菁类比色荧光探针IX的反应式如下式（B）：
合成近红外花菁类比色荧光探针IX的具体步骤：
（1）中间体VIII的合成：VII和乙酸钠按照1:1~1:4的摩尔比溶在无水N,N-二甲基甲酰胺（15 mL）中，70~90℃氩气气氛下加热1~6 h。混合物冷却至室温后，过滤，将得到的溶液经减压浓缩得到油状粗产品。最后，硅胶柱层析分离纯化（二氯甲烷作为洗脱液），得到中间体VIII纯品，收率40~65%。
（2）近红外花菁类比色荧光探针IX的合成：中间体VIII和噻吩酰氯按照1:10~1:1摩尔比溶于N,N-二甲基甲酰胺中；置于恒压滴液漏斗中，缓慢滴加至上述溶液中，于室温下搅拌反应30 min，最后，硅胶柱层析分离纯化（二氯甲烷和甲醇作为洗脱液，其体积比1:0~50:1）。得到所述近红外花菁类比色荧光探针IX，并用质谱、核磁共振氢谱/碳谱表征了其结构。
高分辨质谱（电喷雾，正模式）表征：C
48H
49N
2O
2S
+的分子质量M理论计算值为717.35；实测值[M
+H]
+为717.345（如图3）。
实施例 3 合成近红外花菁类比色荧光探针XII
本实施例的荧光探针XII对应式（I）化合物的R
1为氯，R
2为甲基，n为1。
合成近红外花菁类比色荧光探针XII的反应式如下式（C）：
合成近红外花菁类比色荧光探针XII的具体步骤：
（1）中间体XI的合成：X和乙酸钠按照1:3~1:1.5的摩尔比溶在无水N,N-二甲基甲酰胺（15 mL）中，75~100℃氩气气氛下加热2~5 h。混合物冷却至室温后，过滤，将得到的溶液经减压浓缩得到油状粗产品。最后，硅胶柱层析分离纯化（二氯甲烷作为洗脱液），得到中间体XI纯品，收率65~80%。
（2）近红外花菁类比色荧光探针XII的合成：中间体XI和噻吩酰氯按照1:8~1:2摩尔比溶于N,N-二甲基甲酰胺中；置于恒压滴液漏斗中，缓慢滴加至上述溶液中，于室温下搅拌反应30 min，最后，硅胶柱层析分离纯化（二氯甲烷和甲醇作为洗脱液，其体积比1:0~80:1)。得到所述近红外花菁类比色荧光探针XII，并用质谱、核磁共振氢谱/碳谱表征了其结构。
高分辨质谱（电喷雾，正模式）表征：C
37H
37Cl
2N
2O
2S
+的分子质量M理论计算值为643.19；实测值[M
+H]
+为644.20（如图4）。
实施例 4 合成近红外花菁类比色荧光探针XV
本实施例的荧光探针XV对应式（I）化合物的R
1为氢，R
2为丙烷磺酸基，n为1。
合成近红外花菁类比色荧光探针XV的反应式如下式（D）：
合成近红外花菁类比色荧光探针XV的具体步骤：
（1）中间体XIV的合成：XIII和乙酸钠按照1:4~1:1的摩尔比溶在无水N,N-二甲基甲酰胺(15 mL)中，70~100℃氩气气氛下加热1~5 h。混合物冷却至室温后，过滤，将得到的溶液经减压浓缩得到油状粗产品。最后，硅胶柱层析分离纯化（二氯甲烷作为洗脱液），得到中间体XIV纯品，收率45~75%。
（2）近红外花菁类比色荧光探针XV的合成：中间体XIV和噻吩酰氯按照1:6~1:2摩尔比溶于N,N-二甲基甲酰胺中；置于恒压滴液漏斗中，缓慢滴加至上述溶液中，于室温下搅拌反应30 min，最后，硅胶柱层析分离纯化（二氯甲烷和甲醇作为洗脱液，其体积比1:0~60:1)。得到所述近红外花菁类比色荧光探针XIV，并用质谱、核磁共振氢谱/碳谱表征了其结构。
高分辨质谱（电喷雾，正模式）表征：C
41H
45N
2O
8S
3
+的分子质量M理论计算值为789.23；实测值[M
+H]
+为789.22（如图5）。
实施例 5 合成近红外花菁类比色荧光探针XVII
本实施例的荧光探针XVII对应式（I）化合物的R
1为氢，R
2为乙基，n为0。
合成近红外花菁类比色荧光探针XVII的反应式如下式（E）：
合成近红外花菁类比色荧光探针XVII的具体步骤：
（1）中间体XVI的合成：XV和乙酸钠按照1:3~1:1的摩尔比溶在无水N,N-二甲基甲酰胺（15 mL）中，70~95℃氩气气氛下加热1~4 h。混合物冷却至室温后，过滤，将得到的溶液经减压浓缩得到油状粗产品。最后，硅胶柱层析分离纯化（二氯甲烷作为洗脱液），得到中间体XVI纯品，收率50~75%。
（2）近红外花菁类比色荧光探针XVII的合成：中间体XVI和噻吩酰氯按照1:6~1:1摩尔比溶于N,N-二甲基甲酰胺中；置于恒压滴液漏斗中，缓慢滴加至上述溶液中，于室温下搅拌反应30 min，最后，硅胶柱层析分离纯化（二氯甲烷和甲醇作为洗脱液，其体积比1:0~100:1)。得到所述近红外花菁类比色荧光探针XVII，并用质谱、核磁共振氢谱/碳谱表征了其结构。
高分辨质谱（电喷雾，正模式）表征：C
38H
41N
2O
2S
+的分子质量M理论计算值为589.29；实测值[M
+H]
+为589.36（如图6）。
实施例 6 本发明的荧光探针对N
2H
4的选择性检测
10
µmol/L近红外花菁类比色荧光探针（实施例1制备的荧光探针VI）的无水乙醇和10 mol/L、pH = 4的PBS缓冲液的体积比为99:1的混合溶液，分别加入100 µmol/L的金属离子（Mg
2+，Al
3+，Ca
2+，Cr
3+，Mn
2+，Fe
2+，Fe
3+，Co
2+，Ni
2+，Zn
2+，Ag
+，Cd
2+，Sn
2+，Ba
2+，Hg
2+，Pb
2+，VO
2+，La
3+，Ce
3+，Pr
3+，Nd
3+，Sm
3+，Eu
3+，Dy
3+，Er
3+，Th
4+，Cu
2+），15 min后检测溶液的荧光发射光谱变化及紫外可见吸收光谱变化，检测结果如图7和图8所示，近红外花菁类比色荧光探针自身在627 nm处几乎没有发射峰，在537 nm处几乎没有吸收峰；当加入N
2H
4后，近红外花菁类比色荧光探针溶液在627 nm处出现强发射峰；然而其它金属离子，如Mg
2+，Al
3+，Ca
2+，Cr
3+，Mn
2+，Fe
2+，Fe
3+，Co
2+，Ni
2+，Zn
2+，Ag
+，Cd
2+，Sn
2+，Ba
2+，Hg
2+，Pb
2+，La
3+，Ce
3+，Pr
3+，Nd
3+，Sm
3+，Eu
3+，Dy
3+，Er
3+，Th
4+加入后，近红外花菁类比色荧光探针溶液在627 nm处的发射峰无明显增强，在537 nm处的吸收峰亦无明显变化；实验结果表明，只有加入N
2H
4，才能引起近红外花菁类比色荧光探针溶液的显著荧光增强；该近红外花菁类比色荧光探针在无水乙醇和10 mol/L、pH = 4的PBS缓冲液的体积比为99:1的混合溶液中对N
2H
4具有良好的选择性。
实施例 7 近红外花菁类比色荧光探针对N
2H
4的荧光滴定实验
10
µmol/L近红外花菁类比色荧光探针的无水乙醇和30 mol/L、pH = 7的PBS缓冲液的体积比为1:1的混合溶液，逐步增加加入的N
2H
4的浓度，15 min后测试各试样的荧光光谱及吸收光谱变化检测结果如图9、图10和图11所示，随着N
2H
4浓度的逐渐增大，近红外花菁类比色荧光探针溶液在627 nm处的荧光强度逐渐增大，在537 nm处的吸收强度也逐渐增大，当N
2H
4的浓度分别增大到100 µmol/L左右时，滴定达到饱和，发射强度及吸收强度不再增大；这也说明该近红外花菁类比色荧光探针对N
2H
4具有较好的传感性质。
实施例 8 近红外花菁类比色荧光探针对N
2H
4识别的竞争实验
10
µmol/L近红外花菁类比色荧光探针的无水乙醇和PBS缓冲液，分别加入100 µmol/L的其它胺分子（Cys，GSH，Hcy，Urea，NH
3H
2O，PhNH
2，TEA，DETA，NH
2OH，BA，ButNH
2，NO,H
2O
2，H
2S，Cy-N
2H
4），15 min后测试各溶液的荧光光谱；然后向以上各个含胺分子的溶液中分别加入100 µmol/L的N
2H
4，放置15 min后再分别测试各个溶液的荧光光谱和紫外可见吸收光谱。结果如图12所示，共存的其它含胺分子对N
2H
4的荧光识别、对N
2H
4的紫外可见吸收识别没有显著干扰。
实施例 9 近红外花菁类比色荧光探针对N
2H
4检测限的计算
检测限根据荧光光谱或紫外可见吸收光谱滴定数据计算。以I/I
0为纵坐标，N
2H
4浓度为横坐标作图。I为各试样的荧光强度，I
0为不加N
2H
4时探针溶液的荧光强度。结果如图13所示，在N
2H
4为0~40 µmol/L的浓度范围内，线性关系较好（R
2 = 0.9938），直线方程为y = 0.5436*10
6C
N2H4+1.4383。利用公式DL = 3σ/S（σ为空白实验标准方差，S为直线斜率），经计算检测限为0.5 ppb。探针对N
2H
4具有超低的检测限。
实施例 10 近红外花菁类比色荧光探针的原位快速检测N
2H
4
将探针负载到普通滤纸，制备成便携式纸基薄膜。另外，配制N
2H
4体积分数为0%、0.1%、0.5%、1%、5%、10%、20%、30%、40%、50%的水溶液，利用探针薄膜检测其气态肼（溶液自然挥发），利用智能手机扫描与气态肼作用后的纸基薄膜颜色，得出相对应浓度的RGB值。根据R/RGB值做出线性关系图，结果如图14所示，N
2H
4在0.1%到40%的浓度范围内，线性关系较好（R
2 = 0.99），直线方程y = 0.013x+0.302。由此该探针可实现便携式现场检测。
实施例 11 近红外花菁类比色荧光探针的活细胞荧光示踪成像
首先，与商业线粒体靶向染料Mito-tracker green对比，该荧光探针具有很好的靶向性（皮尔森共定位系数为0.93）。其次，将该探针和不同浓度的肼（10 μM、20 μM、50 μM）在宫颈癌细胞（Hela细胞）共同孵育之后进行荧光共聚焦显微镜成像，与空白对照组比，随着肼浓度的增加荧光信号逐渐增强；随后，将探针与宫颈癌细胞孵育之后，分别加入异烟肼、异烟肼和利福平、利福平。图15是荧光探针VI在Hela细胞中，与线粒体染料Mito-tracker FM488的共染图。其中，A为明场图；B为荧光探针VI在细胞中的共聚焦荧光图；C为线粒体染料Mito-tracker FM488在细胞中共聚焦荧光图，D为A，B，C的叠加图。图16是荧光探针VI在Hela细胞中，药物分解的共聚焦荧光图；其中A是异烟肼和荧光探针VI的共染图；B是异烟肼和利福平与荧光探针VI的共染图；C是利福平和荧光探针VI的共染图。图17是荧光探针VI在Hela细胞中，加入不同浓度肼之后的共聚焦荧光图；其中，A是仅有荧光探针VI在海拉细胞的共聚焦荧光图；B是荧光探针VI和10 μM肼的共染图；C是荧光探针VI和20 μM肼的共染图；D是荧光探针VI和50 μM肼的共染图。结果显示，单独加异烟肼孵育之后的宫颈癌细胞中可以观察到荧光信号，而同时加异烟肼和利福平孵育之后的宫颈癌细胞荧光信号增强，单独加利福平孵育之后的宫颈癌细胞与对照组均观察不到荧光信号。由此说明该探针可以实现抗结核药物异烟肼代谢产物（肼）的示踪成像检测。
综合以上实施例，本发明提供了一种近红外花菁类比色荧光探针及其制备方法和应用，该探针的结构式如式（I）所示。该荧光探针在无水乙醇和PBS缓冲液的混合溶液中，利用荧光和紫外可见吸收光谱可识别检测N
2H
4。本发明的荧光探针对N
2H
4的识别有优良的选择性，与其它常见胺类物质和阳离子作用时荧光信号或吸收信号基本没有变化，抗干扰能力强，具有较好的传感性质和高灵敏度，具有0.5 ppb的超低检测限。该探针具有近红外荧光优点同时可实现肼的可便携式现场分析可视化检测，不仅可实现对外源性肼的成像检测，更重要的是可实现抗结核药物异烟肼代谢产物（肼）的示踪成像检测。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (10)
- 一种近红外花菁类比色荧光探针，其特征在于，所述荧光探针的结构式如式（I）所示：其中，R 1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；R 2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；n为0或1。
- 一种近红外花菁类比色荧光探针的制备方法，其特征在于，包括如下步骤：S1：合成第一化合物；第一化合物的结构式如式（II）所示；S2：由所述第一化合物合成近红外花菁类比色荧光探针，所述近红外花菁类比色荧光探针的结构式如式（I）所示：其中，所述第一化合物和所述近红外花菁类比色荧光探针的结构式中R 1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；R 2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；n为0或1。
- 根据权利要求2所述的制备方法，其特征在于，所述步骤S1中将第二化合物溶解在N,N-二甲基甲酰胺中，随后加入无水醋酸钠，在75~90℃氩气气氛下，生成所述第一化合物；所述第二化合物的结构式如式（III）所示；其中，R 1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；R 2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；n为0或1。
- 根据权利要求2所述的制备方法，其特征在于，所述步骤S2中将所述第一化合物溶于无水二氯甲烷中，依次加入三乙胺、噻吩酰氯进行反应，反应结束后分离纯化，得到所述近红外花菁类比色荧光探针。
- 一种利用近红外花菁类比色荧光探针识别N 2H 4的方法，其特征在于，所述近红外花菁类比色荧光探针在无水乙醇和PBS缓冲液的混合溶液中，利用检测光谱识别N 2H 4；所述近红外花菁类比色荧光探针的结构式如式（I）所示：其中，R 1为氢、氟、氯、溴、碘、甲基、苯环中的任意一种；R 2为甲基、乙基、丙基、丁基、丙烷磺酸基中的任意一种；n为0或1。
- 根据权利要求5所述的方法，其特征在于，所述混合溶液为无水乙醇与10~50 mmol/L的PBS缓冲液的混合溶液，所述无水乙醇与所述10~50 mmol/L的PBS缓冲液的体积比为99:1~1:99，所述PBS缓冲液的pH值为4~10。
- 根据权利要求5所述的方法，其特征在于，所述检测光谱是荧光光谱。
- 根据权利要求5所述的方法，其特征在于，所述检测光谱是紫外-可见吸收光谱。
- 权利要求1所述的近红外花菁类比色荧光探针在环境中现场原位快速检测N 2H 4中的应用。
- 权利要求1所述的近红外花菁类比色荧光探针在生物体外以及生物体内抗结核药物代谢物N 2H 4的线粒体靶向示踪成像检测中的应用。
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| CN108982447A (zh) * | 2018-07-19 | 2018-12-11 | 曲阜师范大学 | 一种用于检测肼的比率式荧光探针的制备方法及应用 |
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| CN108982447A (zh) * | 2018-07-19 | 2018-12-11 | 曲阜师范大学 | 一种用于检测肼的比率式荧光探针的制备方法及应用 |
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