CN113234068A - Near-infrared cyanine analog color fluorescent probe and preparation method and application thereof - Google Patents
Near-infrared cyanine analog color fluorescent probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a near-infrared cyanine analog color fluorescent probe and a preparation method and application thereof. The fluorescent probe can identify and detect N in a mixed solution of absolute ethyl alcohol and PBS buffer solution by using fluorescence and ultraviolet visible absorption spectrum2H4. The probe pair N2H4The identification has high selectivity and sensitivity, strong anti-interference capability and very low detection limit; the fluorescent probe can be applied to the metabolite N of the antituberculosis drug in environmental pollution or in vitro and in vivo2H4Detection of (3).
Description
Technical Field
The invention relates to the technical field of toxic substance detection and identification, in particular to a near-infrared cyanine analog color fluorescent probe and a preparation method and application thereof.
Background
Antituberculosis drugs are the most common pharmaceutical agents and can cause a range of liver damage in patients. Isoniazid is the first choice of effective tuberculosis treatment agent, and is widely used for inhibiting the activity of mycobacterium tuberculosis. Studies have shown that the hepatotoxicity of isoniazid depends on the metabolites (hydrazine) produced by various enzymes. Rifampicin and isoniazid are widely used in the treatment of tuberculosis, increasing hydrazine levels by enzyme induction. There is experimental evidence that isoniazid and its major metabolite hydrazine may be associated with mitochondrial dysfunction. The effects of hydrazine (commonly referred to as hydrazine) on mitochondrial metabolic activity are largely divided into two, the first requiring the process of arresting the conversion of amine neurotransmitters by inhibiting monoamine oxidase in the mitochondria. The second is the associated morphological changes, including mitochondrial swelling and low density, leading to disruption of homeostasis. In addition, hydrazine is an important chemical organic material, has strong reducibility and nucleophilicity, and has wide application in the production of pesticides, medicines, emulsifiers, corrosion inhibitors, related materials and photographic materials. However, with its importance and wide application range, the discharge of hydrazine-containing industrial waste into the environment in large quantities inevitably destroys the ecological balance, seriously pollutes the land and water resources, seriously damages important organs such as human liver, lung and kidney, and induces high toxicity and carcinogenicity through absorption into the skin and lung. Due to the high toxicity and carcinogenicity of hydrazine, the U.S. environmental protection agency sets a maximum threshold value of hydrazine of 10ppb in drinking water. Therefore, there is a need for a reliable and efficient method for the analytical detection of hydrazine (N)2H4)。
The fluorescent molecular probe has the advantages of good selectivity, high sensitivity, low price, simple and convenient operation, rapid, real-time and in-situ quantitative detection and analysis and the like, has wide application in various aspects of industry, agriculture, medicine, environmental detection and the like, and has attracted extensive attention of researchers in the chemical field. In recent years, designing and synthesizing a high-selectivity fluorescent probe to detect hydrazine, a high-toxicity pollutant, is one of important research directions in the field of chemical research.
The near-infrared fluorescent probe can reduce absorption and scattering of biomolecules, improve the signal-to-noise ratio of fluorescence imaging, realize visualization of physiological processes in cells and living bodies in a non-invasive manner, and be widely researched in the field of fluorescence imaging. At present, a great number of organic small-molecule fluorescent probes for detecting hydrazine are reported, but the near-infrared fluorescent probes for detecting hydrazine are rare, and the existing near-infrared hydrazine fluorescent probes can only be used for detecting non-targeting exogenous hydrazine in organisms, and have the advantages of low selectivity and sensitivity, poor interference resistance and insufficient detection limit, so that a near-infrared fluorescent probe which has the advantages of high selectivity and sensitivity, strong interference resistance and low detection limit and can be used for visually detecting the targeting endogenous exogenous hydrazine in the organisms is urgently needed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a near-infrared cyanine analog color fluorescent probe and a preparation method and application thereof. The structural formula of the probe is shown as a formula (I). The probe can identify N of liquid phase and gas phase through two channels of fluorescence and ultraviolet visible absorption2H4And the fluorescent probe has good sensitivity and strong anti-interference capability, and can be applied to the fluorescent imaging detection of exogenous hydrazine and drug metabolites in living cells.
The invention provides a near-infrared cyanine analog color fluorescent probe, which has a structural formula shown in a formula (I):
wherein R is1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is any one of methyl, ethyl, propyl, butyl and propane sulfonic group;
n is 0 or 1.
The invention also provides a preparation method of the near-infrared cyanine analog color fluorescent probe, which comprises the following steps:
s1: synthesizing a first compound; the structural formula of the first compound is shown as a formula (II);
s2: synthesizing a near-infrared cyanine analog color fluorescent probe by using the first compound, wherein the structural formula of the near-infrared cyanine analog color fluorescent probe is shown as a formula (I):
wherein R in structural formulas of the first compound and the near-infrared cyanine colorimetric fluorescent probe1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is any one of methyl, ethyl, propyl, butyl and propane sulfonic group;
n is 0 or 1.
Further, in the step S1, dissolving a second compound in N, N-dimethylformamide, and then adding anhydrous sodium acetate to generate the first compound at 75-90 ℃ under an argon atmosphere; the structural formula of the second compound is shown as a formula (III);
wherein R is1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is any one of methyl, ethyl, propyl, butyl and propane sulfonic group;
n is 0 or 1.
Further, in the step S2, the first compound is dissolved in anhydrous dichloromethane, triethylamine and thiophenecarbonyl chloride are sequentially added to react, and after the reaction is finished, separation and purification are performed to obtain the near-infrared cyanine analog color fluorescent probe.
The invention also provides a method for identifying N by using the near-infrared cyanine analog color fluorescent probe2H4The method of (1), wherein the near-infrared cyanine analog color fluorescent probe is used for identifying N by using detection spectrum in a mixed solution of absolute ethyl alcohol and PBS buffer solution2H4(ii) a The structural formula of the near-infrared cyanine analog color fluorescent probe is shown as the formula (I):
wherein R is1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is any one of methyl, ethyl, propyl, butyl and propane sulfonic group;
n is 0 or 1.
Further, the mixed solution is a mixed solution of absolute ethyl alcohol and 10-50 mmol/L PBS buffer solution, the volume ratio of the absolute ethyl alcohol to the 10-50 mmol/L PBS buffer solution is 99: 1-1: 99, and the pH value of the PBS buffer solution is 4-10. Within the parameter range of the test condition, the aim of the invention can be better realized: namely, the probe has obvious detection and identification effects on hydrazine. Under the test condition outside the parameter range, the probe has no obvious detection and identification effect on hydrazine.
Further, the detection spectrum is a fluorescence spectrum.
Further, the detection spectrum is an ultraviolet-visible absorption spectrum.
The invention also provides the near-infrared cyanine analog color fluorescent probe for rapidly detecting N in situ in the environment2H4The use of (1).
The invention also provides a near-infrared cyanine analog color fluorescent probe for resisting tuberculosis drug metabolite N in vitro and in vivo2H4The application in the detection of the mitochondrial targeting tracing imaging.
In summary, compared with the prior art, the invention achieves the following technical effects:
(1) fluorescent probe pair N of the present invention2H4The identification has excellent selectivity, and the fluorescent signal or the absorption signal is basically unchanged when the fluorescent probe reacts with other common amine substances and cations, so that the anti-interference capability is strong.
(2) The fluorescent probe has good sensing property and high sensitivity.
(3) The fluorescent probes of the present invention have an ultra-low detection limit of 0.5 ppb.
(4) The probe has the advantage of near-infrared fluorescence, and can realize portable field analysis and visual detection of hydrazine.
(5) The near-infrared fluorescent probe can realize the imaging detection of exogenous hydrazine, and more importantly, can realize the tracing imaging detection of isoniazid metabolite (hydrazine) of an antituberculous drug.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the fluorescent probe-based identification detection of N according to the present invention2H4Schematic diagram of (1).
FIG. 2 is a high resolution mass spectrum representation of a near infrared cyanine analog color fluorescent probe VI.
FIG. 3 is a high-resolution mass spectrum characterization diagram of a near-infrared cyanine colorimetric fluorescent probe IX.
FIG. 4 is a high-resolution mass spectrum characterization diagram of a near-infrared cyanine colorimetric fluorescent probe XII.
FIG. 5 is a high resolution mass spectrum characterization chart of near infrared cyanine analog color fluorescent probe XV.
FIG. 6 is a high resolution mass spectrum characterization chart of the near infrared cyanine colorimetric fluorescent probe XVII.
FIG. 7 is a graph of the UV-VIS absorption spectra of various analytes.
FIG. 8 is a fluorescence emission spectrum of different analytes.
FIG. 9 is a graph of the UV-VIS absorption spectra of hydrazine at various concentrations.
FIG. 10 is a fluorescence emission spectrum of hydrazine at various concentrations.
FIG. 11 is a linear plot of the titration for various concentrations of hydrazine.
Fig. 12 is a competitive relationship diagram.
Fig. 13 is a linear correlation diagram of the calculated detection limit.
Figure 14 is a RGB line graph of different hydrazine concentrations.
FIG. 15 shows co-staining of fluorescent probe VI with the mitochondrial dye Mito-tracker FM488 in Hela cells.
FIG. 16 is a confocal fluorescence of the metabolic breakdown of drugs in Hela cells with fluorescent probe VI.
FIG. 17 is a confocal fluorescence of fluorescent probe VI in Hela cells after the addition of hydrazine at various concentrations.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.
The invention provides a near-infrared cyanine analog color fluorescent probe, which has a structural formula shown in a formula (I):
wherein R is1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is methyl, ethyl, propyl, butyl, propane sulfonic acid groupAny one of the above;
n is 0 or 1.
The main structure of the near-infrared cyanine analog color fluorescent probe is shown as the formula (I), because R1、R2Respectively represent different benzene ring substituents and side chain groups, and only the modification of the main structure is carried out without influencing the structural and functional characteristics, so that the person skilled in the art can deduce the R1、R2Can realize the identification and detection of N2H4The same applies to n being 0 or 1.
The reaction formula of the near-infrared cyanine analog color fluorescent probe is as follows:
the method comprises the following specific steps:
(1) dissolving the formula III in N, N-dimethylformamide, adding anhydrous sodium acetate, and reacting according to the reaction formula to generate a formula II, wherein the yield is 58-85%;
(2) dissolving the formula II in anhydrous dichloromethane, finally sequentially adding triethylamine and thiophenecarbonyl chloride for reaction, and after the reaction is finished, extracting, carrying out column chromatography separation and purification to obtain the near-infrared cyanine analogue color fluorescent probe formula I, wherein the yield is 52-80%.
The invention also provides application of the near-infrared cyanine analog color fluorescent probe, (1) the probe can identify N by using different detection spectrums in a mixed solution of ethanol and PBS buffer solution2H4(such as fluorescence spectrum and ultraviolet-visible absorption spectrum), the principle of which is shown in figure 1, the probe solution has weak background fluorescence, the color is cyanine green, after hydrazine is added, the probe solution reacts with a thiophene ester recognition group of the probe, the thiophene ester protection group is separated from a fluorescent parent, the PET process is inhibited, so that the fluorescence of the solution is obviously enhanced, and the cyanine green is changed into red, therefore, the probe can carry out colorimetric and fluorescent double-channel recognition detection on the hydrazine; (2) the probe can be used for tracing imaging detection of target exogenous and drug metabolite hydrazine in organisms.
The mixed solution is a mixed solution obtained by mixing absolute ethyl alcohol and 10-50 mmol/L PBS solution according to a volume ratio of 99: 1-1: 99, wherein the pH value of the PBS buffer solution is 4-10. Within the parameter range of the test condition, the aim of the invention can be better realized: namely, the probe has obvious detection and identification effects on hydrazine. Under the test condition outside the parameter range, the probe has no obvious detection and identification effect on hydrazine.
Said identification N2H4The detection spectra of (a) are fluorescence spectra and ultraviolet-visible absorption spectra.
After the fluorescent probe and the cervical cancer cells are incubated together, hydrazine and isoniazid with different concentrations are added respectively, and then the hydrazine in the cell mitochondria is visually monitored by a fluorescence confocal microscope.
Example 1 Synthesis of near-Infrared cyanine colorimetric fluorescent Probe VI
The fluorescent probe VI of this example corresponds to R of the compound of formula (I)1Is hydrogen, R2Is propyl and n is 1.
The reaction formula for synthesizing the near-infrared cyanine analog color fluorescent probe VI is shown as the following formula (A):
the method comprises the following specific steps of synthesizing a near-infrared cyanine colorimetric fluorescent probe VI:
(1) synthesis of intermediate V: IV and sodium acetate are dissolved in anhydrous N, N-dimethylformamide (15mL) according to the molar ratio of 1: 5-1: 2, and the mixture is heated for 2-6 h at the temperature of 75-90 ℃ under the argon atmosphere. After cooling the mixture to room temperature, it was filtered and the resulting solution was concentrated under reduced pressure to give an oily crude product. And finally, separating and purifying by silica gel column chromatography (dichloromethane is used as eluent) to obtain a pure intermediate V product with the yield of 50-70%.
(2) Synthesis of near-infrared cyanine analog color fluorescent probe VI: dissolving the intermediate V and the thiophenecarboxamide in the N, N-dimethylformamide according to the molar ratio of 1: 6-1: 2; and (3) placing the mixture into a constant-pressure dropping funnel, slowly dropping the mixture into the solution, stirring the mixture at room temperature for reaction for 30min, and finally separating and purifying the mixture by silica gel column chromatography (dichloromethane and methanol are used as eluent, and the volume ratio of the dichloromethane to the methanol is 1: 0-100: 1). And obtaining the near-infrared cyanine colorimetric fluorescent probe VI, and representing the structure of the near-infrared cyanine colorimetric fluorescent probe VI by using a mass spectrum and a nuclear magnetic resonance hydrogen spectrum/carbon spectrum.
High resolution mass spectrometry (electrospray, positive mode) characterization: c41H47N2O2The theoretical calculated value of the molecular mass M of S is 631.34; measured value [ M+H]+631.2 (see fig. 2).
Example 2 Synthesis of near-Infrared cyanine analog-color fluorescent Probe IX
The fluorescent probe IX of this example corresponds to R of the compound of formula (I)1Is a benzene ring, R2Is propyl and n is 0.
The reaction formula for synthesizing the near-infrared cyanine analog color fluorescent probe IX is shown as the following formula (B):
the method comprises the following specific steps of synthesizing a near-infrared cyanine colorimetric fluorescent probe IX:
(1) synthesis of intermediate VIII: VII and sodium acetate are dissolved in anhydrous N, N-dimethylformamide (15mL) according to the molar ratio of 1: 1-1: 4, and the mixture is heated for 1-6 hours at 70-90 ℃ under an argon atmosphere. After cooling the mixture to room temperature, it was filtered and the resulting solution was concentrated under reduced pressure to give an oily crude product. And finally, separating and purifying by silica gel column chromatography (dichloromethane is used as eluent) to obtain an intermediate VIII pure product with the yield of 40-65%.
(2) Synthesis of near-infrared cyanine analog color fluorescent probe IX: dissolving the intermediate VIII and thiophenecarboxamide in N, N-dimethylformamide according to a molar ratio of 1: 10-1: 1; and (3) placing the mixture into a constant-pressure dropping funnel, slowly dropping the mixture into the solution, stirring the mixture at room temperature for reaction for 30min, and finally separating and purifying the mixture by silica gel column chromatography (dichloromethane and methanol are used as eluent, and the volume ratio of the dichloromethane to the methanol is 1: 0-50: 1). And obtaining the near-infrared cyanine colorimetric fluorescent probe IX, and representing the structure of the near-infrared cyanine colorimetric fluorescent probe IX by using a mass spectrum and a nuclear magnetic resonance hydrogen spectrum/carbon spectrum.
High resolution mass spectrometry (electrospray, positive mode)) And (3) characterization: c48H49N2O2S+Theoretically calculated molecular mass M of 717.35; measured value [ M+H]+717.345 (see fig. 3).
Example 3 Synthesis of near-Infrared cyanine colorimetric fluorescent Probe XII
The fluorescent probe XII of this example corresponds to R of the compound of formula (I)1Is chlorine, R2Is methyl and n is 1.
The reaction formula for synthesizing the near-infrared cyanine analog color fluorescent probe XII is shown as the following formula (C):
the method comprises the following specific steps of synthesizing a near-infrared cyanine colorimetric fluorescent probe XII:
(1) synthesis of intermediate XI: dissolving the X and the sodium acetate in anhydrous N, N-dimethylformamide (15mL) according to a molar ratio of 1: 3-1: 1.5, and heating for 2-5 h at 75-100 ℃ under an argon atmosphere. After cooling the mixture to room temperature, it was filtered and the resulting solution was concentrated under reduced pressure to give an oily crude product. And finally, separating and purifying by silica gel column chromatography (dichloromethane is used as eluent) to obtain a pure intermediate XI product with the yield of 65-80%.
(2) Synthesis of near-infrared cyanine analog color fluorescent probe XII: dissolving an intermediate XI and thiophenecarbonyl chloride in N, N-dimethylformamide according to a molar ratio of 1: 8-1: 2; and (3) placing the mixture into a constant-pressure dropping funnel, slowly dropping the mixture into the solution, stirring the mixture at room temperature for reaction for 30min, and finally separating and purifying the mixture by silica gel column chromatography (dichloromethane and methanol are used as eluent, and the volume ratio of the dichloromethane to the methanol is 1: 0-80: 1). And obtaining the near-infrared cyanine colorimetric fluorescent probe XII, and representing the structure of the near-infrared cyanine colorimetric fluorescent probe XII by using a mass spectrum and a nuclear magnetic resonance hydrogen spectrum/carbon spectrum.
High resolution mass spectrometry (electrospray, positive mode) characterization: c37H37Cl2N2O2S+Theoretically calculated molecular mass M of 643.19; measured value [ M+H]+644.20 (see fig. 4).
Example 4 Synthesis of near-Infrared cyanine colorimetric fluorescent Probe XV
The fluorescent probe XV of this example corresponds to R of the compound of formula (I)1Is hydrogen, R2Is a propanesulfonic acid group, and n is 1.
The reaction formula for synthesizing the near-infrared cyanine analog color fluorescent probe XV is shown as the following formula (D):
the method comprises the following specific steps of synthesizing a near-infrared cyanine colorimetric fluorescent probe XV:
(1) synthesis of intermediate XIV: XIII and sodium acetate are dissolved in anhydrous N, N-dimethylformamide (15mL) according to the molar ratio of 1: 4-1: 1, and the mixture is heated for 1-5 hours at 70-100 ℃ under an argon atmosphere. After cooling the mixture to room temperature, it was filtered and the resulting solution was concentrated under reduced pressure to give an oily crude product. And finally, separating and purifying by silica gel column chromatography (dichloromethane is used as eluent) to obtain a pure intermediate XIV product with the yield of 45-75%.
(2) Synthesis of near-infrared cyanine analog color fluorescent probe XV: dissolving the intermediate XIV and the thiophenecarboxamide in the N, N-dimethylformamide according to the molar ratio of 1: 6-1: 2; and (3) placing the mixture into a constant-pressure dropping funnel, slowly dropping the mixture into the solution, stirring the mixture at room temperature for reaction for 30min, and finally separating and purifying the mixture by silica gel column chromatography (dichloromethane and methanol are used as eluent, and the volume ratio of the dichloromethane to the methanol is 1: 0-60: 1). And obtaining the near-infrared cyanine colorimetric fluorescent probe XIV, and representing the structure of the probe by using a mass spectrum and a nuclear magnetic resonance hydrogen spectrum/carbon spectrum.
High resolution mass spectrometry (electrospray, positive mode) characterization: c41H45N2O8S3 +Theoretically calculated molecular mass M of 789.23; measured value [ M+H]+789.22 (see fig. 5).
Example 5 Synthesis of near-Infrared cyanine-based colorimetric fluorescent Probe XVII
The fluorescent probe XVII of this example corresponds to R of the compound of formula (I)1Is hydrogen, R2Is ethyl and n is 0.
The reaction formula for synthesizing the near-infrared cyanine analog color fluorescent probe XVII is shown as the following formula (E):
the method comprises the following specific steps of synthesizing a near-infrared cyanine colorimetric fluorescent probe XVII:
(1) synthesis of intermediate XVI: dissolving XV and sodium acetate in anhydrous N, N-dimethylformamide (15mL) according to a molar ratio of 1: 3-1: 1, and heating for 1-4 h at 70-95 ℃ under an argon atmosphere. After cooling the mixture to room temperature, it was filtered and the resulting solution was concentrated under reduced pressure to give an oily crude product. And finally, separating and purifying by silica gel column chromatography (dichloromethane is used as eluent) to obtain a pure product of the intermediate XVI, wherein the yield is 50-75%.
(2) Synthesis of near-infrared cyanine analog color fluorescent probe XVII: dissolving the intermediate XVI and thiophenecarboxamide in N, N-dimethylformamide according to a molar ratio of 1: 6-1: 1; and (3) placing the mixture into a constant-pressure dropping funnel, slowly dropping the mixture into the solution, stirring the mixture at room temperature for reaction for 30min, and finally separating and purifying the mixture by silica gel column chromatography (dichloromethane and methanol are used as eluent, and the volume ratio of the dichloromethane to the methanol is 1: 0-100: 1). The near-infrared cyanine colorimetric fluorescent probe XVII is obtained, and the structure of the near-infrared cyanine colorimetric fluorescent probe XVII is represented by mass spectrum and nuclear magnetic resonance hydrogen spectrum/carbon spectrum.
High resolution mass spectrometry (electrospray, positive mode) characterization: c38H41N2O2S+The theoretical calculated value of the molecular mass M of (A) is 589.29; measured value [ M+H]+589.36 (see fig. 6).
Example 6 fluorescent Probe pairs of the invention N2H4Selective detection of
A mixed solution of 10. mu. mol/L of near-infrared cyanine analogue fluorescent probe (fluorescent probe VI prepared in example 1) in a volume ratio of anhydrous ethanol to 10mol/L, pH-4 in PBS buffer solution of 99:1 was added with 100. mu. mol/L of metal ion (Mg)2+，Al3+，Ca2+，Cr3+，Mn2+，Fe2+，Fe3+，Co2+，Ni2+，Zn2+，Ag+，Cd2+，Sn2+，Ba2+，Hg2+，Pb2+，VO2+，La3 +，Ce3+，Pr3+，Nd3+，Sm3+，Eu3+，Dy3+，Er3+，Th4+，Cu2+) Detecting the change of the fluorescence emission spectrum and the change of the ultraviolet visible absorption spectrum of the solution after 15min, wherein the detection results are shown in fig. 7 and 8, the near-infrared cyanine colorimetric fluorescent probe has almost no emission peak at 627nm and almost no absorption peak at 537 nm; when adding N2H4Then, the near-infrared cyanine analog color fluorescent probe solution has a strong emission peak at 627 nm; however other metal ions, e.g. Mg2+，Al3+，Ca2+，Cr3+，Mn2+，Fe2+，Fe3+，Co2+，Ni2+，Zn2+，Ag+，Cd2+，Sn2+，Ba2+，Hg2+，Pb2+，La3+，Ce3+，Pr3+，Nd3+，Sm3+，Eu3+，Dy3+，Er3+，Th4+After the fluorescent probe solution is added, the emission peak of the near-infrared cyanine analog color fluorescent probe solution at 627nm is not obviously enhanced, and the absorption peak at 537nm is not obviously changed; the experimental results show that only N is added2H4Can cause the obvious fluorescence enhancement of the near-infrared cyanine analog color fluorescent probe solution; the near-infrared cyanine analog color fluorescent probe is applied to N in a mixed solution of absolute ethyl alcohol and 10mol/L, pH-4 PBS buffer solution with the volume ratio of 99:12H4Has good selectivity.
Example 7 near-Infrared cyanine-based colorimetric fluorescent Probe pair N2H4Fluorescence titration experiment of
10 mu mol/L of a mixed solution of anhydrous ethanol of the near-infrared cyanine analogue color fluorescent probe and 30mol/L, pH-7 PBS buffer solution in a volume ratio of 1:1, and gradually increasing the added N2H4Concentration of (3), results of detecting changes in fluorescence spectrum and absorption spectrum of each sample after 15min are shown in FIGS. 9, 10 and 11, with the amount of N2H4Gradual increase in concentration, near-infraredThe fluorescence intensity of the cyanine analog color fluorescent probe solution at 627nm is gradually increased, the absorption intensity at 537nm is also gradually increased, and when N is2H4When the concentration of the compounds is respectively increased to about 100 mu mol/L, the titration reaches saturation, and the emission intensity and the absorption intensity are not increased any more; this also indicates that the near-infrared cyanine analog color fluorescent probe is directed to N2H4Has better sensing property.
Example 8 near-Infrared cyanine-based colorimetric fluorescent Probe pair N2H4Identified competition experiments
10 mu mol/L of near-infrared cyanine analogue color fluorescence probe anhydrous ethanol and PBS buffer solution are respectively added with 100 mu mol/L of other amine molecules (Cys, GSH, Hcy, Urea, NH)3H2O，PhNH2，TEA，DETA，NH2OH，BA，ButNH2，NO,H2O2，H2S，Cy-N2H4) Testing the fluorescence spectrum of each solution after 15 min; then 100 mu mol/L of N is added into each solution containing amine molecules2H4And standing for 15min, and then respectively testing the fluorescence spectrum and the ultraviolet visible absorption spectrum of each solution. As a result, as shown in FIG. 12, other amine-containing molecule pairs N coexisted2H4Fluorescence recognition of (2), for N2H4The identification of the uv-vis absorption is not significantly disturbed.
Example 9 near-Infrared cyanine-based colorimetric fluorescent Probe pair N2H4Calculation of detection limits
The detection limit is calculated according to the titration data of the fluorescence spectrum or the ultraviolet-visible absorption spectrum. By I/I0Is ordinate, N2H4Concentrations are plotted on the abscissa. I is the fluorescence intensity of each sample, I0To add no N2H4The fluorescence intensity of the probe solution. The results are shown in FIG. 13 at N2H4The concentration is in the range of 0-40 mu mol/L, and the linear relation is good (R)20.9938), the equation of the straight line is y 0.5436 x 106CN2H4+1.4383. The detection limit was calculated to be 0.5ppb using the formula DL-3 σ/S (σ is the blank experiment standard deviation, S is the slope of the line). Probe pair N2H4Has an ultra-low detection limit.
Example 10 in situ Rapid detection of N with near Infrared cyanine colorimetric fluorescent probes2H4
And loading the probe to common filter paper to prepare the portable paper-based film. In addition, preparation N2H4The method comprises the steps of using an aqueous solution with volume fractions of 0%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40% and 50%, detecting gaseous hydrazine (natural volatilization of the solution) by using a probe film, and scanning the color of a paper-based film after the paper-based film reacts with the gaseous hydrazine by using a smart phone to obtain RGB values with corresponding concentrations. A linear relationship graph is made from the R/RGB values, with the results shown in FIG. 14, N2H4In the concentration range of 0.1% to 40%, the linear relationship is good (R)20.99), and the equation y of the straight line is 0.013x + 0.302. Therefore, the probe can realize portable field detection.
Example 11 fluorescent tracing imaging of Living cells with near Infrared cyanine-based colorimetric fluorescent probes
First, the fluorescent probe has very good targeting (Pearson co-localization coefficient of 0.93) compared to the commercial mitochondrion targeting dye Mito-tracker green. Secondly, incubating the probe and hydrazine (10 mu M, 20 mu M and 50 mu M) with different concentrations in a cervical cancer cell (Hela cell) together, and then carrying out fluorescence confocal microscope imaging, wherein compared with a blank control group, the fluorescence signal is gradually enhanced along with the increase of the hydrazine concentration; subsequently, after the probe was incubated with cervical cancer cells, isoniazid and rifampicin, rifampicin were added, respectively. FIG. 15 shows co-staining of fluorescent probe VI with the mitochondrial dye Mito-tracker FM488 in Hela cells. Wherein, A is a bright field diagram; b is a confocal fluorescence diagram of the fluorescent probe VI in the cell; c is a confocal fluorescence image of a mitochondrial dye Mito-tracker FM488 in cells, and D is an overlay image of A, B and C. FIG. 16 is a confocal fluorescence image of the drug dissociation of fluorescent probe VI in Hela cells; wherein A is a co-staining pattern of isoniazid and fluorescent probe VI; b is a co-staining pattern of isoniazid and rifampicin with fluorescent probe VI; c is a co-staining pattern of rifampicin and fluorescent probe VI. FIG. 17 is a confocal fluorescence of fluorescent probe VI in Hela cells after the addition of hydrazine at various concentrations; wherein A is a confocal fluorescence diagram of only the fluorescent probe VI in the HeLa cells; b is a co-staining pattern of fluorescent probe VI and 10. mu.M hydrazine; c is a co-staining pattern of fluorescent probe VI and 20. mu.M hydrazine; d is a co-staining pattern of fluorescent probe VI and 50. mu.M hydrazine. The result shows that the fluorescence signal can be observed in the cervical cancer cell after the incubation of the isoniazid alone, the fluorescence signal of the cervical cancer cell after the incubation of the isoniazid and the rifampicin is enhanced, and the fluorescence signal can not be observed in the cervical cancer cell after the incubation of the rifampicin alone and a control group. Therefore, the probe can realize the tracing imaging detection of the isoniazid metabolite (hydrazine) of the antituberculous drug.
By combining the above embodiments, the invention provides a near-infrared cyanine analog color fluorescent probe, a preparation method and an application thereof, wherein the structural formula of the probe is shown as formula (I). The fluorescent probe can identify and detect N in a mixed solution of absolute ethyl alcohol and PBS buffer solution by using fluorescence and ultraviolet visible absorption spectrum2H4. Fluorescent probe pair N of the present invention2H4The fluorescent signal or the absorption signal is basically unchanged when the fluorescent signal or the absorption signal is acted with other common amine substances and cations, the anti-interference capability is strong, the sensing property and the sensitivity are good, and the ultra-low detection limit of 0.5ppb is realized. The probe has the advantage of near-infrared fluorescence, can realize portable field analysis and visual detection of hydrazine, can realize imaging detection of exogenous hydrazine, and more importantly can realize tracing imaging detection of isoniazid metabolite (hydrazine) of an antituberculous drug.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A near-infrared cyanine analog color fluorescent probe is characterized in that the structural formula of the fluorescent probe is shown as the formula (I):
wherein R is1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is any one of methyl, ethyl, propyl, butyl and propane sulfonic group;
n is 0 or 1.
2. A preparation method of a near-infrared cyanine analog color fluorescent probe is characterized by comprising the following steps:
s1: synthesizing a first compound; the structural formula of the first compound is shown as a formula (II);
s2: synthesizing a near-infrared cyanine analog color fluorescent probe by using the first compound, wherein the structural formula of the near-infrared cyanine analog color fluorescent probe is shown as a formula (I):
wherein R in structural formulas of the first compound and the near-infrared cyanine colorimetric fluorescent probe1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is any one of methyl, ethyl, propyl, butyl and propane sulfonic group;
n is 0 or 1.
3. The preparation method according to claim 2, wherein the second compound is dissolved in N, N-dimethylformamide in step S1, and then anhydrous sodium acetate is added to generate the first compound under an argon atmosphere at 75 to 90 ℃; the structural formula of the second compound is shown as a formula (III);
wherein R is1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is any one of methyl, ethyl, propyl, butyl and propane sulfonic group;
n is 0 or 1.
4. The preparation method according to claim 2, wherein in step S2, the first compound is dissolved in anhydrous dichloromethane, triethylamine and thiophenecarbonyl chloride are sequentially added to react, and after the reaction is completed, the near-infrared cyanine analog-color fluorescent probe is obtained by separation and purification.
5. N identification by near-infrared cyanine analog color fluorescent probe2H4The method is characterized in that the near-infrared cyanine analog color fluorescent probe is used for identifying N by using detection spectrum in a mixed solution of absolute ethyl alcohol and PBS buffer solution2H4(ii) a The structural formula of the near-infrared cyanine analog color fluorescent probe is shown as the formula (I):
wherein R is1Is any one of hydrogen, fluorine, chlorine, bromine, iodine, methyl and benzene ring;
R2is any one of methyl, ethyl, propyl, butyl and propane sulfonic group;
n is 0 or 1.
6. The method according to claim 5, wherein the mixed solution is a mixed solution of anhydrous ethanol and 10-50 mmol/L PBS buffer, the volume ratio of the anhydrous ethanol to the 10-50 mmol/L PBS buffer is 99: 1-1: 99, and the pH value of the PBS buffer is 4-10.
7. The method of claim 5, wherein the detection spectrum is a fluorescence spectrum.
8. The method of claim 5, wherein the detection spectrum is an ultraviolet-visible absorption spectrum.
9. The near-infrared cyanine analog color fluorescent probe of claim 1 for in-situ rapid detection of N in environment2H4The use of (1).
10. The near-infrared cyanine analog-color fluorescent probe of claim 1, which is the anti-tuberculosis drug metabolite N in vitro and in vivo2H4The application in the detection of the mitochondrial targeting tracing imaging.
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| CN115806544A (en) * | 2021-09-14 | 2023-03-17 | 中国科学院深圳先进技术研究院 | Fluorescent compounds and probes |
| CN116332923A (en) * | 2023-04-06 | 2023-06-27 | 大连理工大学 | Carbazole and phenazine compound meso-position substituted cyanine dye, and preparation method and application thereof |
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| WO2022242147A1 (en) * | 2021-05-17 | 2022-11-24 | 中国科学院深圳先进技术研究院 | Near-infrared cyanine colorimetric fluorescent probe, preparation method therefor and use thereof |
| CN115806544A (en) * | 2021-09-14 | 2023-03-17 | 中国科学院深圳先进技术研究院 | Fluorescent compounds and probes |
| WO2023040089A1 (en) * | 2021-09-14 | 2023-03-23 | 中国科学院深圳先进技术研究院 | Fluorescent compound and probe |
| CN116332923A (en) * | 2023-04-06 | 2023-06-27 | 大连理工大学 | Carbazole and phenazine compound meso-position substituted cyanine dye, and preparation method and application thereof |
| CN116332923B (en) * | 2023-04-06 | 2024-09-10 | 大连理工大学 | Carbazole and phenazine compound meso-position substituted cyanine dye, and preparation method and application thereof |
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