CN112521413B - Two-channel fluorescent probe for detecting viscosity and hydrogen peroxide as well as preparation and application thereof - Google Patents
Two-channel fluorescent probe for detecting viscosity and hydrogen peroxide as well as preparation and application thereof Download PDFInfo
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- HXITXNWTGFUOAU-UHFFFAOYSA-N phenylboronic acid Chemical compound OB(O)C1=CC=CC=C1 HXITXNWTGFUOAU-UHFFFAOYSA-N 0.000 description 3
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- DXCRBMYIDDYCDQ-UHFFFAOYSA-N (5-formylthiophen-2-yl)oxyboronic acid Chemical compound OB(O)OC1=CC=C(C=O)S1 DXCRBMYIDDYCDQ-UHFFFAOYSA-N 0.000 description 1
- IFQZRPLRRQMRNU-UHFFFAOYSA-N (6-methylidenecyclohexa-2,4-dien-1-yl)boronic acid Chemical compound OB(O)C1C=CC=CC1=C IFQZRPLRRQMRNU-UHFFFAOYSA-N 0.000 description 1
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- DLTDKNZISWUVBJ-UHFFFAOYSA-N 5-[4-(n-phenylanilino)phenyl]thiophene-2-carbaldehyde Chemical compound S1C(C=O)=CC=C1C1=CC=C(N(C=2C=CC=CC=2)C=2C=CC=CC=2)C=C1 DLTDKNZISWUVBJ-UHFFFAOYSA-N 0.000 description 1
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- FRGTUKOCHJUJIU-UHFFFAOYSA-N n,n-diphenylaniline;thiophene Chemical group C=1C=CSC=1.C1=CC=CC=C1N(C=1C=CC=CC=1)C1=CC=CC=C1 FRGTUKOCHJUJIU-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a fluorescent probe Mito-TPB for detecting viscosity and hydrogen peroxide through two channels, and preparation and application thereof, and belongs to the technical field of fluorescent probes. The fluorescent probe Mito-TPB for detecting viscosity and hydrogen peroxide through two channels provided by the invention has an aggregation-induced emission characteristic, and can be used as a detection reagent for simultaneously detecting viscosity and H in an inflammatory cell model or a tumor tissue model2O2The change and the detection means are simple and sensitive.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe for detecting viscosity and hydrogen peroxide through two channels, and preparation and application thereof.
Background
Cell viscosity, an important microenvironment parameter, plays a crucial role in many diffusion-mediated biological processes, such as signal transmission and electron transmission. Mitochondria are considered as energy factories of cells, and are closely related to cell viability and physiological activity. Abnormal changes in viscosity can cause mutations in mitochondrial network tissues, which further affect the diffusion of mitochondrial metabolites, and finally cause inflammation, diabetes, neurodegenerative diseases, even cancer, and the like.
Hydrogen peroxide (H)2O2) Is a physiologically active oxygen molecule mainly produced in mitochondria, and plays a regulatory role in cell growth, immune response, host defense, signal pathways, and the like. However, in mitochondria H2O2Can lead to mitochondrial swelling, fragmentation and fine structural changes, thereby inducing mitochondrial dysfunction and several diseases including cancer, inflammation, obesity, neurodegenerative diseases, and the like. To get a better understanding of mitochondrial viscosity and H2O2The inherent relationship between levels and mitochondrial-related diseases, and the development of a simple, reliable, accurate method for simultaneously detecting mitochondrial viscosity and H is highly desirable2O2A variant method.
The small molecular fluorescent probe is widely applied to fluorescent imaging because of the advantages of high sensitivity, real-time detection, rapid nondestructive analysis and the like. At present, many have been reported for the single detection of mitochondrial viscosity or H2O2But can be used for both viscosity and H2O2Fluorescent probes have been rarely reported. In addition, the probe is mostly based on a mechanism of quenching luminescence caused by aggregation, namely, luminescence at low concentration, and fluorescence quenching can occur when the probe is at high concentration or in an aggregation state, so that the effectiveness of the probe as a luminescent material is seriously influenced. On the contrary, the molecules with aggregation-induced (AIE) luminescence property have good photostability, large stokes shift, high signal-to-noise ratio and long-time labeling capability due to the properties of no luminescence during dissolution, high luminescence during aggregation, and are used as ideal bioluminescent probes for cell and living body imaging and tracking. In addition, the probes reported so far have mainly focused on mitochondrial viscosity and H in living cells or neurodegenerative diseases (such as Alzheimer's disease or Parkinson's disease models)2O2While simultaneously detecting. However, the limited disease models limit us to viscosity and H2O2The level and the intrinsic relationship of the related diseases. Therefore, a mitochondrial fluorescent probe with aggregation-induced emission characteristics is developed, and the viscosity and H of various diseases such as inflammation, malignant tumor models and the like are detected in a dual channel manner2O2The level change is very urgent for the deep understanding of mitochondrial-related diseases.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a mitochondrion-targeted fluorescent probe (Mito-TPB) with aggregation-induced fluorescence emission characteristics. Due to The Intramolecular Charge Transfer (TICT) quenching effect of free rotation and distortion of the AIE molecules, the probes do not fluoresce or fluoresce weakly, the movement of the AIE molecules is limited and the TICT effect is inhibited along with the increase of the viscosity of an environmental system, and the probe molecules show strong Near Infrared (NIR) fluorescence emission at 666nm, so that the viscosity can be detected. Meanwhile, the phenylboronic acid recognition unit in the probe molecule can react with H2O2And reacting to release a substance with strong orange fluorescence emission, wherein the position of an emission peak is 586 nm. Realize NIR and orange dual-channel simultaneous detection of viscosity and H in grains2O2And (4) horizontal.
In order to achieve the purpose, the invention adopts the following technical scheme:
a dual-channel fluorescent probe for detecting viscosity and hydrogen peroxide is characterized in that the structural formula is as follows:
a preparation method of a fluorescent probe for detecting viscosity and hydrogen peroxide through two channels is characterized by comprising the following steps:
step 1: reacting 4-bromo-N, N-diphenylaniline with Pd (dppf) Cl2Dissolving in anhydrous THF; adding K2CO3Heating the solution for reflux reaction; then slowly adding (5-formylthiophene-2-yl) boric acid dissolved in anhydrous tetrahydrofuran into the reaction solution, and heating for reaction; cooling to room temperature after the reaction is finished, concentrating the solvent in vacuum, and purifying by silica gel column chromatography to obtain a yellow solid, namely a compound 2(5- (4- (diphenylamino) phenyl) thiophene-2-formaldehyde);
step 2: dissolving t-BuOK, mono (4- (((triphenyl-lambda 4-phosphono) methyl) pyridine-1-onium) dichloride and a compound 2 in THF, stirring for reaction at room temperature, cooling to room temperature after the reaction is finished, concentrating the solvent in vacuum, and purifying by silica gel column chromatography to obtain an orange solid compound, namely pre-Mito ((E) -N, N-diphenyl-4- (5- (2- (pyridin-4-yl) vinyl) thiophen-2-yl) aniline);
and step 3: dissolving the compound pre-Mito and 4- (bromomethyl) phenylboronic acid pinacol ester in anhydrous toluene, and stirring overnight; after the reaction is finished, cooling to room temperature, concentrating the solvent in vacuum, and purifying by silica gel column chromatography to obtain a green solid;
and 4, step 4: dissolving the green solid obtained in the step 3 in acetone, and adding KPF6Reacting at room temperature overnight, cooling the system to room temperature after the reaction is finished, carrying out rotary evaporation under reduced pressure, and removing the solvent to obtain a crude product;
and 5: and (3) purifying the crude product prepared in the step (4) by silica gel column chromatography to obtain a purple red solid, namely a fluorescent viscosity probe Mito-TPB ((E) -1- (4-boron benzyl) -4- (2- (5- (4- (diphenylamino) phenyl) thiophene-2-yl) vinyl) pyridine-1-onium).
The preparation reaction formula of the fluorescent probe Mito-TPB is as follows:
the preparation process of the compound 2 provided by the invention refers to the prior art with similar structure, and the references are D.Wang, M.M.S.Lee, G.G.Shan, R.T.K.Kwok, J.W.Y.Lam, H.F.Su, Y.C.Cai and B.Z.Tang, adv.Mater.2018 and 30,1802105.
Further, the molar ratio of compound 2 to mono (4- (((triphenyl- λ 4-phosphono) methyl) pyridin-1-ium) dichloride and t-BuOK in step 2 was 1: 1: 2.2.
Further, the reaction time in step 2 is 9 h.
Further, the molar ratio of the compound pre-Mito and the 4- (bromomethyl) phenylboronic acid pinacol ester in the step 3 is 1: 1.
further, the reaction time in the step 3 is 12-20 hours, and the reaction temperature is 110 ℃.
Further, the silica gel column chromatography in the step 3 is eluted with dichloromethane/anhydrous methanol at a volume ratio of 30: 1.
Further, the compound green solid and KPF in the step 46In a molar ratio of 1: 9.
further, the reaction time in the step 4 is 12-20 hours.
Further, the silica gel column chromatography in the step 5 is eluted with dichloromethane/anhydrous methanol at a volume ratio of 20: 1.
The fluorescent probe provided by the invention has the characteristic of aggregation-induced emission, the Near Infrared (NIR) fluorescence intensity of the probe is gradually enhanced along with the increase of the environmental viscosity, and the orange fluorescence intensity is increased along with H2O2The concentration increases gradually.
Preparation of fluorescent probe for detecting viscosity and hydrogen peroxide through two channels and simultaneously detecting viscosity and H in inflammatory cell model2O2Varying the application in the reagent.
Fluorescent probe for detecting viscosity and hydrogen peroxide through two channels and simultaneously detecting swelling during preparationViscosity and H in tumor tissue model2O2Varying the application in the reagent.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention relates to a dual-channel fluorescence probe for detecting viscosity and hydrogen peroxide, which takes a triphenylamine thiophene structure as an aggregation-induced fluorescence nucleus and has an electron-donating group (D), and a typical mitochondrion targeting group pyridine positive charge structure is an electron-withdrawing group (A) to form a D-pi-A configuration; the fluorescence of the probe is gradually enhanced along with the increase of the content of the normal hexane in the ethanol/normal hexane mixed solvent, and the probe has typical aggregation-induced fluorescence emission characteristics;
(2) the recognition principle of the probe on the viscosity change is as follows: in a dissolved state or a low-viscosity solution, due to the fact that ethylene single bonds in molecules rotate freely, the whole molecules have a TICT effect, and the fluorescence of the probe is weak; when the probe is in a solution with high viscosity, the free rotation motion in the molecule is limited, the TICT effect is inhibited, so that the whole molecule is coplanar, and the probe can emit strong near-infrared fluorescence;
(3) detection of the change in viscosity by the probe: in the methanol/glycerol system, when the viscosity coefficient eta of the solution is increased from 0.59cP to 950cP, the near infrared fluorescence intensity at 666nm is remarkably enhanced; and log I666There is a good linear relationship (R) to log η20.9909), the slope is 0.3725, and the high-sensitivity quantitative or qualitative detection characteristic of the environmental viscosity is realized;
(4) the probe pair H2O2The recognition principle of the changes: h2O2Can react with the phenylboronic acid group of the probe to remove the pyridine-linked methylenephenylboronic acid, thereby releasing a new fluorophore (pre-Mito) with a strong orange fluorescent emission.
(5) The probe pair H2O2Detection of the change: the molecular probe is dissolved in a mixed system of dimethyl sulfoxide (DMSO) and Phosphate Buffer Solution (PBS) along with H2O2The concentration is increased, and the orange fluorescence at 586nm is obviously enhanced; to H2O2The linear range of detection of (2) is 0-30. mu.M (R)20.9891), detection limit of 0.141 μ M, far lowPhysiological H in inflammatory tissues2O2Level (10-100. mu.M). Therefore, the probe is very suitable for H of water bodies and biological systems2O2And (6) detecting.
(6) The probe pair viscosity and H2O2The difference of the detected maximum fluorescence emission wavelength is about 80nm, so that the spectrum interference of simultaneous detection of two channels can be effectively avoided, and the detection sensitivity is improved.
(7) The detection means is simple and only comprises a fluorescence spectrophotometer and a laser confocal microscope.
Description of the drawings:
FIG. 1 shows the nuclear magnetic characterization of the probe Mito-TPB of the invention,1H-NMR spectrum;
FIG. 2 shows the nuclear magnetic characterization of the probe Mito-TPB of the invention,13C-NMR spectrum;
FIG. 3 is a nuclear magnetic characterization, HR-MS spectrum, of probe Mito-TPB of the present invention;
FIG. 4 is a fluorescence emission spectrogram of the probe Mito-TPB of the invention in an ethanol/n-hexane mixed solvent along with the volume content of n-hexane;
FIG. 5 is a graph showing the variation of the fluorescence intensity of the probe Mito-TPB in the mixed system of ethanol and n-hexane with the volume content;
FIG. 6 is a fluorescence emission spectrogram of the probe Mito-TPB of the invention in a methanol/glycerol mixed system, which varies with the volume content of glycerol;
FIG. 7 is log I of Mito-TPB of the probe of the present invention666A plot of variation with log η in a methanol/glycerol mixed system;
FIG. 8 shows the mixing system of the probe Mito-TPB of the invention in DMSO/PBS with H2O2Fluorescence emission spectrum of concentration change;
FIG. 9 shows the fluorescence intensity (I) of the probe Mito-TPB of the present invention586) And H2O2A linear plot of concentration change;
FIG. 10 shows the invention probe Mito-TPB pair H2O2Selectivity of (a);
FIG. 11 shows the recognition of H by Mito-TPB of the probe of the present invention2O2A mass spectrometry pattern of (a);
FIG. 12 is a photograph of fluorescence co-localization of probe Mito-TPB of the present invention and MitoTracker Deep Red (MTDR), a commercially available mitochondrial-specific dye;
FIG. 13 shows that the probe Mito-TPB of the invention detects viscosity and H through two channels after Lipopolysaccharide (LPS) induces cell inflammation2O2A change in horizontal fluorescence imaging;
FIG. 14 shows the dual channel measurement of viscosity and H for the probe Mito-TPB of the present invention in tumor tissue models2O2A change in horizontal fluorescence imaging;
Detailed Description
Example 1
Preparation and characterization of a dual-channel fluorescent probe for detecting viscosity and hydrogen peroxide:
(1) 4-bromo-N, N-diphenylaniline (810mg, 2.5mmol) and Pd (dppf) Cl2(183mg, 0.25mmol) was dissolved in anhydrous THF (35mL) and K was added2CO3/H2O (2.07g, 15mmol/25 mL). The reaction mixture was stirred under reflux for 1 hour under argon. (5-Formylthiophen-2-yl) boronic acid (780mg, 5.0mmol) dissolved in anhydrous tetrahydrofuran (15mL) was then slowly added to the reaction solution and the reaction mixture was stirred at 80 ℃ for 12 h. Cooled to room temperature, the solvent was concentrated in vacuo and purified by silica gel column chromatography (petroleum ether/ethyl acetate, 50:1, v/v) to give compound 2(899mg, 79% yield) as a yellow solid.1H NMR(400MHz,CDCl3):δ9.85(s,1H),7.69(d,J＝4.0Hz,1H),7.51(d,J＝8.8Hz,2H),7.30–7.20(m,5H),7.17–7.11(m,4H),7.11–7.03(m,4H)。
(2) t-BuOK (138mg, 1.23mmol) and mono (4- (((triphenyl-. lamda.4-phosphono) methyl) pyridin-1-ium) dichloride (25mL) were added to anhydrous THF (25mL), the mixture was stirred at 0 deg.C for 30min, then Compound 2(200mg, 0.56mmol) was added, stirring was continued at room temperature for 9h, cooled to room temperature, the solvent was concentrated in vacuo, and purified by silica gel column chromatography (petroleum ether/ethyl acetate)50:1 to 25:1, v/v) gave the compound as an orange solid, pre-Mito (111mg, 46% yield).1H NMR(400MHz,DMSO)δ8.51(d,J＝6.0Hz,2H),7.72(d,J＝16.4Hz,1H),7.59(d,J＝8.8Hz,2H),7.52(d,J＝6.0Hz,2H),7.40(d,J＝3.6Hz,1H),7.34(t,J＝8.0Hz,4H),7.30(d,J＝4.0Hz,1H),7.13–7.05(m,6H),6.98(d,J＝8.8Hz,2H),6.90(d,J＝16.4Hz,1H).
(3) The compound pre-Mito (70mg, 0.16mmol) and 4- (bromomethyl) phenylboronic acid pinacol ester (47mg, 0.16mmol) were dissolved in anhydrous toluene (3.5mL) under argon, and the reaction was stirred at 110 ℃ for 15 hours. Cooling to room temperature, vacuum concentrating the solvent, purifying by silica gel column Chromatography (CH)2Cl2MeOH, 30/1, v/v) gave a green solid. Dissolving the solid in acetone, and reacting with KPF6/H2A solution of O (270mg, 1.47mmol/0.5mL) was mixed and the reaction stirred at room temperature for 15 hours. Cooling to room temperature, vacuum concentrating the solvent, purifying by silica gel column Chromatography (CH)2Cl2MeOH, 20/1, v/v) gave the compound as a purple-red solid as Mito-TPB (55mg, 54% yield).1H NMR(400MHz,DMSO-d6) (fig. 1) δ 8.95(d, J ═ 7.2Hz,2H),8.19(d, J ═ 16.0Hz,1H),8.18(d, J ═ 6.4Hz,2H),8.15(s,2H),7.83(d, J ═ 8.4Hz,2H), 7.65-7.60 (m,2H),7.51(s,2H),7.43(d, J ═ 8.0Hz,2H), 7.39-7.33 (m,4H),7.12(d, J ═ 16.0Hz,1H), 7.15-7.06 (m,6H),6.97(d, J ═ 8.8Hz,2H),5.69(s,2H).13C NMR(100MHz,CD3OD) (FIG. 2) delta 155.6,150.6,145.0,148.5,145.0,140.1,136.6,135.9,135.6,130.6,129.0,127.9,126.2,125.0,124.8,123.6,121.6,64.4.HR-MS m/z (FIG. 3): [ M + H ]]+calculated for C36H30BN2O2S+,565.2116；measured,565.2110.
Example 2
Aggregation-induced fluorescence emission characteristic of fluorescent probe Mito-TPB in ethanol/n-hexane mixed solvent
The fluorescent probe in example 1 was diluted with an ethanol/n-hexane mixed solvent to a final concentration of 5. mu. mol/L, the fixed excitation wavelength was 480nm, the fluorescence emission spectrum of the probe as a function of the volume content of n-hexane was recorded (FIG. 4), and a curve of the fluorescence intensity of the probe as a function of the volume content of n-hexane in an ethanol/n-hexane mixed system was plotted (FIG. 5). The probe has typical aggregation-induced emission characteristics as the 695nm fluorescence intensity gradually increases and blueshifts to 655nm as the n-hexane volume content increases from 0% to 95%, and reaches a maximum at 90% n-hexane volume content.
Example 3
Fluorescent response characteristic of fluorescent probe Mito-TPB to viscosity in methanol/glycerol mixed solvent
The probe of example 1 was diluted with a methanol/glycerol mixed solvent to a final concentration of 5. mu. mol/L, the fixed excitation wavelength was 488nm, the fluorescence emission spectrum of the probe as a function of the volume content of glycerol (or the viscosity coefficient. eta.) (FIG. 6) was recorded, and the value of the fluorescence intensity of the probe at 666nm (log I/L) was plotted666) Linear dependence as a function of viscosity coefficient (log η) in methanol/glycerol mixed systems (fig. 7). As the volume ratio of glycerol increased from 0% (0.59cP) to 99% (950cP), the near infrared fluorescence emission at 666nm increased in turn, indicating that the probe can achieve highly sensitive detection of ambient viscosity.
Example 4
The probe Mito-TPB from example 1 was diluted with DMSO/PBS mixed solvent (1/1, v/v, pH 8.4) to a final concentration of 5. mu. mol/L, fixed excitation wavelength 405nm, and the recording probe was recorded with H2O2Fluorescence emission spectrum of varying concentration (FIG. 8), with H2O2The concentration is increased from 0 mu mol/L to 100 mu mol/L, and the orange fluorescence emission at 586nm is sequentially enhanced; to H2O2The linear range of detection of (2) is 0-30. mu.M (R)20.9891) (fig. 9), detection limit was 0.141 μ M, indicating that the probe can achieve the pair H2O2High sensitivity detection.
Example 5
The concentration of the fluorescent probe Mito-TPB in example 1 is kept at 5 mu mol/L, the fluorescence spectrum of the probe in the presence of common ions and bioactive small molecules is respectively examined, and the probe pair H is examined2O2Selectivity of (2). As shown in FIG. 10, in DMSO/PBS (1/1, v/v) system at pH 8.4, the following substances (100. mu. mol/L) were added, respectively, except for H2O2Can obviously enhance the fluorescence intensity of the probeInstruction probe pair H2O2Has good selectivity. In fig. 10, the substances are: (1) blank, (2) H2O2,(3)Met,(4)Tyr,(5)GSH,(6)Gln,(7)Pro,(8)Cys,(9)Phe,(10)Thr,(11)His,(12)Trp,(13)Arg,(14)Ser,(15)Hg2+,(16)Cd2+,(17)Ba2+,(18)K+,(19)CO3 2-(20)Cu2+,(21)Ca2+,(22)Na+,(23)Fe3+,(24)Fe2+,(25)Cl-,(26)ClO4 -,(27)ClO-,(28)NO3-,(29)NO2-,(30)·OH,(31)NO.
Example 6
The fluorescent probes Mito-TPB, probes Mito-TPB and H of example 1 were used2O2The solution after 2H reaction and the compound pre-Mito are respectively subjected to mass spectrometry to verify probes Mito-TPB and H2O2The mechanism of action of (c). As shown in FIG. 11, the peak at m/z-565.2110 is the mass spectrum peak of probe Mito-TPB (theoretical value: 565.2116), and the peak at m/z-431.1587 can be assigned to probe Mito-TPB and H2O2The mass spectrum peak of the new fluorophore released after 2H of reaction and almost matched (theoretical value: 430.1504) with the mass spectrum peak of the pure compound pre-Mito (m/z: 431.1576), indicating that probes Mito-TPB and H are present2O2After the reaction, the pyridine-linked methyleneboronic acid is removed, releasing a new fluorophore, the compound pre-Mito.
Example 7
To see if the probe Mito-TPB could be targeted to localise in mitochondria, a co-localisation experiment of the probe with the commercially available mitochondrial-specific dye MTDR was performed. Adherent HeLa cells were incubated with MTDR (final concentration 0.3. mu. mol/L) at pH 7.4, 5% CO at 37 ℃2After incubation in the incubator for 30min, excess dye was removed by gently washing 3 times with phosphate buffer (pH 7.4). Then adding probe Mito-TPB (final concentration is 5 mu mol/L) to continue co-incubation for 30min, and observing co-localization of the probe Mito-TPB and the probe Mito-TPB under a laser confocal microscope. Considering that the near-infrared fluorescence emission of the probe Mito-TPB somewhat overlaps with the near-infrared red emission range of the commercially available MTDR, in order to obtain a probe with suitable fluorescence emissionA signal-to-noise ratio co-location image, wherein the fixed excitation wavelength of the probe Mito-TPB is 488nm, and the range of a pseudo-green fluorescence imaging channel is 560-670 nm; the fixed excitation wavelength of MTDR is 633nm, and the collection red channel range is 680-770 nm. As can be seen from FIG. 12, the fluorescent probe Mito-TPB is in a typical rod-like mitochondrial morphology and can be well overlapped with MTDR to obtain yellow overlapped fluorescence, and the software processed average co-localization coefficient (A) of the fluorescent probe Mito-TPB and MTDR is as high as 0.94. The fluorescent probe Mito-TPB and MTDR have obvious co-localization imaging and can be targeted and localized in mitochondria.
Example 8
Adherent HeLa cells were incubated with the fluorescent probe Mito-TPB of example 1 (final concentration 5. mu. mol/L) at pH 7.4, 5% CO at 37 ℃2After incubation for 30min in the incubator, two-channel fluorescence imaging of the probe under a laser confocal microscope, wherein: orange channel (Ex 405nm, Em 540-2O2Detecting; NIR channels (Ex 488nm, Em 650-. As shown in FIG. 13, the probe itself emits weak fluorescence in both channels, Lipopolysaccharide (LPS) is added to stimulate the cells for 2H, so that the cells produce inflammation, and the enhancement of both channel fluorescence emission in the cells can be observed, which indicates that LPS can induce intracellular viscosity and H2O2The level is obviously increased, and the probe Mito-TPB can realize the effect on the intracellular viscosity and H of an inflammation model2O2While simultaneously detecting.
Example 9
The fluorescent probe Mito-TPB (final concentration 10. mu. mol/L) in example 1 was incubated in normal tissues (e.g., heart, liver, spleen, lung, kidney and thymus) and tumor tissues, respectively, and two-channel fluorescence imaging of the probe was performed under a confocal laser microscope, in which: orange channel (Ex 405nm, Em 540-2O2Detecting; NIR channels (Ex 488nm, Em 650-. As shown in FIG. 14, the probe emits weak fluorescence in both channels in normal tissue, whereas bright fluorescence emission is observed in both channels in tumor tissue, indicating tumor tissueInternal viscosity and H2O2The level is obviously increased, and the probe Mito-TPB can realize the viscosity and H in tumor tissues2O2And simultaneously high-sensitivity detection.
Claims (10)
1. A dual-channel fluorescent probe for detecting viscosity and hydrogen peroxide is characterized in that the structural formula is as follows:
2. the method for preparing the fluorescent probe for detecting viscosity and hydrogen peroxide by two channels as claimed in claim 1, which comprises the following steps:
step 1: ((E) -N, N-diphenyl-4- (5- (2- (pyridin-4-yl) vinyl) thiophen-2-yl) aniline) (i.e., the compound pre-Mito) and 4- (bromomethyl) phenylboronic acid pinacol ester were dissolved in anhydrous toluene and stirred overnight; after the reaction is finished, cooling to room temperature, concentrating the solvent in vacuum, and purifying by silica gel column chromatography to obtain a green solid;
step 2: dissolving the green solid obtained in the step 1 in acetone, and adding KPF6Reacting at room temperature overnight, cooling the system to room temperature after the reaction is finished, carrying out rotary evaporation under reduced pressure, and removing the solvent to obtain a crude product;
and step 3: and (3) purifying the crude product prepared in the step (2) by silica gel column chromatography to obtain a purple red solid, namely a fluorescent viscosity probe Mito-TPB ((E) -1- (4-boron benzyl) -4- (2- (5- (4- (diphenylamino) phenyl) thiophene-2-yl) vinyl) pyridine-1-onium).
3. The method for preparing a dual-channel fluorescence probe for detecting viscosity and hydrogen peroxide according to claim 2, wherein the molar ratio of the compound pre-Mito and the 4- (bromomethyl) phenylboronic acid pinacol ester in the step 1 is 1: 1.
4. the method for preparing the fluorescent probe for detecting viscosity and hydrogen peroxide through two channels as claimed in claim 2, wherein the reaction time in the step 1 is 12-20 h, and the reaction temperature is 110 ℃.
5. The method for preparing a fluorescent probe for detecting viscosity and hydrogen peroxide through two channels as claimed in claim 2, wherein the silica gel column chromatography in step 1 is eluted with dichloromethane/anhydrous methanol at a volume ratio of 30: 1.
6. The method for preparing a fluorescent probe for detecting viscosity and hydrogen peroxide through two channels as claimed in claim 2, wherein the green solid and KPF in step 26In a molar ratio of 1: 9.
7. the method for preparing the fluorescent probe for detecting viscosity and hydrogen peroxide through two channels as claimed in claim 2, wherein the reaction time in the step 2 is 12-20 h.
8. The method for preparing a fluorescent probe for dual-channel detection of viscosity and hydrogen peroxide as claimed in claim 2, wherein the silica gel column chromatography in step 3 is eluted with dichloromethane/anhydrous methanol at a volume ratio of 20: 1.
9. The fluorescent probe for dual-channel detection of viscosity and hydrogen peroxide as claimed in claim 1, which is prepared for simultaneous detection of viscosity and H in inflammatory cell model2O2Varying the application in the reagent.
10. The dual-channel viscosity and hydrogen peroxide detection fluorescent probe as claimed in claim 1, which is prepared and used for simultaneously detecting viscosity and H in tumor tissue model2O2Varying the application in the reagent.
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