CN114315770B - Near infrared two-region fluorescence imaging molecular probe and preparation method and application thereof - Google Patents
Near infrared two-region fluorescence imaging molecular probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a near infrared two-region fluorescence imaging molecular probe and a preparation method and application thereof, wherein the near infrared two-region fluorescence imaging molecular probe is a molecular probe LET-7, and the chemical structural formula of the molecular probe LET-7 is as follows:the fluorescent signal of the molecular probe LET-7 provided by the invention is weak, when GSH exists, the molecular probe LET-7 can be specifically combined with the GSH, and the generated product LET-G has a strong fluorescent signal, and can realize the opening and amplification of the fluorescent signal, so that the specific detection of the GSH can be realized. The molecular probe LET-7 provided by the invention is a GSH probe capable of realizing near infrared two-region fluorescence imaging for the first time, and has the advantages of simple preparation method, definite detection mechanism, high sensitivity, strong specificity and wide application prospect.
Description
Technical Field
The invention relates to the technical field of fluorescent probe detection, in particular to a near infrared two-region fluorescent imaging molecular probe and a preparation method and application thereof.
Technical Field
Reduced Glutathione (GSH) is a cysteine-containing small molecule thiol that is most abundant in cells and is involved in many physiological and pathological processes. GSH has important roles in maintaining redox balance in cells, inhibiting the occurrence of many diseases such as Alzheimer's disease, immune dysfunction, parkinson's disease, diabetes, septicemia, liver and cardiovascular diseases, etc. due to its redox characteristics and nucleophilicity. Therefore, the development of an effective way to detect GSH in vivo is of great importance for understanding the pathophysiological processes associated with GSH.
Compared with the traditional methods for detecting glutathione, such as capillary electrophoresis, high Performance Liquid Chromatography (HPLC), voltammetry and the like, the fluorescent imaging (FLI) is an imaging technology which is mature and widely applied to the field of living organisms, does not need complex pretreatment process and longer detection time, and has the advantages of high sensitivity and good selectivity. However, most of the probes reported for GSH fluorescence imaging are near infrared one-region fluorescent probes (emission wavelength is in the range of 700-900 nm), whose emitted fluorescence is interfered with tissue autofluorescence, and the tissue penetration capacity is limited (1-2 cm), and thus limited in living applications. Near infrared two-region fluorescence (emission wavelength is in the range of 1000-1700 nm) has deeper tissue penetration capability, lower tissue scattering capability and lower autofluorescence capability, and higher signal-to-noise ratio (SBR) is provided by increasing signal quantity and reducing background noise, which is more beneficial to in-vivo fluorescence imaging. Therefore, there is an urgent need to develop a near infrared two-region fluorescence imaging probe with high selectivity and high sensitivity to detect GSH in vivo.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a near infrared two-region fluorescence imaging molecular probe and a preparation method and application thereof, and aims to solve the problems of poor tissue penetration, poor sensitivity and poor selectivity of the existing fluorescence imaging molecular probe.
The technical scheme of the invention is as follows:
a near infrared two-region fluorescence imaging molecular probe, wherein the near infrared two-region fluorescence imaging molecular probe is a molecular probe LET-7, and the chemical structural formula of the molecular probe LET-7 is as follows:
a preparation method of a near infrared two-region fluorescence imaging molecular probe comprises the following steps:
sequentially injecting anhydrous DMF, phosphorus oxychloride, a first solvent and cyclohexanone into a closed reaction solvent under the ice bath condition to obtain a first mixed solution; removing the ice bath, stirring for a first preset time after the first mixed solution is warmed to room temperature, heating to the first preset temperature for reacting for a second preset time, and then reacting for a third preset time at the second preset temperature to obtain a reaction solution; after the reaction liquid is cooled to room temperature, adding ice water, stirring overnight, filtering, washing and drying the generated precipitate to obtain 2-chloro-3-hydroxymethylene-cyclohexen-1-eneformaldehyde;
under inert atmosphere, 3-hydroxy-3-methyl-2-butanone, malononitrile and sodium acetate are mixed and dissolved in a second solvent, stirring is carried out at room temperature for a fourth preset time, the second solvent is added, the reaction is carried out at a third preset temperature for a fifth preset time, after the reaction liquid is cooled to room temperature, hydrochloric acid is dropwise added to adjust the pH value to 4-5, and the generated precipitate is filtered, washed and dried to obtain 2- (3-cyano-4, 5-trimethylfuran-2 (5H) -alkylene) malononitrile;
under inert atmosphere, mixing the 2-chloro-3-hydroxymethylene-cyclohexen-1-eneformaldehyde, 2- (3-cyano-4, 5-trimethylfuran-2 (5H) -alkylene) malononitrile and sodium acetate into a third solvent, reacting for a sixth preset time at a fourth preset temperature, and distilling under reduced pressure to remove the solvent to obtain a green solid intermediate product; dissolving the green solid intermediate product and tetrabutylammonium chloride in anhydrous dichloromethane according to one time equivalent, stirring at room temperature for a seventh preset time, washing with water, drying with anhydrous magnesium sulfate to obtain a crude product, separating with a chromatographic column, and finally obtaining the anion sevenMethine cyanine dye Cy 925 having a chemical structural formula:
under inert atmosphere, dissolving the anion heptamethine cyanine dye Cy 925 in a fourth solvent, dropwise adding 3, 5-bis (trifluoromethyl) thiophenol, stirring for eighth preset time at a fifth preset temperature, and reacting to obtain the near infrared two-region fluorescence imaging molecular probe, wherein the chemical structural formula is as follows:
the preparation method of the near infrared two-region fluorescence imaging molecular probe comprises the following steps of; the first preset time is 0-2h; the first preset temperature is 40-60 ℃; the second preset time is 1-5h; the second preset temperature is 70-90 ℃; the third predetermined time is 1-2 hours.
The preparation method of the near infrared two-region fluorescence imaging molecular probe comprises the steps that the second solvent is an absolute alcohol solvent; the fourth preset time is 0.5-4h; the third preset temperature is 70-90 ℃; the fifth predetermined time is 0.5-4 hours.
The preparation method of the near infrared two-region fluorescence imaging molecular probe comprises the following steps that the third solvent is acetic anhydride or an absolute alcohol solvent; the fourth preset temperature is 70-90 ℃; the sixth preset time is 8-12h; the seventh predetermined time is 5-15 hours.
The preparation method of the near infrared two-region fluorescence imaging molecular probe comprises the following steps of preparing a fourth solvent, wherein the fourth solvent is anhydrous N, N-dimethylformamide; the fifth preset temperature is 10-90 ℃; the eighth predetermined time is 5-15 hours.
The preparation method of the near infrared two-region fluorescence imaging molecular probe comprises the step of preparing the near infrared two-region fluorescence imaging molecular probe, wherein the inert atmosphere is one of nitrogen atmosphere, helium atmosphere, argon atmosphere and neon atmosphere.
Use of a near infrared two-region fluorescence imaging molecular probe according to claim 1 for detecting reduced GSH.
The application of the near infrared two-region fluorescence imaging molecular probe, wherein the near infrared two-region fluorescence imaging molecular probe is specifically combined with the reducing GSH to generate a product LET-G with a fluorescence signal, and the chemical structural formula of the product LET-G is as follows:
the application of the near infrared two-region fluorescence imaging molecular probe, wherein after the near infrared two-region fluorescence imaging molecular probe is specifically combined with the reducing GSH, the color is changed from yellow green to green.
The beneficial effects are that: the preparation method of the near infrared two-region fluorescence imaging molecular probe provided by the invention is simple and safe, high in yield, low in raw materials and easy to realize industrial production. Meanwhile, the molecular probe LET-7 prepared by the method can realize the accurate detection of GSH through near infrared two-region fluorescence imaging, has strong tissue penetrating power, high sensitivity and strong selectivity, and has good application prospect in GSH living body fluorescence imaging.
Drawings
FIG. 1 is a response mechanism diagram of molecular probes LET-7 and GSH.
FIG. 2 is a synthetic scheme of molecular probe LET-7LET-7 in example 1.
In FIG. 3, a is an ESI-MS mass spectrum of the molecular probe LET-7, b is a nuclear magnetic resonance hydrogen spectrum of the molecular probe LET-7, and c is a nuclear magnetic resonance carbon spectrum of the molecular probe LET-7.
FIG. 4 a is a graph showing the change of the ultraviolet-visible absorption spectrum of the molecular probe LET-7 before and after in vitro specific reaction with GSH; b is a fluorescence emission spectrum change chart before and after the specific reaction of the molecular probe LET-7 and GSH; c is a color change chart before and after the specific reaction of the molecular probe LET-7 and GSH; d is a GSH concentration dependent fluorescence emission spectrum; e is a linear relation graph of fluorescence intensity at 928nm and GSH concentration; f is a time dependent fluorescence emission spectrum; g is a graph of fluorescence intensity at 928nm over time; h is a time dependent near infrared two-zone fluorescence imaging.
FIG. 5 a is a graph showing the comparison of fluorescence intensity of the molecular probe LET-7 reacted with different amino acids in vitro; b is a near infrared two-region fluorescence imaging diagram of the molecular probe LET-7 reacting with different amino acids in vitro; c is an inhibition efficiency graph of GSH inhibitor response to molecular probe LET-7; d is a near infrared two-region fluorescence imaging diagram of the GSH inhibitor with different concentrations in the reaction liquid of the molecular probe LET-7 and the GSH.
FIG. 6 a is a schematic diagram of LET-G and ICG chemical structures; b is an ultraviolet-visible absorption spectrum change chart before and after LET-G and ICG illumination; c is a fluorescence spectrum change chart before and after LET-G and ICG illumination; d is a near infrared two-region fluorescence imaging diagram of LET-G and ICG changing along with illumination time; e is a near infrared two-region fluorescence quantification chart of LET-G and ICG according to the change of illumination time.
FIG. 7 a is a schematic diagram of a molecular probe LET-7 imaging scheme; b is a near infrared two-region fluorescence image of the back of the animal at different time after injecting the molecular probe LET-7 solution; c is a near infrared two-region fluorescence intensity quantitative graph of the normal part and the tumor part of the mice in the imaging group and the GSH inhibition group; d is a near infrared two-region fluorescence image of the back of the animal after the mice in the imaging group are injected with the LET-7 solution for 3 hours; e is a near infrared two-region fluorescence image of the back of the animal after 3h of injection of LET-7 solution into GSH-inhibited mice.
FIGS. 8A, b and c are signal to noise ratios of fluorescence signals from normal and tumor sites in mice in imaging and GSH inhibition groups; d is an H & E staining pattern of the major organs of mice after injection of LET-7 solution.
Detailed Description
The invention provides a near infrared two-region fluorescence imaging molecular probe and a preparation method and application thereof, and the invention is further described in detail below in order to make the purposes, technical schemes and effects of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides a near infrared two-region fluorescence imaging molecular probe, wherein the near infrared two-region fluorescence imaging molecular probe is a molecular probe LET-7, and the molecular probe LET-7 has a chemical structural formula as follows:
the fluorescence signal of the molecular probe LET-7 provided by the embodiment is weak, when GSH exists, the molecular probe LET-7 can be specifically combined with the GSH as shown in figure 1, and the generated product LET-G has a strong fluorescence signal, and can realize the opening and amplification of the fluorescence signal, so that the specific detection of the GSH can be realized. The molecular probe LET-7 provided by the embodiment is a GSH probe capable of realizing near infrared two-region fluorescence imaging for the first time, and has the advantages of simple preparation method, definite detection mechanism, high sensitivity, strong specificity and wide application prospect.
In this example, the fluorescence signal of the molecular probe LET-7 solution was significantly enhanced with increasing GSH concentration, and the emission wavelength was at 928nm (excitation wavelength 808 nm). As an example, when the concentration of the molecular probe LET-7 solution is 2. Mu.M, the fluorescence intensity thereof has a good linear relationship with the concentration of GSH in the solution in the range of 0.2 to 20. Mu.M.
In this example, the molecular probe LET-7 is specific for GSH and is not interfered by other amino acids such as L-cysteine (Cys), L-homocysteine (Hcy), L-methionine (Met), L-phenylalanine (Phe), glycine (Gly), L-lysine (Lys), L-glutamine (Gln), L-glutamic acid (Glu), L-histidine (His), L-serine (Ser), L-alanine (Ala), L-arginine (Arg), L-Dithiothreitol (DTT), etc.
In this example, the molecular probe LET-7 undergoes a distinct color change after specific reaction with GSH, and the solution changes from yellow-green to green.
In some embodiments, there is also provided a method of preparing a near infrared two-region fluorescence imaging molecular probe, comprising the steps of:
sequentially injecting anhydrous DMF, phosphorus oxychloride, a first solvent and cyclohexanone into a closed reaction solvent under the ice bath condition to obtain a first mixed solution; removing the ice bath, stirring for a first preset time after the first mixed solution is warmed to room temperature, heating to the first preset temperature for reacting for a second preset time, and then reacting for a third preset time at the second preset temperature to obtain a reaction solution; after the reaction liquid is cooled to room temperature, adding ice water, stirring overnight, filtering, washing and drying the generated precipitate to obtain 2-chloro-3-hydroxymethylene-cyclohexen-1-eneformaldehyde;
under inert atmosphere, 3-hydroxy-3-methyl-2-butanone, malononitrile and sodium acetate are mixed and dissolved in a second solvent, stirring is carried out at room temperature for a fourth preset time, the second solvent is added, the reaction is carried out at a third preset temperature for a fifth preset time, after the reaction liquid is cooled to room temperature, hydrochloric acid is dropwise added to adjust the pH value to 4-5, and the generated precipitate is filtered, washed and dried to obtain 2- (3-cyano-4, 5-trimethylfuran-2 (5H) -alkylene) malononitrile;
under inert atmosphere, mixing the 2-chloro-3-hydroxymethylene-cyclohexen-1-eneformaldehyde, 2- (3-cyano-4, 5-trimethylfuran-2 (5H) -alkylene) malononitrile and sodium acetate into a third solvent, reacting for a sixth preset time at a fourth preset temperature, and distilling under reduced pressure to remove the solvent to obtain a green solid intermediate product; dissolving the green solid intermediate product and tetrabutylammonium chloride in anhydrous dichloromethane according to one time equivalent, stirring at room temperature for a seventh preset time, washing with water, drying with anhydrous magnesium sulfate to obtain a crude product, and separating by a chromatographic column to finally obtain the anion heptamethine cyanine dye Cy 925, wherein the chemical structural formula is as follows:
under inert atmosphere, dissolving the anion heptamethine cyanine dye Cy 925 in a fourth solvent, dropwise adding 3, 5-bis (trifluoromethyl) thiophenol, stirring for eighth preset time at a fifth preset temperature, and reacting to obtain the near infrared two-region fluorescence imaging molecular probe, wherein the chemical structural formula is as follows:
in the embodiment, firstly, an anionic heptamethine cyanine dye Cy 925 is prepared, and the anionic heptamethine cyanine dye Cy 925 and 3, 5-bis (trifluoromethyl) thiophenol undergo nucleophilic reaction to prepare the molecular probe LET-7. The anion heptamethine cyanine dye Cy 925 has a maximum absorption peak at 900nm, a maximum emission wavelength at 925nm and can be trailing to more than 1000nm, and has good light stability, thus being an ideal near infrared two-region fluorescent parent structure. The fluorescent signal of the molecular probe LET-7 is weak, when GSH exists, the molecular probe LET-7 can be specifically combined with the GSH as shown in figure 1, and the generated product LET-G has a strong fluorescent signal. This allows the molecular probe LET-7 to be used as GSH probe for near infrared two-region fluorescence imaging, while near infrared two-region fluorescence (emission wavelength is in the range of 1000-1700 nm) has deeper tissue penetration capability, lower tissue scattering capability and lower autofluorescence capability, and provides higher signal to noise ratio (SBR) by increasing signal quantity and reducing background noise, which is more beneficial to in vivo fluorescence imaging.
In some embodiments, the first solvent is anhydrous tetrahydrofuran; the first preset time is 0-2h; the first preset temperature is 40-60 ℃; the second preset time is 1-5h; the second preset temperature is 70-90 ℃; the third predetermined time is 1-2 hours.
In some embodiments, the second solvent is an anhydrous alcoholic solvent; the fourth preset time is 0.5-4h; the third preset temperature is 70-90 ℃; the fifth predetermined time is 0.5-4 hours.
In some embodiments, the third solvent is acetic anhydride or an anhydrous alcohol solvent; the fourth preset temperature is 70-90 ℃; the sixth preset time is 8-12h; the seventh predetermined time is 5-15 hours.
In some embodiments, the fourth solvent is anhydrous N, N-dimethylformamide; the fifth preset temperature is 10-90 ℃; the eighth predetermined time is 5-15 hours.
In some embodiments, the inert atmosphere is one of a nitrogen atmosphere, a helium atmosphere, an argon atmosphere, and a neon atmosphere, but is not limited thereto.
In some embodiments, there is also provided an application of the near infrared two-region fluorescence imaging molecular probe, which is characterized in that the near infrared two-region fluorescence imaging molecular probe is used for detecting reduced GSH. In this example, the near infrared two-region fluorescence imaging molecular probe (molecular probe LET-7) can specifically bind to GSH as shown in FIG. 1, and the resulting product LET-G has a high degree of specificityThe strong fluorescent signal can realize the start and amplification of the fluorescent signal, thereby realizing the specific detection of GSH. Wherein, the near infrared two-region fluorescence imaging molecular probe is specifically combined with the reducing GSH to generate a product LET-G with fluorescent signals, and the chemical structural formula of the product LET-G is as follows:
in some embodiments, the molecular probe LET-7 is dispersed in a mixed solvent of PBS and DMSO to prepare a LET-7 solution, and then the LET-7 solution is reacted with the reduced GSH, and after the near infrared two-region fluorescence imaging molecular probe is specifically combined with the reduced GSH, the color is changed from yellow green to green.
In some embodiments, when the molecular probe LET-7 is used to detect reduced GSH, the animal model employed is a tumor-bearing nude mouse, and the tumor cells are 4T1 cells.
The invention is further illustrated by the following examples:
example 1
As shown in FIG. 2, anhydrous DMF (8 mL), phosphorus oxychloride (6.4 mL,68.8 mmol), anhydrous THF (4 mL) and cyclohexanone (4 g,40.8 mmol) were slowly injected into a closed reaction vessel at 0deg.C. After warming to room temperature, stirring for 30min, heating to 50 ℃ for reaction for 3.0h, reacting at 80 ℃ for 1.5h, cooling the reaction liquid to room temperature, adding ice water (500 mL), stirring overnight to generate precipitate, filtering, washing and drying to obtain a tan solid product 1 (6.6 g, yield 96%).
N 2 3-hydroxy-3-methyl-2-butanone (1.0 g,10 mmol), malononitrile (1.35 g,20.3 mmol) and sodium acetate (101 mg,1.58 mmol) were dissolved in absolute ethanol (2 mL) under protection, stirred at room temperature for 1h, absolute ethanol (6 mL) was added, reacted at 80℃for 1h, after the reaction solution cooled to room temperature, hydrochloric acid (6M) was added dropwise to adjust pH to 4-5 to form precipitate, filtered, washed 3 times with cold ethanol, and dried to give solid product 2 (0.98 g, yield 50%).
N 2 Under protection, compound 1 (0.433 g,2.5 mmol), compound 2 (1 g)5.0 mmol) and sodium acetate (0.412 g,5.0 mmol) were dissolved in absolute ethanol (20 mL) and reacted at 80℃for 10h, the solvent was distilled off under reduced pressure to give a green solid intermediate. The green solid intermediate was dissolved in one equivalent of tetrabutylammonium chloride in anhydrous dichloromethane, stirred at room temperature for 15min, washed with water (3 times), dried over anhydrous magnesium sulfate, and the crude product was isolated using a chromatography column (dichloromethane/methanol=10:1) to give dark green solid product 3 (1 g, yield 51%).
N 2 Compound 3 (200 mg,0.26 mmol) was dissolved in anhydrous DMF (5 mL) and 3, 5-bis (trifluoromethyl) thiophenol (76 mg,0.26 mmol) was slowly added dropwise, stirred at room temperature for 5h, the solvent was distilled off under reduced pressure, and the crude product was isolated using a chromatography column (dichloromethane/methanol=20:1) to give the dark green solid product DTCF (210 mg, 82% yield) as molecular probe LET-7.
As shown in FIG. 1, LET-7 specifically reacts with GSH to generate LET-G, thereby realizing near infrared two-region fluorescence response.
Example 2
As shown in FIG. 3a, the molecular probe LET-7 in example 1 was dissolved in methanol, and the mass-to-charge ratio was 743.17 as measured by ESI-MS mass spectrum.
As shown in FIG. 3b, 5mg of molecular probe LET-7 of example 1 was dissolved in 600. Mu.L of deuterated DMSO, and tested to obtain a nuclear magnetic resonance hydrogen spectrum.
As shown in FIG. 3c, 10mg of molecular probe LET-7 of example 1 was dissolved in 600. Mu.L of deuterated DMSO and tested to obtain a nuclear magnetic resonance carbon spectrum.
Example 3
As shown in fig. 4 a and b, the molecular probe LET-7 of example 1 was dispersed in a mixed solvent (PBS/dmso=7:3) to prepare a 2 μm solution, GSH (20 μm) was added, and incubated at 37 ℃ for 60min, and absorbance spectra and fluorescence spectra before and after incubation of the solution with GSH were measured, respectively. From this, it can be seen that LET-7 has a weak blue shift in its absorption spectrum (from 900nm to 892 nm) in the presence of GSH. And the fluorescence emission (928 nm) signal is obviously enhanced. The feasibility of LET-7 for fluorescence imaging to detect GSH was demonstrated.
As shown in FIG. 4 c, the molecular probe LET-7 of example 1 was dispersed in a mixed solvent (PBS/DMSO=7:3) to prepare a 10. Mu.M solution. Two sets of the above LET-7 solutions were taken, the first set was not treated at all, the second set was incubated with GSH (100. Mu.M) at 37℃for 60min, and the color changes of the two sets were photographed and compared. It can be seen that the color of the solution changed from yellow-green to green, further demonstrating that this reaction process can take place effectively.
As shown in fig. 4 d and e, the molecular probe LET-7 of example 1 was dispersed in a mixed solvent (PBS/dmso=7:3) to prepare a 2 μm solution. GSH (0.2,4,8,12,16, 20 mu M) with different concentrations was added, and after incubation at 37 ℃ for 60min, the fluorescence spectra of each solution were measured separately, and the linear relationship between the fluorescence intensity at 928nm and GSH concentration was counted, from which it can be seen that the two had a good linear relationship.
As shown in fig. 4 f and g, the molecular probe LET-7 of example 1 was dispersed in a mixed solvent (PBS/dmso=7:3) to prepare a 2 μm solution. After addition of GSH (20. Mu.M), the solution was incubated at 37℃for 70min, the fluorescence spectrum of the solution was measured after each incubation for 2min, and the relationship between the fluorescence intensity at 928nm and the incubation time was counted. From this, it can be seen that after 60min incubation, LET-7 has reacted fully with GSH, demonstrating that LET-7 has the property of detecting GSH rapidly.
As shown in fig. 4h, the molecular probe LET-7 of example 1 was dispersed in a mixed solvent (PBS/dmso=7:3) to prepare a 2 μm solution. Two sets of solutions were taken, one with GSH (20. Mu.M) and one with equal volumes of PBS and incubated at 37℃for 60min. And (3) under 808nm laser irradiation, performing near infrared two-region fluorescence imaging on the two groups of solutions after the reactions of 0, 10, 20, 30, 40, 50 and 60 minutes by using a near infrared two-region fluorescence imager. From the above, it can be seen that the detection process of LET-7 on GSH can realize near infrared two-region fluorescence visualization, and the feasibility of using the detection process for in-vivo GSH detection is proved.
Example 4
As shown in fig. 5 a and b, the molecular probe LET-7 of example 1 was dispersed in a mixed solvent (PBS/dmso=7:3) to prepare a 2 μm solution. Various amino acid solutions (PBS, GSH, cys, hcy, met, phe, gly, gln, lys, glu, his, ser, ala, arg and DTT) were added respectively, incubated at 37℃for 60min, the fluorescence intensity at 928nm of each solution was measured under 808nm laser irradiation, and near infrared two-region fluorescence imaging was performed on each reaction solution. From this, it can be seen that LET-7 is not susceptible to interference from other amino acids, demonstrating that LET-7 is specific for GSH detection.
As shown in fig. 5 c and d, the molecular probe LET-7 of example 1 was dispersed in a mixed solvent (PBS/dmso=7:3) to prepare a 2 μm solution. Twelve sets of LET-7 solutions were taken, the first set was not treated, after adding GSH (20. Mu.M) to the remaining eleven sets, respectively, and then adding N-methylmaleimide (NMM) (0, 6, 12, 18, 24, 30, 36, 42, 48, 54, and 60. Mu.M) at various concentrations, and incubated at 37℃for 60min. And (3) carrying out near infrared two-region fluorescence imaging images on each reaction liquid under 808nm laser irradiation. The fluorescence spectrum of each solution is measured, and the inhibition efficiency of NMM on the fluorescence intensity of each solution is counted. From this, it can be seen that the fluorescence emission intensity of the solution can be effectively inhibited after NMM is added, further proving the specific detection of LET-7 on GSH.
Example 5
As shown in FIG. 6 a, LET-G is a fluorescent signal molecule generated by LET-7 in response to GSH. ICG served as a reference for its light stability experiments.
As shown in fig. 6 b and c, LET-G and ICG were dispersed in a mixed solvent (PBS/dmso=7:3), respectively, to prepare a 10 μm solution. After illumination under the same laser conditions, absorption spectra before and after illumination and fluorescence spectra of the two groups of solutions are measured. From this, it can be seen that LET-G has more excellent optical stability than ICG, demonstrating its excellent photobleaching resistance.
As shown in fig. 6 d and e, LET-G and ICG were dispersed in a mixed solvent (PBS/dmso=7:3), respectively, to prepare a 10 μm solution. After illumination under the same laser conditions, near infrared two-region fluorescence imaging is carried out on the two groups of solutions. From this, it can be seen that the near infrared two-region fluorescence signal of ICG is drastically reduced with the increase of irradiation time, while the near infrared two-region fluorescence signal of LET-G is not substantially changed, further demonstrating that LET-G has excellent photobleaching resistance.
Example 6
As shown in FIGS. 7, a, b, c, d and e, the molecular probe LET-7 of example 1 was dispersed in a mixed solvent (PBS/DMSO=98:2) to prepare a 10. Mu.M solution. Taking one group of nude mice with tumor as an experimental group, taking another group of nude mice with tumor after 30min of tail vein injection of NMM solution (10 mM,40 mu L) as GSH inhibition group, and injecting 50 mu L of LET-7 solution into the normal part of the left side of the nude mice subcutaneously and the right side of the nude mice intratumorally respectively; and shooting near infrared two-region fluorescence imaging images of the back of the nude mice in real time, and counting the fluorescence intensity. From the results, the LET-7 can detect GSH in living tumors, and the feasibility of real-time visualization of the LET-7 at the living level GSH is proved.
As shown in fig. 8 a, b and c, near infrared two-region fluorescence imaging was performed after injection of the LET-7 solution for 3 hours in the tumor-bearing nude mice of the above experimental group and GSH inhibition group, and the signal to noise ratio of the fluorescence signal at each administration site was calculated by fluorescence spectrum. From this, it can be seen that LET-7 has high sensitivity for detection of GSH in living body, proving superiority of LET-7 for near infrared two-region imaging detection of GSH.
As shown in FIG. 8 d, the mice injected with the LET-7 solution for 24 hours were dissected to obtain H & E staining patterns of major organs (heart, liver, spleen, lung and kidney), from which it can be seen that no lesions were formed in the major organs, demonstrating excellent biosafety of LET-7.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (10)
1. The near infrared two-region fluorescence imaging molecular probe is characterized in that the near infrared two-region fluorescence imaging molecular probe is a molecular probe LET-7, and the molecular probe LET-7 has a chemical structural formula as follows:
2. the preparation method of the near infrared two-region fluorescence imaging molecular probe is characterized by comprising the following steps:
sequentially injecting anhydrous DMF, phosphorus oxychloride, a first solvent and cyclohexanone into a closed reaction solvent under the ice bath condition to obtain a first mixed solution; removing the ice bath, stirring for a first preset time after the first mixed solution is warmed to room temperature, heating to the first preset temperature for reacting for a second preset time, and then reacting for a third preset time at the second preset temperature to obtain a reaction solution; after the reaction liquid is cooled to room temperature, adding ice water, stirring overnight, filtering, washing and drying the generated precipitate to obtain 2-chloro-3-hydroxymethylene-cyclohexen-1-eneformaldehyde;
under inert atmosphere, 3-hydroxy-3-methyl-2-butanone, malononitrile and sodium acetate are mixed and dissolved in a second solvent, stirring is carried out at room temperature for a fourth preset time, the second solvent is added, the reaction is carried out at a third preset temperature for a fifth preset time, after the reaction liquid is cooled to room temperature, hydrochloric acid is dropwise added to adjust the pH value to 4-5, and the generated precipitate is filtered, washed and dried to obtain 2- (3-cyano-4, 5-trimethylfuran-2 (5H) -alkylene) malononitrile;
under inert atmosphere, mixing the 2-chloro-3-hydroxymethylene-cyclohexen-1-eneformaldehyde, 2- (3-cyano-4, 5-trimethylfuran-2 (5H) -alkylene) malononitrile and sodium acetate into a third solvent, reacting for a sixth preset time at a fourth preset temperature, and distilling under reduced pressure to remove the solvent to obtain a green solid intermediate product; dissolving the green solid intermediate product and tetrabutylammonium chloride in anhydrous dichloromethane according to one time equivalent, stirring at room temperature for a seventh preset time, washing with water, drying with anhydrous magnesium sulfate to obtain a crude product, and separating by a chromatographic column to finally obtain the anion heptamethine cyanine dye Cy 925, wherein the chemical structural formula is as follows:
under inert atmosphere, dissolving the anion heptamethine cyanine dye Cy 925 in a fourth solvent, dropwise adding 3, 5-bis (trifluoromethyl) thiophenol, stirring for eighth preset time at a fifth preset temperature, and reacting to obtain the near infrared two-region fluorescence imaging molecular probe, wherein the chemical structural formula is as follows:
3. the method for preparing the near infrared two-region fluorescence imaging molecular probe according to claim 2, wherein the first solvent is anhydrous tetrahydrofuran; the first preset time is 0-2h; the first preset temperature is 40-60 ℃; the second preset time is 1-5h; the second preset temperature is 70-90 ℃; the third predetermined time is 1-2 hours.
4. The method for preparing a near infrared two-region fluorescence imaging molecular probe according to claim 2, wherein the second solvent is an absolute alcohol solvent; the fourth preset time is 0.5-4h; the third preset temperature is 70-90 ℃; the fifth predetermined time is 0.5-4 hours.
5. The method for preparing the near infrared two-region fluorescence imaging molecular probe according to claim 2, wherein the third solvent is acetic anhydride or an absolute alcohol solvent; the fourth preset temperature is 70-90 ℃; the sixth preset time is 8-12h; the seventh predetermined time is 5-15 hours.
6. The method for preparing the near infrared two-region fluorescence imaging molecular probe according to claim 2, wherein the fourth solvent is anhydrous N, N-dimethylformamide; the fifth preset temperature is 10-90 ℃; the eighth predetermined time is 5-15 hours.
7. The method for preparing a near infrared two-zone fluorescence imaging molecular probe according to claim 2, wherein the inert atmosphere is one of a nitrogen atmosphere, a helium atmosphere, an argon atmosphere and a neon atmosphere.
8. The use of a near infrared two-region fluorescence imaging molecular probe according to claim 1 for detecting reduced GSH.
9. According to the weightsThe use of a near infrared two-region fluorescence imaging molecular probe according to claim 8, wherein the near infrared two-region fluorescence imaging molecular probe specifically binds to the reducing GSH to generate a product LET-G having a fluorescent signal, the chemical structural formula of the product LET-G being:
10. the use of a near infrared two-region fluorescence imaging molecular probe according to claim 8, wherein the color changes from yellow-green to green after the near infrared two-region fluorescence imaging molecular probe specifically binds to the reducing GSH.
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