CN115181073B - Red fluorescent probe for detecting elastase and preparation method and application thereof - Google Patents
Red fluorescent probe for detecting elastase and preparation method and application thereof Download PDFInfo
- Publication number
- CN115181073B CN115181073B CN202210762057.XA CN202210762057A CN115181073B CN 115181073 B CN115181073 B CN 115181073B CN 202210762057 A CN202210762057 A CN 202210762057A CN 115181073 B CN115181073 B CN 115181073B
- Authority
- CN
- China
- Prior art keywords
- elastase
- fluorescent probe
- detecting
- red fluorescent
- preparing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
- C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a red fluorescent probe for detecting elastase, a preparation method and application thereof, and the molecular formula is C 19 H 9 F 5 N 2 O 3 The structural formula is shown as formula (I),
the fluorescent probe has high sensitivity, good selectivity and strong affinity to elastase, and can reduce background fluorescence interference in the process of detecting elastase.
Description
Technical Field
The invention relates to the field of chemical analysis and detection, in particular to an elastase red fluorescent probe and a preparation method and application thereof.
Background
Elastase is a hydrolase capable of cleaving peptide bonds of macromolecular proteins, and plays an important role in the life of the human body. When it is over-expressed in vivo, it can lead to the development of a range of physiological diseases such as acute lung injury, acute respiratory distress syndrome, chronic obstructive pulmonary disease, scleroderma, in addition to which abnormal expression of elastase in vivo can lead to degradation of tissue matrix and exacerbation of inflammation, and can accelerate tumor growth and promote tumor invasive metastasis, so it is of great importance to develop a fluorescent probe capable of specifically detecting excessive elastase in vivo.
The detection method of elastase mainly comprises an electrochemical method, a mass spectrometry, a chromatography, an enzyme-linked immunosorbent assay and a fluorescence analysis method, wherein most of the four methods have high detection cost, complicated sample pretreatment, long detection time and incapability of imaging elastase in living cells in real time.
Most of the fluorescent probes developed at present emit green light, so that the tissue autofluorescence interference is serious, the damage to cells and tissues is large, and the living cells cannot be monitored and imaged in real time, so that the development of the fluorescent probes emitting red light for detecting the elastase living body in the living body with high selectivity is particularly important.
Disclosure of Invention
In view of the above, the application provides a red fluorescent probe for detecting elastase, a preparation method and application thereof, and the fluorescent probe has high sensitivity, good selectivity and strong affinity for elastase, and can reduce background fluorescence interference in the process of detecting elastase.
In order to achieve the technical purpose, the application adopts the following technical scheme:
in a first aspect, the present application provides a red fluorescent probe for detecting elastase, having the formula C 19 H 9 F 5 N 2 O 3 The structural formula is shown as formula (I),
in a second aspect, the present application provides a method for detecting elastaseThe preparation method of the red fluorescent probe comprises the following steps: taking phenothiazine derivative and pentafluoropropionic anhydride as raw materials, reacting in a solvent at normal temperature in the presence of pyridine, and separating and purifying to obtain the red fluorescent probe for detecting elastase, wherein the molecular formula of the phenothiazine derivative is C 16 H 10 N 2 O 2 The structural formula is shown as formula (II),
preferably, the phenothiazine derivative is obtained by column chromatography of cresol purple and then vacuum drying.
Preferably, the molar ratio of phenothiazine derivative to pyridine is 1:3-4.
Preferably, the molar ratio of phenothiazine derivative to pentafluoropropionic anhydride is 1:1-4.
Preferably, the volume ratio of the eluent used for column chromatography is 100:1 with dichloromethane.
In a third aspect, the present application provides the use of a red fluorescent probe for detecting elastase, the fluorescent probe being for in vitro or in vivo detection of elastase.
Preferably, the fluorescent probe is used for qualitative and quantitative detection of the elastase in the water environment.
Preferably, the fluorescent probes are used for detection and imaging of endogenous elastase in living cells and living zebra fish, and the cells are used for non-therapeutic purposes.
Preferably, the fluorescent probe is used for qualitative and quantitative detection of elastase with the concentration of 0-10U/mL.
The beneficial effects of this application are as follows: according to the invention, phenothiazine is adopted as a parent fluorophore, and the fluorescent dye has red light property, so that the self-luminous interference of tissues is effectively avoided, and the damage to cells and light is reduced; the fluorescent probe in the scheme has strong selectivity and high sensitivity to elastase; the fluorescent probe prepared by the invention has low Mie constant and good affinity to elastase; the fluorescent probe prepared by the invention can be used for monitoring and imaging the endogenous elastase of cells and zebra fish; the target fluorescent probe can be obtained by a one-step method, and the preparation method is simple and quick.
Drawings
FIG. 1 shows a fluorescent probe obtained in example 1 of the present invention 1 H NMR spectrum;
FIG. 2 is a HRMS spectrum of the fluorescent probe obtained in example 1 of the present invention;
FIG. 3 shows the fluorophores obtained in example 1 of the present invention 1 H NMR spectrum;
FIG. 4 shows the fluorophores obtained in example 1 of the present invention 13 C NMR spectrum;
FIG. 5 is a HRMS spectrum of the fluorophore obtained in example 1 of the present invention;
FIG. 6 is a graph showing the relationship between the fluorescence intensity and response time of elastase detected by the fluorescent probe according to example 1 of the present invention;
FIG. 7 is a graph showing the titration of elastase at various concentrations with respect to the fluorescent probe obtained in example 1 of the present invention;
FIG. 8 is a diagram showing the detection limit of elastase in the low concentration range by using the fluorescent probe obtained in example 1 of the present invention;
FIG. 9 is a fitted graph of the Michaelis equation for the enzymatic kinetics of elastase detection in a low concentration range using the fluorescent probe obtained in example 1 of the present invention;
FIG. 10 is a graph showing the selectivity of the response of the fluorescent probe obtained in example 1 to elastase and other analytes according to the present invention;
FIG. 11 is a high performance liquid chromatography for detecting elastase by using the fluorescent probe obtained in example 1 of the present invention;
FIG. 12 is a fluorescence imaging diagram of the fluorescent probe obtained in example 1 of the present invention for detecting endogenous elastase in different living cells;
FIG. 13 is a fluorescence imaging diagram of the detection of endogenous elastase in plaque HepG2 cells by the fluorescent probe of example 1 of the present invention;
FIG. 14 is a fluorescence imaging diagram of the detection of endogenous elastase in zebra fish by using the fluorescent probe of example 1 of the present invention;
FIG. 15 shows the mechanism of action of fluorescent probe and elastase.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. 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 application provides a red fluorescent probe for detecting elastase, which has a molecular formula of C 19 H 9 F 5 N 2 O 3 The structural formula is shown as formula (I),
in the scheme, the fluorescent probe for red light emission takes a phenothiazine derivative as a fluorescent reporter group, and detection of elastase under physiological conditions is realized by using pentafluoropropionamide (a substrate of elastase), and the fluorescent probe has the advantages of low background fluorescence, high sensitivity and selectivity, strong affinity and the like; the fluorescent probe of the invention is non-fluorescent and emits strong fluorescence after reacting with elastase, and the fluorescence multiple is enhanced by about 208 times; the fluorescent probe can also monitor the activity of the elastase endogenous to living cells and zebra fish in real time, and can be used as a potential tool for detecting elastase related diseases.
In addition, the application provides a preparation method of the red fluorescent probe for detecting elastase, which comprises the following steps: dissolving 1 molar equivalent of phenothiazine derivative in anhydrous dichloromethane to dissolve the phenothiazine derivative completely, adding 3-4 molar equivalents of pyridine into the mixed solution, stirring the mixed solution for 30-60 minutes at normal temperature, adding anhydrous dichloromethane containing 1-4 molar equivalents of pentafluoropropionic anhydride into the mixed solution, stirring the mixed solution for 12-24 hours at normal temperature, and obtaining a target probe compound LX-2 through extraction, washing and column chromatography after the reaction is completed, wherein the synthesis route is as follows:
wherein the molecular formula of the phenothiazine derivative is C 16 H 10 N 2 O 2 The structural formula is shown as formula (II),
The fluorescent probe capable of identifying elastase can be obtained through a one-step method, and the method is simple and convenient, and raw materials are easy to obtain.
Meanwhile, the application of the red fluorescent probe for detecting elastase is provided, the fluorescent probe is used for in-vitro or in-vivo detection of elastase, and particularly can detect the catalytic activity of elastase in vitro and the activity of living cells in vivo and zebra fish endogenous elastase, wherein the in-vitro detection of the fluorescent probe for elastase comprises qualitative and quantitative detection of elastase in water environment, particularly the qualitative and quantitative detection of elastase with the detection concentration of 0-10U/mL, the in-vivo detection of the fluorescent probe for elastase comprises detection and imaging of living cells and zebra fish endogenous elastase, and the cells are used for non-therapeutic purposes; the product of the fluorescent probe (I) and elastase after response is a compound (II), the compound (II) has red fluorescence, it can be understood that the fluorescent probe (I) has weak fluorescence, the compound (II) belongs to strong fluorescence, the fluorescence intensity of the compound (II) at 615nm is gradually increased, the compound (II) belongs to a fluorescence enhancement type fluorescent probe, specifically, the action mechanism of the fluorescent probe (I) and elastase is shown in figure 15, the amino group at the 9 th position of the fluorescent group is an electron donating group, when the fluorescent probe is excited by light, electrons of the fluorescent probe are transferred to carbonyl oxygen, an intramolecular charge transfer mechanism is generated, the fluorescence is enhanced, the amino group at the 9 th position of the fluorescent probe is replaced by a pentafluoropropionyl electron withdrawing group, the intramolecular charge transfer mechanism is blocked, fluorescence quenching is performed, and after the elastase is added, an amide bond on the fluorescent probe is broken, the fluorescence intensity is gradually enhanced at 615nm, and the activity and the content of the elastic protease in vitro buffer solution, living cells and endogenous zebra cells can be qualitatively and quantitatively detected.
The present embodiment will be described below by way of specific examples.
Example 1
A fluorescent probe for detecting elastase, which is prepared by the following steps:
synthesis of phenothiazine derivative Crohn (1000 mg3.81 mmol) was weighed for column chromatography (ethyl acetate: dichloromethane=100:1, v/v) and dried under vacuum to give 400mg of a pink solid, i.e. phenothiazine derivative as fluorophore compound 1 in about 40% yield.
The fluorophore Compound 1 obtained in the present example 1 The H NMR is shown in figure 3, 1 H NMR(400MHz,DMSO-d 6 ) Bap δ8.54 (d, j=8.0 hz, 1H), 8.13 (d, j=8.0 hz, 1H), 7.83 (t, j=16 hz, 1H), 7.73 (t, j=12 hz, 1H), 7.56 (d, j=8 hz, 1H), 6.71 (s, 2H), 6.68 (s, 1H), 6.53 (s, 1H), 6.31 (s, 1H); the fluorophore Compound 1 obtained in the present example 13 The C NMR is shown in FIG. 4, 13 C NMR(101MHz,DMSO-d 6 ) Delta 182.43,154.52,152.22,146.79,138.63,132.07,132.05,131.69,131.58,130.44,125.49,125.00,123.78,113.22,105.07,98.08 HRMS of fluorophore Compound 1 obtained in this example is shown in FIG. 5, HRMS (ESI) calculated for C 16 H 11 N 2 O 2 + [M+H] + 263.08150；Found:263.08104.。
The fluorescent probe LX-2 is synthesized by weighing fluorophore compound 1 (200.00mg 0.76mmol) and pyridine (240.97mg 3.05mmol) and dissolving in 10mL of anhydrous dichloromethane, dropwise adding 10mL of anhydrous dichloromethane solution containing pentafluoropropionic anhydride (472.80mg 1.53mmol) under the condition of ice-water bath after complete dissolution, stirring at normal temperature for 12h, extracting with dichloromethane, and anhydrous Na 2 SO 4 Drying, column chromatography (ethyl acetate: dichloromethane=100:1, v/v), and vacuum drying gave 233.35mg of a reddish solid, namely fluorescent probe LX-2, in about 75% yield.
Fluorescent probe LX-2 obtained in this example 1 H NMR is shown in FIG. 1. 1 H NMR(400MHz,DMSO-d 6 ) δ11.76 (s, 1H), 8.64 (d, j=4 hz, 1H), 8.16 (d, j=8 hz, 1H), 7.98-7.80 (m, 4H), 7.76 (d, j=8 hz, 1H), 6.48 (s, 1H). As shown in FIG. 2, the HRMS (ESI) calculated for C of the fluorescent probe LX-2 obtained in this example 19 H 10 F 5 N 2 O 3 + [M+H] + 409.06039；Found:409.06061.。
Test example 1
The effect of the fluorescent probe obtained in example 1 on the response to elastase was measured: in a 1mL plastic EP tube, a mass of fluorescent probe LX-2 obtained in example 1 was weighed, a 10mM stock solution was prepared with DMSO (dimethyl sulfoxide), and another 1mL plastic EP tube was removed with a pipette, and a 1500U/mL elastase stock solution was prepared with 50% PBS (pH=7.4), 50% glycerol, and 0.5% BAS. The fluorescence spectrum of the system was measured over time by adding 1. Mu.L of the stock solution of the above-mentioned probe molecule and 999. Mu.L of PBS (pH=7.4) buffer to a cuvette, detecting the fluorescence spectrum at 615nm, and simultaneously adding 1. Mu.L of the stock solution of the above-mentioned probe molecule and 932.33. Mu.L of PBS (pH=7.4) buffer and 66.67. Mu.L of elastase stock solution, and the fluorescence spectrum of the system was measured over time, and as shown in FIG. 6, the fluorescence probe itself was not fluorescent, the fluorescence intensity was enhanced after the reaction with elastase, and the fluorescence intensity at 615nm was increased by about 390 times. The fact that the fluorescence intensity of the system reached a maximum and remained almost unchanged when the reaction time reached 120 minutes, indicated that the optimal incubation time of the fluorescent probe LX-2 for elastase was 120 minutes.
Test example 2
Determination of the change in fluorescence intensity with concentration of the fluorescent probe obtained in example 1 in response to elastase at different concentrations: to the cuvette, 0.5. Mu.L stock solution of fluorescent probe molecules was added, followed by different volumes (999, 992.33, 985.67, 972.33, 959, 932.33. Mu.L) of PBS buffer pH=7.4, and finally different volumes (0,6.67, 13.33, 26.67, 40, 66.67. Mu.L) of elastase were added to give working concentrations of 0, 10, 20, 40, 60, 80, 100U/mL, respectively. After the reaction was carried out at 37℃for 120 minutes, the fluorescence spectrum of the reaction system was measured. As shown in FIG. 7, the fluorescent probe LX-2 itself had a weak fluorescent background, and the fluorescence intensity was enhanced after the elastase was added, and as the concentration of elastase was increased, the fluorescence intensity was also enhanced continuously, and when the concentration of elastase reached 100U/mL, the fluorescence intensity reached a maximum value, and reached a plateau, indicating that the optimal concentration of elastase was 100U/mL.
Test example 3
Assay of the fluorescent probe obtained in example 1 with elastase limit of detection assay: to the cuvette were added the above-mentioned 0.5. Mu.L fluorescent probe molecule, different volumes (999, 992.33, 985.67, 972.33, 959, 932.33. Mu.L) of PBS buffer solution with pH=7.4 and different volumes (0,0.13,0.33,0.67,1.33,2,2.67,3.33,4,4.67,5.33,6,6.67. Mu.L) of elastase stock solutions, corresponding to working concentrations of 0, 0.2, 0.5, 1,2, 3,4, 5, 6, 7, 8, 9, 10U/mL of elastase, respectively, and the fluorescence intensity at 615nm was tested after 120 minutes of reaction. As shown in FIG. 8, it can be seen from the graph that the fluorescence intensity and the probe concentration show a good linear relationship in the range of low concentration of elastase, and the detection limit of the elastase detected by the probe is 0.015U/mL as calculated from the formula 3σ/k, indicating that the elastase can be quantitatively detected by the fluorescent probe.
Test example 4
Determination of the enzymatic kinetic parameters of the fluorescent probe obtained in example 1 and elastase: the above-mentioned different volumes (0,0.1,0.02,0.03,0.05,0.1,0.25,0.5,0.75,1,1,25. Mu.L) of the above-mentioned stock solution of fluorescent probe molecules and 6.67. Mu.L of elastase were added to the cuvette at the corresponding working concentrations of 0,0.1, 0.2, 0.3, 0.5, 1, 2.5, 5, 7.5, 10, 12.5. Mu.M, and further different volumes (993.33, 993.32, 993.31, 993.30, 993.28, 993.23, 993.08, 992.83, 992.58, 992.33, 992.08. Mu.L) of PBS buffer solution having pH=7.4 and, after 120 minutes of reaction, the change in fluorescence intensity at 615nm was measured with the reaction time, respectively. The slope of the fluorescence intensity versus reaction time curve is the reaction rate, which is plotted against the probe concentration to test the elastase for K on the probe m And V max As shown in FIG. 9, it can be seen from the figure that the fluorescence probe is followedThe increase in needle concentration gradually increased the reaction rate, with the maximum reaction rate of 1.49.+ -. 0.012min when a greater concentration of fluorescent probe (12.5. Mu.M) was added -1 Mies constant 0.65.+ -. 0.046. Mu.M, indicating good affinity and catalytic rate of the probe for elastase.
Test example 5
Determination of the specificity of the fluorescent probe obtained in example 1 for recognition of elastase: to the cuvette, the fluorescent probe obtained in example 1 (working concentration 5 μm), PBS (ph=7.4) and each of the usual interfering ions or substances were added, respectively, and the samples corresponding to a-v numbers were: blank, calcium ion (100. Mu.M), sodium ion (100. Mu.M), magnesium ion (100. Mu.M), zinc ion (100. Mu.M), sulfate (100. Mu.M), bicarbonate (100. Mu.M), L-alanine (20. Mu.M), L-serine (100. Mu.M), glycine (100. Mu.M), cly (100. Mu.M), arginine (100. Mu.M), leucine (100. Mu.M), cysteine (100. Mu.M), homocysteine (100. Mu.M), glutathione (100. Mu.M), L-tryptophan (100. Mu.M), alpha-chymotrypsin (100U/mL), trypsin (100U/mL), acetylcholinesterase (2U/mL), elastase (100U/mL) were tested for fluorescence intensity at 615nm at 120 minutes, as shown in FIG. 10, the addition of elastase No. a-U produced no significant fluorescence, whereas the addition of elastase No. 100U/mL to v showed a strong fluorescence response, demonstrating that the resulting fluorescent probe in example 1 was capable of specifically recognizing elastase, with high selectivity for elastase.
Test example 6
The mechanism of detection of elastase by the fluorescent probe obtained in example 1 was verified: the above 10. Mu.L of fluorescent probe molecule (working concentration: 100. Mu.M), 100. Mu.L of fluorescent group (working concentration: 100. Mu.M), 10. Mu.L of fluorescent probe molecule (working concentration: 100. Mu.M) and 66.67. Mu.L of elastase (working concentration: 100U/mL) were each added to a 1mL plastic EP tube, reacted at 37℃for 120 minutes, and then subjected to HPLC. As shown in FIG. 11, the retention time of the fluorescent group compound 1 was 7.5 minutes, the retention time of the fluorescent probe was 4.9 minutes, and a new peak was generated at the retention time of 7.5 minutes after the addition of elastase, which was consistent with the retention time of the fluorescent group, indicating that the fluorescent probe reacted with elastase to generate the fluorescent group.
Test example 7
Assay of the fluorescent probes obtained in example 1 for the detection of elastase activity in different living cells: SKOV3, heLa, hepG2 cells were selected as cell model, 1 μl of the probe obtained in example 1 (working concentration 10 μΜ) was incubated with the cell model for 30 min, and washed with PBS for fluorescence imaging. As shown in FIG. 12, the probe obtained in example 1, which was able to distinguish HepG2 cells from other cells, showed weak red fluorescence in SKOV3 and HeLa cells, and strong red fluorescence in HepG2 cells, showed a higher elastase content in HepG2 cells than in other two cells.
Test example 8
Assay of the fluorescent probes obtained in example 1 for the detection of the activity of endogenous elastase in HepG2 cells: using HepG2 cells as a cell model, 1. Mu.L of the probe obtained in example 1 (working concentration 10. Mu.M) was incubated with HepG2 cells for various times, and then washed with PBS, and fluorescence imaging was performed on the HepG2 cells. As shown in FIG. 13, after the probe obtained in example 1 was added, the red fluorescence was gradually increased with the lapse of the reaction time, and when the reaction time reached 120 minutes, the red fluorescence intensity reached a plateau, and under the same conditions, the lipopolysaccharide inducer was added, and the cells showed more intense red light. The above results indicate that the probe obtained in example 1 is capable of detecting the activity of endogenous elastase in HepG2 cells.
Test example 9
Assay of the fluorescent probes obtained in example 1 for the detection of the activity of endogenous elastase in zebra fish: zebra fish were divided into 3 groups, and 1. Mu.L of the probe obtained in example 1 (working concentration: 10. Mu.M) was incubated with zebra fish for 30 minutes, washed with PBS, and fluorescence imaging was performed on the zebra fish. The second group was pre-incubated with lipopolysaccharide inducer for 720 minutes, and the fluorescent probe obtained in example 1 (working concentration 10. Mu.M) was incubated with zebra fish for 30 minutes, and the zebra fish was imaged after washing off the remaining fluorescent probe and lipopolysaccharide inducer with PBS. The third group was pre-incubated with lipopolysaccharide inducer for 720 min, then with cilexetilapia for 30 min, and finally with the fluorescent probe obtained in example 1 (working concentration 10. Mu.M) for 30 min, and imaged after washing with PBS. As shown in fig. 14, there was substantially no fluorescence in the first group of zebra fish, while the second group was seen as a distinct red fluorescence after further addition of lipopolysaccharide inducer, while the third group was seen as a distinct decrease in red fluorescence after further addition of cevalance. The probe is capable of detecting endogenous elastase in animals.
In addition, compared with the detection of a fluorescent probe recognizing elastase in the related art, the differences between the present application are shown in Table 1:
TABLE 1 characterization of different fluorescent probes
In the prior art, the fluorescent probe for detecting elastase has the problems that (1) a fluorescent group emitted by red light is used instead, so that the background fluorescence interference is reduced. (2) The biocompatibility is improved, and the probe is used for detecting and imaging the endogenous elastase of living cells and living models. Compared with the reported elastase fluorescent probe, the fluorescent probe has the following advantages: (1) The preparation method of the fluorescent probe is simple and can be completed in one step. (2) The Michaelis constant of the fluorescent probe is as low as 0.65+/-0.046 mu M, which shows that the probe has strong affinity to elastase. (3) The fluorescent probe has red light, and can detect the activity of endogenous elastase in living cells and zebra fish.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (10)
1. A red fluorescent probe for detecting elastase is characterized in that the molecular formula is C 19 H 9 F 5 N 2 O 3 The structural formula is shown as formula (I),
2. a method for preparing the red fluorescent probe for detecting elastase according to claim 1, comprising the following steps: taking a phenothiazine derivative and pentafluoropropionic anhydride as raw materials, reacting in a solvent at normal temperature in the presence of pyridine, and separating and purifying to obtain the red fluorescent probe for detecting elastase, wherein the molecular formula of the phenothiazine derivative is C 16 H 10 N 2 O 2 The structural formula is shown as formula (II),
3. the method for preparing the red fluorescent probe for detecting elastase according to claim 2, wherein the phenothiazine derivative is obtained by subjecting cresol purple to column chromatography and then vacuum drying.
4. The method for preparing the red fluorescent probe for detecting elastase according to claim 2, wherein the molar ratio of the phenothiazine derivative to pyridine is 1:3-4.
5. The method for preparing the red fluorescent probe for detecting elastase according to claim 2, wherein the molar ratio of the phenothiazine derivative to the pentafluoropropionic anhydride is 1:1-4.
6. The method for preparing the red fluorescent probe for detecting elastase according to claim 3, wherein the volume ratio of the eluent used in the column chromatography is 100:1 with dichloromethane.
7. Use of the red fluorescent probe for detecting elastase according to claim 1, wherein the fluorescent probe is used for preparing an in vitro or in vivo detection reagent for elastase.
8. The use of the red fluorescent probe for detecting elastase according to claim 7, wherein the fluorescent probe is used for preparing qualitative and quantitative detection reagents for elastase in an aqueous environment.
9. The use of the red fluorescent probe for detecting elastase according to claim 7, wherein the fluorescent probe is used for preparing detection and imaging reagents for endogenous elastase of living cells and living zebra fish.
10. The use of the red fluorescent probe for detecting elastase according to claim 1, wherein the fluorescent probe is used for preparing a qualitative and quantitative detection reagent of elastase with a concentration of 0-10U/mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210762057.XA CN115181073B (en) | 2022-06-30 | 2022-06-30 | Red fluorescent probe for detecting elastase and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210762057.XA CN115181073B (en) | 2022-06-30 | 2022-06-30 | Red fluorescent probe for detecting elastase and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115181073A CN115181073A (en) | 2022-10-14 |
| CN115181073B true CN115181073B (en) | 2023-06-23 |
Family
ID=83515547
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210762057.XA Active CN115181073B (en) | 2022-06-30 | 2022-06-30 | Red fluorescent probe for detecting elastase and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115181073B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114380808A (en) * | 2021-12-21 | 2022-04-22 | 深圳大学 | Molecular probe for bimodal imaging detection of neutrophil elastase, preparation method and application |
-
2022
- 2022-06-30 CN CN202210762057.XA patent/CN115181073B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114380808A (en) * | 2021-12-21 | 2022-04-22 | 深圳大学 | Molecular probe for bimodal imaging detection of neutrophil elastase, preparation method and application |
Non-Patent Citations (4)
| Title |
|---|
| Activity-Based Near-Infrared Fluorogenic Probe for Enabling in Vitro and in Vivo Profiling of Neutrophil Elastase;Shi-Yu Liu et al.;《 Analytical Chemistry》;第91卷(第6期);第3877-3884页 * |
| Enhanced Colorimetric Differentiation between Staphylococcus aureus and Pseudomonas aeruginosa Using a Shape-Encoded Sensor Hydrogel;Zhiyuan Jia et al.;《 ACS Applied Bio Materials》;第3卷(第7期);第4398-4407页 * |
| Non-Peptide-Based Fluorogenic Small-Molecule Probe for Elastase;Qi Sun et al.;《Analytical Chemistry》;第85卷(第23期);第11304-11311页 * |
| Rationally Designed Two-Photon Ratiometric Elastase Probe for Investigating Inflammatory Bowel Disease;Sourav Sarkar et al.;《 Analytical Chemistry》;第94卷(第2期);第1373-1381页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115181073A (en) | 2022-10-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Monitoring γ-glutamyl transpeptidase activity and evaluating its inhibitors by a water-soluble near-infrared fluorescent probe | |
| Zhang et al. | A low dose, highly selective and sensitive colorimetric and fluorescent probe for biothiols and its application in bioimaging | |
| JPH0137693B2 (en) | ||
| Chen et al. | A low background D–A–D type fluorescent probe for imaging of biothiols in living cells | |
| CN110357865B (en) | Near-infrared fluorescent probe for detecting hNQO1 enzyme, and synthetic method and application thereof | |
| CN110563650B (en) | Ratio type two-photon fluorescent probe of sulfatase, synthetic method and application thereof | |
| CN113185506B (en) | Synthesis of multi-signal fluorescent probe and application thereof in detection of Cys, GSH and Hcy | |
| US20210269421A1 (en) | Water-soluble fluorescent probe and nanoparticals with aggregation-induced emission effect for ovarian cancer and preparation method and use thereof | |
| Li et al. | Recent progress of oxidative stress associated biomarker detection | |
| CN110526908B (en) | Cys/Hcy fluorescent probe capable of being distinguished and detected based on long wave emission of 2-styryl indole salt derivative and application thereof | |
| CN108752373B (en) | Fluorescent probe for identifying hydrogen peroxide based on phenylboronate | |
| CN114105927B (en) | Construction of benzopyran nitrile fluorescent molecular probe and in-vitro diagnosis application thereof | |
| CN115181073B (en) | Red fluorescent probe for detecting elastase and preparation method and application thereof | |
| CN112745340A (en) | Selective detection of H based on BODIPY dye targeting lysosome2Fluorescent probe of S, preparation and application | |
| CN113004258A (en) | Preparation method and application of hydrogen sulfide ratio type fluorescent molecular probe based on ESIPT effect | |
| Zou et al. | A non-peptide probe for detecting chymotrypsin activity based on protection–deprotection strategy in living systems | |
| CN113200975B (en) | Specific response ONOO based on isatin derivatives - Water-soluble fluorescent probe, its preparation method and application | |
| Li et al. | A novel gold nanoprobe for a simple electrochemiluminescence determination of a prostate-specific antigen based on a peptide cleavage reaction | |
| CN109734710A (en) | A kind of fluorescence probe detecting cysteine and its synthetic method and application | |
| CN114634464A (en) | Lysosome targeted near-infrared fluorescent probe for detecting hypochlorous acid and preparation method and application thereof | |
| CN114349717A (en) | Benzothiazole derivative cysteine-based fluorescent probe and preparation method thereof | |
| CN109400616B (en) | Sulfur dioxide derivative fluorescent probe and preparation method and application thereof | |
| CN114907302B (en) | Fluorescent probe for rapidly detecting elastase and preparation method and application thereof | |
| CN115477641A (en) | Glutamyl transpeptidase fluorescent/photoacoustic probe and preparation method and application thereof | |
| CN111039866A (en) | Golgi-targeted hydrogen sulfide fluorescent probe, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |