CN111187247A - Preparation method of microenvironment sensitive fluorescent probe and application of microenvironment sensitive fluorescent probe to HSA/BSA (human serum albumin/bovine serum albumin) detection - Google Patents
Preparation method of microenvironment sensitive fluorescent probe and application of microenvironment sensitive fluorescent probe to HSA/BSA (human serum albumin/bovine serum albumin) detection Download PDFInfo
- Publication number
- CN111187247A CN111187247A CN202010037437.8A CN202010037437A CN111187247A CN 111187247 A CN111187247 A CN 111187247A CN 202010037437 A CN202010037437 A CN 202010037437A CN 111187247 A CN111187247 A CN 111187247A
- Authority
- CN
- China
- Prior art keywords
- bsa
- hsa
- fluorescent probe
- microenvironment
- probe
- 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.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/94—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems condensed with rings other than six-membered or with ring systems containing such 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
-
- 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
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention belongs to the technical field of fluorescent probes for protein detection, and provides a microenvironment sensitive fluorescent probe, a preparation method thereof and application of the microenvironment sensitive fluorescent probe to HSA/BSA detection. The molecular formula of the fluorescent probe is C31H23NO2. The preparation method comprises the following steps: salicylaldehyde, 2-cyclopentene-1-ketone and imidazole in the presence of nitrogen to obtain compound 1. Reacting the compound 1 with 4-benzhydrylaldehyde to obtain the molecular probe. The probe is extremely sensitive to a microenvironment, and the fluorescence spectrum is obviously changed along with the change of the polarity and the viscosity of the solvent. The probe reacts with HSA/BSA in a solvent system of PBS: DMF =8:2, the emission wavelength is blue-shifted and the fluorescence is enhanced, and the change of the fluorescence intensity can be used for detecting the concentration of HSA/BSA and imaging the HSA/BSA cells. The fluorescent probe of the invention has simple synthesis and is sensitive to micro environmentThe kit has the advantages of good selectivity for HSA/BSA detection, high sensitivity and wide linear range.
Description
Technical Field
The invention belongs to the technical field of fluorescent probes for protein detection, and particularly relates to a microenvironment sensitive fluorescent probe, a preparation method thereof and application of the microenvironment sensitive fluorescent probe to HSA/BSA detection.
Background
Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) are homologous proteins, have high similarity in structure and amino acid sequence, and both contain cysteine residues. HSA, the most abundant protein in human plasma, can be involved in enzymatic activities, maintaining plasma osmolality, immunomodulation, and transport of drug molecules. The content abnormality can be used as early warning for renal dysfunction and related renal diseases. BSA, as a stabilizer for enzymes, prevents decomposition and nonspecific adsorption of enzymes, and alleviates denaturation due to adverse environmental factors such as heat, surface tension and chemical factors. Bovine serum is generally adopted as a main component of cell culture in the current vaccine production, which causes BSA (bovine serum albumin) to remain in the vaccine to different degrees, wherein the BSA is a heterologous protein in organisms, and if the BSA remains too much, adverse reactions such as anaphylactic shock and the like can be caused after the vaccine is injected. Therefore, it is very important to develop a method for detecting HSA and BSA efficiently, accurately and rapidly.
In recent years, fluorescent probes have attracted great attention due to the advantages of high response speed, high sensitivity, simplicity in operation, real-time monitoring and the like, and can be used for protein detection.
Disclosure of Invention
The invention aims to provide a microenvironment sensitive fluorescent probe, a preparation method thereof and application of the microenvironment sensitive fluorescent probe to HSA/BSA detection. The invention utilizes the microenvironment of a protein structure and combines with a sulfhydryl reaction site to design and synthesize a fluorescent probe which can be used for detecting HSA/BSA in cells.
The invention is realized by the following technical scheme: a microenvironment sensitive fluorescent probe, the molecular formula of which is C31H23NO2The structural formula is as follows:
。
the method for preparing the microenvironment sensitive fluorescent probe comprises the following specific steps:
(1) at H2Adding salicylaldehyde, 2-cyclopentene-1-one and imidazole into a mixed solvent of O/THF, and stirring the mixture for 12-14 h at room temperature under the protection of nitrogen; the resulting reaction solution was extracted with dichloromethane three times, washed twice with saturated brine and anhydrous Na2SO4Drying, removing the solvent under reduced pressure, and purifying the product by column chromatography to give compound 1 as a yellow solid of formula:
(ii) a Wherein the solvent used in column chromatography is dichloromethane/petroleum ether = 5/1, v/v;
(2) dissolving the compound 1, p-diphenylaminobenzaldehyde and piperidine in ethanol, refluxing for 24-26 h, adding water into a mixed solution after reaction, extracting with dichloromethane, washing an organic phase twice with saturated saline solution, and carrying out anhydrous Na2SO4Drying, removing the solvent under reduced pressure, separating and purifying by silica gel column dichloromethane/methanol =500/1 and v/v, and drying in vacuum to obtain a red solid compound 2, namely the microenvironment sensitive HAS/BSA fluorescent probe molecule.
H in the mixed reaction solvent in the step (1)2The volume ratio of O to THF is 1: 1, using 1mL of solvent for every 1 mmol of salicylaldehyde; for every 1ml of reaction mixture, 1ml of water, salicylaldehyde: 2-cyclopenten-1-one: the molar ratio of imidazole is 1: 1-1.2: 1 to 1.2.
In the step (2), the molar ratio of the compound 1 to the p-diphenylaminobenzaldehyde to the piperidine is 1: 0.8-1: 0.2.
the application of the micro-environment sensitive HSA/BSA fluorescent probe is characterized in that the fluorescent probe is applied to PBS: detection of HSA and BSA in DMF =8:2 systems and cell tissue environments.
In a system of PBS: DMF =8:2, a fluorescent probe was reacted with HSA/BSA, and the concentration of HSA/BSA was detected by the change in fluorescence intensity: before HSA/BSA is added, the fluorescent signal of the probe is weak, the emission wavelength is 610 nm, after HSA/BSA is added, the fluorescent signal of the probe is enhanced, and the emission wavelength is blue-shifted to 580 nm.
In a cellular tissue environment: the linear range of the detection of the human serum albumin is 100-2850 mg/L, the detection limit is 13.65 mg/L, the linear range of the detection of the bovine serum albumin is 0-900mg/L, and the detection limit is 3.82 mg/L.
The micro-environment sensitive HSA/BSA prepared by the invention has the fluorescence characteristics that in a mixed system of glycerol and water: with the increase of the proportion of the glycerol, the fluorescence emission wavelength is blue-shifted, and the fluorescence intensity is enhanced; in the mixed system of 1, 4-dioxane and water, the fluorescence emission wavelength is red-shifted from 576nm to 602 nm as the proportion of water increases.
In PBS: HSA and BSA were detected in DMF =8:2 systems and HeLa cells. Reacting a fluorescent probe with HSA/BSA in a system with PBS: DMF =8:2, and detecting the concentration of HSA/BSA by using the change of fluorescence intensity; before HSA/BSA is added, the fluorescent signal of the probe is weaker, the emission wavelength is 610 nm, and after HSA/BSA is added, the fluorescent signal of the probe is enhanced, and the emission wavelength is blue-shifted to 580 nm. The linear range of the fluorescent probe for detecting HSA is 100-2850 mg/L, the detection limit is 13.65 mg/L, the linear range for detecting BSA is 0-900mg/L, and the detection limit is 3.82 mg/L.
The fluorescent probe prepared by the invention has N, N-diphenyl electron donating groups and carbonyl electron withdrawing groups, has obvious intramolecular charge transfer characteristics, longer emission wavelength and larger tosrak shift, and the recognition mechanism of the fluorescent probe to HSA/BSA comprises two aspects, namely 1, a molecular structure of the probe has α -unsaturated carbonyl structure which can generate addition reaction with free sulfydryl in protein, and 2, the fluorescent intensity is enhanced and blue shift is realized by utilizing a microenvironment of the protein, so the probe can detect the HSA/BSA, and HSA and BSA imaging is realized in a HeLa cell.
The fluorescence emission wavelength and intensity of the fluorescent probe are sensitive to the change of the polarity and the viscosity of the solvent. The mechanism for detecting HAS/BSA by the fluorescent probe molecule is novel. The fluorescent probe molecule realizes the imaging of HSA/BSA in HeLa cells.
Drawings
FIG. 1 is a graph showing the change of fluorescence spectrum with polarity and viscosity of probe molecules in example seven; wherein: (a) is a schematic diagram of the change of the fluorescence spectrum of the probe molecule along with the polarity; (b) is a schematic diagram of the change of the fluorescence spectrum of the probe molecule along with the viscosity;
FIG. 2 is a graph showing the fluorescence spectra of the probe molecules of example eight as a function of the HSA/BSA concentration; wherein: (a) is a diagram showing the change of the fluorescence spectrum of the probe molecule with the HSA concentration; (b) is a diagram showing the change of the fluorescence spectrum of the probe molecule with the concentration of BSA;
FIG. 3 is a graph showing the change of fluorescence intensity with time in the reaction of the probe molecule with HSA/BSA in example nine;
FIG. 4 is a graph showing the change in fluorescence intensity before and after the reaction of a probe molecule with different interfering analytes in example ten;
FIG. 5 shows the imaging study of HSA/BSA by the probe molecules in example eleven in HELA cells; wherein: (a) imaging the HSA cells for the probe molecule; (b) imaging BSA cells for probe molecules;
FIG. 6 is a scheme showing the synthesis of the probe according to the present invention.
Detailed Description
The present invention is further illustrated by, but is not limited to, the following examples.
The synthetic route of the micro-environment sensitive HSA/BSA fluorescent probe is shown in FIG. 6. Specific examples are as follows:
the first embodiment is as follows: synthesis of Compound 1: in a 25 mL three-necked flask equipped with a magnetic stirrer, salicylaldehyde (10 mmol, 1.065 mL), 2-cyclopenten-1-one (11 mmol, 0.918 mL) and imidazole (11 mmol, 0.750g) were added, respectively, followed by 10mL H2O/THF (v/v = 1: 1), and the mixture was stirred at room temperature for 12 hours under nitrogen. Will be reversedThe solution was extracted three times with dichloromethane, washed twice with saturated brine and anhydrous Na2SO4Drying, removal of the solvent under reduced pressure and further purification of the product by column chromatography (dichloromethane/petroleum ether, 5/1, v/v) gave compound 1 as a yellow solid (0.210 g, 11.29%).
1H NMR (600 MHz, DMSO) δ 7.42 (d, J = 7.4 Hz, 1H), 7.30 (dd, J =21.6, 13.9 Hz, 2H), 7.00 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 5.30(t, J = 8.1 Hz, 1H), 2.61 (dd, J = 19.6, 8.2 Hz, 1H), 2.48 – 2.34 (m, 2H),2.09 – 1.97 (m, 1H).13C NMR (151 MHz, DMSO) δ 201.54, 155.19, 132.78, 132.71,131.06, 126.72, 122.68, 122.33, 116.65, 75.76, 37.12, 28.09。
Example two: synthesis of Compound 1: in a 25 mL three-necked flask equipped with a magnetic stirrer, salicylaldehyde (10 mmol, 1.065 mL), 2-cyclopenten-1-one (10 mmol, 0.835 mL) and imidazole (10 mmol, 0.682g) were added, respectively, followed by 10mL H2O/THF (v/v = 1: 1), and the mixture was stirred at room temperature for 13 hours under nitrogen blanket. The reaction solution was extracted three times with dichloromethane, washed twice with saturated brine and anhydrous Na2SO4Drying, removal of the solvent under reduced pressure and further purification of the product by column chromatography (dichloromethane/petroleum ether, 5/1, v/v) gave compound 1 as a yellow solid (0.182 g, 9.78%).
1H NMR (600 MHz, DMSO) δ 7.42 (d, J = 7.4 Hz, 1H), 7.30 (dd, J =21.6, 13.9 Hz, 2H), 7.00 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 5.30(t, J = 8.1 Hz, 1H), 2.61 (dd, J = 19.6, 8.2 Hz, 1H), 2.48 – 2.34 (m, 2H),2.09 – 1.97 (m, 1H).13C NMR (151 MHz, DMSO) δ 201.54, 155.19, 132.78, 132.71,131.06, 126.72, 122.68, 122.33, 116.65, 75.76, 37.12, 28.09。
Example three: synthesis of Compound 1: in a 25 mL three-necked flask equipped with a magnetic stirrer, salicylaldehyde (10 mmol, 1.065 mL), 2-cyclopenten-1-one (11 mmol, 0.918 mL) and imidazole (11 mmol, 0.750g) were added, respectively, followed by 10mL H2O/THF (v/v = 1: 1), and the mixture was stirred at room temperature for 14 h under nitrogen. Will be provided withThe reaction solution was extracted three times with dichloromethane, washed twice with saturated brine and anhydrous Na2SO4Drying, removal of the solvent under reduced pressure and further purification of the product by column chromatography (dichloromethane/petroleum ether, 5/1, v/v) gave compound 1 as a yellow solid (0.198 g, 10.64%).
1H NMR (600 MHz, DMSO) δ 7.42 (d, J = 7.4 Hz, 1H), 7.30 (dd, J =21.6, 13.9 Hz, 2H), 7.00 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 5.30(t, J = 8.1 Hz, 1H), 2.61 (dd, J = 19.6, 8.2 Hz, 1H), 2.48 – 2.34 (m, 2H),2.09 – 1.97 (m, 1H).13C NMR (151 MHz, DMSO) δ 201.54, 155.19, 132.78, 132.71,131.06, 126.72, 122.68, 122.33, 116.65, 75.76, 37.12, 28.09。
Example four: synthesis of probe molecules: in a 25 mL three-necked flask equipped with a magnetic stirrer, compound 1(0.5mmol, 0.093 g), 4-diphenylaminobenzaldehyde (0.45 mmol, 0.123g) and piperidine solution (0.1 mmol,0.010mL) were dissolved in 12 mL ethanol and refluxed for 24 h. After the reaction, 12 mL of water was added to the mixed solution, extraction was performed with methylene chloride, and the organic phase was washed twice with saturated brine and anhydrous Na2SO4Drying, removal of solvent under reduced pressure, further purification by column chromatography (dichloromethane/methanol, 500/1, v/v), drying in vacuo gave compound 2 as a red solid (0.040 g, 20.16%).
1H NMR (600 MHz, CDCl3) δ 7.57 (d,J= 9.2 Hz, 2H), 7.46 (d,J= 7.7Hz, 2H), 7.41 – 7.36 (m, 6H), 7.35 – 7.31 (m, 1H), 7.17 (t,J= 7.8 Hz, 2H),7.13 (d,J= 8.7 Hz, 4H), 7.03 (t,J= 7.6 Hz, 1H), 6.98 (d,J= 8.4 Hz, 1H),6.93 (d,J= 9.1 Hz, 2H), 5.34 (s, 1H), 3.65 (dd,J= 18.0, 9.0 Hz, 1H).13CNMR (151 MHz, DMSO) δ 189.94, 155.29, 149.44, 146.58, 134.40, 134.18, 133.97,132.94, 132.72, 130.94, 130.36, 127.85, 127.41, 126.02, 125.10, 122.98,122.87,120.77, 116.79, 73.99, 34.72。
Example five: synthesis of probe molecules: in a 25 mL three-necked flask equipped with a magnetic stirrer, Compound 1(0.5mmol, 0.093 g), 4-diphenylaminobenzaldehyde(0.5mmol, 0.123g) and piperidine solution (0.1 mmol,0.010mL) were dissolved in 12 mL ethanol and refluxed for 25 h. After the reaction, 12 mL of water was added to the mixed solution, extraction was performed with methylene chloride, and the organic phase was washed twice with saturated brine and anhydrous Na2SO4Drying, removal of solvent under reduced pressure, further purification by column chromatography (dichloromethane/methanol, 500/1, v/v), drying in vacuo gave compound 2 as a red solid (0.036 g, 18.14%).
1H NMR (600 MHz, CDCl3) δ 7.57 (d,J= 9.2 Hz, 2H), 7.46 (d,J= 7.7Hz, 2H), 7.41 – 7.36 (m, 6H), 7.35 – 7.31 (m, 1H), 7.17 (t,J= 7.8 Hz, 2H),7.13 (d,J= 8.7 Hz, 4H), 7.03 (t,J= 7.6 Hz, 1H), 6.98 (d,J= 8.4 Hz, 1H),6.93 (d,J= 9.1 Hz, 2H), 5.34 (s, 1H), 3.65 (dd,J= 18.0, 9.0 Hz, 1H).13CNMR (151 MHz, DMSO) δ 189.94, 155.29, 149.44, 146.58, 134.40, 134.18, 133.97,132.94, 132.72, 130.94, 130.36, 127.85, 127.41, 126.02, 125.10, 122.98,122.87,120.77, 116.79, 73.99, 34.72。
Example six: synthesis of probe molecules: in a 25 mL three-necked flask equipped with a magnetic stirrer, compound 1(0.5mmol, 0.093 g), 4-diphenylaminobenzaldehyde (0.4 mmol, 0.123g) and a piperidine solution (0.1 mmol,0.010mL) were dissolved in 12 mL of ethanol and refluxed for 26 hours. After the reaction, 12 mL of water was added to the mixed solution, extraction was performed with methylene chloride, and the organic phase was washed twice with saturated brine and anhydrous Na2SO4Drying, removal of solvent under reduced pressure, further purification by column chromatography (dichloromethane/methanol, 500/1, v/v), and drying in vacuo afforded compound 2 as a red solid (0.033 g, 16.63%).
1H NMR (600 MHz, CDCl3) δ 7.57 (d,J= 9.2 Hz, 2H), 7.46 (d,J= 7.7Hz, 2H), 7.41 – 7.36 (m, 6H), 7.35 – 7.31 (m, 1H), 7.17 (t,J= 7.8 Hz, 2H),7.13 (d,J= 8.7 Hz, 4H), 7.03 (t,J= 7.6 Hz, 1H), 6.98 (d,J= 8.4 Hz, 1H),6.93 (d,J= 9.1 Hz, 2H), 5.34 (s, 1H), 3.65 (dd,J= 18.0, 9.0 Hz, 1H).13CNMR (151 MHz, DMSO) δ 189.94, 155.29, 149.44, 146.58, 134.40, 134.18, 133.97,132.94, 132.72, 130.94, 130.36, 127.85, 127.41, 126.02, 125.10, 122.98,122.87,120.77, 116.79, 73.99, 34.72。
Example seven; study of the sensitivity of the probe molecules to the microenvironment: in order to test the sensitivity of probe molecules to microenvironment, the spectral properties of probes in systems with different polarities and viscosities are researched. In the mixed system of glycerol and water, as the viscosity increases (the proportion of glycerol increases), the fluorescence maximum emission wavelength slightly shifts blue and the fluorescence intensity gradually increases, as shown in fig. 1 (a); in the mixed system of 1, 4-dioxane and water, as the polarity is increased (the proportion of water is increased), the maximum emission wavelength of fluorescence is changed from 576nm to 602 nm, and red shift occurs, as shown in FIG. 1 (b).
Example eight: study of probe molecules on changes in fluorescence intensity under different concentrations of HSA and BSA: to test the response of the probe molecules to the HSA and BSA concentrations, the change in fluorescence intensity of the probe molecules under different concentrations of HSA (0, 0.05, 0.15, 0.20, 0.35, 0.40, 0.53, 0.78, 0.90, 1.15, 1.40, 1.65, 1.90, 2.15, 2.40, 2.65, 2.90, 3.15, 3.40, 3.65 mg/ml) and BSA (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0, 1.4, 1.8, 2.2, 2.5 mg/ml) was studied (see FIG. 2). As can be seen from FIG. 2 (a), the linear range for detecting HSA is 2850 mg/L, and the detection limit is 13.65 mg/L, and as can be seen from FIG. 2 (b), the linear range for detecting BSA is 0-900mg/L, and the detection limit is 3.82mg/L, which indicates that the fluorescent probe molecule of the present invention exhibits higher sensitivity to different concentrations of HSA and BSA.
Example nine: time response study of probe molecules to HSA and BSA: in order to test the time response of the probe molecules to HSA and BSA, the change of the fluorescence intensity of the reaction of the probe molecules with HSA and BSA along with the time (0-30 min) was studied. As can be seen from FIG. 3, the probe molecules showed a high response speed to HSA and BSA.
Example ten: selective study of probe molecules for HSA and BSA: to test the selectivity of the probe molecules for HSA and BSA, the probe molecules were tested with different metal ions (Cd)2+，Co2+，Cr3+，Ba2+，Mn2+，Ni2+，Zn2+，Fe3+，Al3+，Ca2+，k+，Mg2+) Anion (HSO)3 -，SO4 2-，CO3 2-，SO3 2-，SCN-，HCO3 -，C2O4 2-，Cl-，I-，CH3COO-，ClO4 -) Experimental study is carried out on the change of fluorescence intensity before and after the reaction of amino acids (Histine, Glutamate, Asparagine, Lysine, Phenylalanine, Tyrosine, Alanine, Serine, Leucine, Arginine, Cysteine, Homocysteine, Glutathione), the concentration of HSA and BSA is 10 μ M, and the concentration of the rest interferents is 100 μ M. As can be seen from fig. 4, the detection of HSA and BSA by the probe molecules is not interfered by other analytes, and has high selectivity.
Example eleven: imaging studies of probe molecules on HSA and BSA in HELA cells: to test the imaging of probe molecules on HSA and BSA in HELA cells, cellular imaging studies were performed on the changes after reaction of the probe molecules with HSA (20 μ M) and BSA (20 μ M), respectively, as shown in fig. 5 (a) and (b). As can be seen from FIG. 5, the probe molecules can image cells of HSA and BSA.
Claims (8)
1. A microenvironment-sensitive fluorescent probe, comprising: the molecular formula of the fluorescent probe is C31H23NO2The structural formula is as follows:
。
2. the method of making the microenvironment-sensitive fluorescent probe of claim 1, wherein: the method comprises the following specific steps:
(1) at H2Adding salicylaldehyde, 2-cyclopentene-1-one and imidazole into a mixed solvent of O/THF, and stirring the mixture for 12-14 h at room temperature under the protection of nitrogen; methylene dichloride for the obtained reaction liquidAlkane extraction three times, saturated brine washing two times, anhydrous Na2SO4Drying, removing the solvent under reduced pressure, and purifying the product by column chromatography to give compound 1 as a yellow solid of formula:
(ii) a Wherein the solvent used in column chromatography is dichloromethane/petroleum ether = 5/1, v/v;
(2) dissolving the compound 1, p-diphenylaminobenzaldehyde and piperidine in ethanol, refluxing for 24-26 h, adding water into a mixed solution after reaction, extracting with dichloromethane, washing an organic phase twice with saturated saline solution, and carrying out anhydrous Na2SO4Drying, removing the solvent under reduced pressure, separating and purifying by silica gel column dichloromethane/methanol =500/1, v/v, and drying in vacuum to obtain red solid compound 2, namely the microenvironment sensitive HSA/BSA fluorescent probe molecule.
3. The method of preparing a microenvironment-sensitive fluorescent probe of claim 2, wherein: h in the mixed reaction solvent in the step (1)2The volume ratio of O to THF is 1: 1, using 1mL of solvent for every 1 mmol of salicylaldehyde; for every 1ml of reaction mixture, 1ml of water, salicylaldehyde: 2-cyclopenten-1-one: the molar ratio of imidazole is 1: 1-1.2: 1 to 1.2.
4. The method of preparing a microenvironment-sensitive fluorescent probe of claim 2, wherein: in the step (2), the molar ratio of the compound 1 to the p-diphenylaminobenzaldehyde to the piperidine is 1: 0.8-1: 0.2.
5. the use of the microenvironment-sensitive fluorescent probe of claim 1, wherein: the fluorescent probe is applied to a mixed system of glycerol and water, a mixed system of 1, 4-dioxane and water, and PBS: detection of HSA and BSA in DMF =8:2 systems and cell tissue environments.
6. The use of the microenvironment-sensitive fluorescent probe of claim 5, wherein: in a glycerol and water mixed system: with the increase of the proportion of the glycerol, the fluorescence emission wavelength is blue-shifted, and the fluorescence intensity is enhanced; in the mixed system of 1, 4-dioxane and water, the fluorescence emission wavelength is red-shifted from 576nm to 602 nm as the proportion of water increases.
7. The use of the microenvironment-sensitive fluorescent probe of claim 5, wherein: in a system of PBS: DMF =8:2, a fluorescent probe was reacted with HSA/BSA, and the concentration of HSA/BSA was detected by the change in fluorescence intensity: before HSA/BSA is added, the fluorescent signal of the probe is weak, the emission wavelength is 610 nm, after HSA/BSA is added, the fluorescent signal of the probe is enhanced, and the emission wavelength is blue-shifted to 580 nm.
8. The use of the microenvironment-sensitive fluorescent probe of claim 5, wherein: in a cellular tissue environment: the linear range of the detection of the human serum albumin is 100-2850 mg/L, the detection limit is 13.65 mg/L, the linear range of the detection of the bovine serum albumin is 0-900mg/L, and the detection limit is 3.82 mg/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010037437.8A CN111187247A (en) | 2020-01-14 | 2020-01-14 | Preparation method of microenvironment sensitive fluorescent probe and application of microenvironment sensitive fluorescent probe to HSA/BSA (human serum albumin/bovine serum albumin) detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010037437.8A CN111187247A (en) | 2020-01-14 | 2020-01-14 | Preparation method of microenvironment sensitive fluorescent probe and application of microenvironment sensitive fluorescent probe to HSA/BSA (human serum albumin/bovine serum albumin) detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111187247A true CN111187247A (en) | 2020-05-22 |
Family
ID=70706220
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010037437.8A Pending CN111187247A (en) | 2020-01-14 | 2020-01-14 | Preparation method of microenvironment sensitive fluorescent probe and application of microenvironment sensitive fluorescent probe to HSA/BSA (human serum albumin/bovine serum albumin) detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111187247A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961673A (en) * | 2021-03-11 | 2021-06-15 | 山西大学 | Targeted lipid drop fluorescent probe and preparation method and application thereof |
| CN113735757A (en) * | 2021-09-30 | 2021-12-03 | 陕西师范大学 | Microenvironment sensitive fluorescent organic small molecule compound and synthetic method and application thereof |
| CN113845519A (en) * | 2021-09-18 | 2021-12-28 | 山西大学 | Microenvironment sensitive type fluorescent probe and preparation method and application thereof |
| CN114716401A (en) * | 2022-05-06 | 2022-07-08 | 山西大学 | Organic small-molecule fluorescent probe and preparation method and application thereof |
| CN114836202A (en) * | 2022-05-25 | 2022-08-02 | 西北师范大学 | Application of Bola type amphiphilic AIE fluorescent probe based on TPE in detection of bovine serum albumin |
| CN117088835A (en) * | 2023-08-24 | 2023-11-21 | 华东理工大学 | Luminous probe and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018020976A (en) * | 2016-08-04 | 2018-02-08 | キヤノン株式会社 | Organic compound, photoelectric conversion element using the same, optical area sensor, photoelectric conversion device, imaging element and imaging apparatus |
| CN110372681A (en) * | 2019-07-29 | 2019-10-25 | 天津医科大学 | A kind of application of the self-assembled nanometer fluorescence probe for selective enumeration method human serum albumins |
| CN110386931A (en) * | 2018-04-20 | 2019-10-29 | 南京大学 | A kind of human albumin's fluorescence probe and its preparation method and purposes |
-
2020
- 2020-01-14 CN CN202010037437.8A patent/CN111187247A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018020976A (en) * | 2016-08-04 | 2018-02-08 | キヤノン株式会社 | Organic compound, photoelectric conversion element using the same, optical area sensor, photoelectric conversion device, imaging element and imaging apparatus |
| CN110386931A (en) * | 2018-04-20 | 2019-10-29 | 南京大学 | A kind of human albumin's fluorescence probe and its preparation method and purposes |
| CN110372681A (en) * | 2019-07-29 | 2019-10-25 | 天津医科大学 | A kind of application of the self-assembled nanometer fluorescence probe for selective enumeration method human serum albumins |
Non-Patent Citations (3)
| Title |
|---|
| XIAO CHENG，ET AL.: "Microscopic Visualization and Mechanism Investigation on Crystal Jumping Behavior of a Cyclic Chalcone Derivative", 《MATERIALS CHEMISTRY FRONTIERS》 * |
| 亢娜，等: "一种灵敏检测巯基蛋白的荧光探针的设计及细胞成像", 《第二十届全国有机分析及生物分析学术研讨会》 * |
| 霍方俊，等: "基于色烯分子的硫醇识别研究", 《中国化学会•第八届有机化学学术会议暨首届重庆有机化学国际研讨会》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961673A (en) * | 2021-03-11 | 2021-06-15 | 山西大学 | Targeted lipid drop fluorescent probe and preparation method and application thereof |
| CN113845519A (en) * | 2021-09-18 | 2021-12-28 | 山西大学 | Microenvironment sensitive type fluorescent probe and preparation method and application thereof |
| CN113845519B (en) * | 2021-09-18 | 2022-05-31 | 山西大学 | Microenvironment sensitive type fluorescent probe and preparation method and application thereof |
| CN113735757A (en) * | 2021-09-30 | 2021-12-03 | 陕西师范大学 | Microenvironment sensitive fluorescent organic small molecule compound and synthetic method and application thereof |
| CN113735757B (en) * | 2021-09-30 | 2024-03-15 | 陕西师范大学 | Micro-environment sensitive fluorescent organic small molecular compound and synthetic method and application thereof |
| CN114716401A (en) * | 2022-05-06 | 2022-07-08 | 山西大学 | Organic small-molecule fluorescent probe and preparation method and application thereof |
| CN114716401B (en) * | 2022-05-06 | 2024-03-12 | 山西大学 | Organic small molecule fluorescent probe and preparation method and application thereof |
| CN114836202A (en) * | 2022-05-25 | 2022-08-02 | 西北师范大学 | Application of Bola type amphiphilic AIE fluorescent probe based on TPE in detection of bovine serum albumin |
| CN117088835A (en) * | 2023-08-24 | 2023-11-21 | 华东理工大学 | Luminous probe and preparation method and application thereof |
| CN117088835B (en) * | 2023-08-24 | 2024-02-27 | 华东理工大学 | Luminous probe and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111187247A (en) | Preparation method of microenvironment sensitive fluorescent probe and application of microenvironment sensitive fluorescent probe to HSA/BSA (human serum albumin/bovine serum albumin) detection | |
| JP5807025B2 (en) | Fluorescent probe | |
| CN104860879B (en) | Malononitrile isophorone copper ion fluorescent probe and preparation method thereof | |
| CN108070092B (en) | Supermolecular gel based on functionalized column [5] arene and application thereof in identifying iron ions and L-Cys | |
| Liu et al. | A novel near-infrared fluorescent probe with a large Stokes shift for biothiol detection and application in in vitro and in vivo fluorescence imaging | |
| Du et al. | Highly selective fluorescent recognition of histidine by a crown ether–terpyridine–Zn (II) sensor | |
| CN101440062A (en) | Synthesis of N-acyl-8-amino quinoline derivatives and use thereof as fluorescent molecular probe | |
| CN111892552A (en) | Triphenylamine derivative, preparation method thereof and application thereof in double-channel fluorescence detection of hydrogen sulfide | |
| CN106188102B (en) | A kind of water-soluble dendroid list imide compound fluorescence probe and its preparation method and application | |
| CN109928940B (en) | Preparation of near-infrared fluorescent probe molecule for detecting hypochlorous acid based on basic blue-3 | |
| CN108048075B (en) | Calcium ion fluorescent probe based on aggregation induction effect and preparation method and application thereof | |
| KR101179513B1 (en) | Methionine amino acid based chemical sensor for selective detecting mercury ion, and preparation method thereof | |
| US5958673A (en) | Fluorescent dye | |
| CN111533761B (en) | Ratio type pH probe with organelle or protein targeting function and application thereof | |
| CN111138431B (en) | Reactive fluorescent probe for detecting thiophenol and synthetic method and application thereof | |
| CN110746423B (en) | Synthesis of aryl imidazophenanthroline fluorescent dye and identification of metal ions | |
| CN109836414B (en) | Pentapylene derivative, preparation method thereof and application thereof in polyamine detection | |
| CN111253309A (en) | Acridinone fluorescent amine compound labeling reagent and synthesis method and application thereof | |
| CN113845655B (en) | Water-soluble fluorescein polymer probe for mercury ion detection and preparation and application thereof | |
| CN112979533B (en) | Ratio type fluorescent probe for detecting sulfur dioxide and preparation method and application thereof | |
| CN106010507A (en) | Dye-molecule-marked tetrazine probe, and preparation method and application thereof | |
| CN108218822A (en) | A kind of ratio type fluorescence probe for detecting azanol and its synthetic method and application | |
| CN109651336B (en) | Fluorescent probe for detecting hydrogen sulfide based on drug molecules and preparation method thereof | |
| CN110746339B (en) | Pyrrole dihydrazone derivative fluorescent probe and preparation method and application thereof | |
| CN110143985B (en) | Iridium complex probe for identifying bisulfite and preparation method 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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200522 |