CN117402141A - Fluorescent probe and preparation method and application thereof - Google Patents
Fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN117402141A CN117402141A CN202311145515.6A CN202311145515A CN117402141A CN 117402141 A CN117402141 A CN 117402141A CN 202311145515 A CN202311145515 A CN 202311145515A CN 117402141 A CN117402141 A CN 117402141A
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- serum albumin
- human serum
- fluorescent probe
- fluorescence
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- 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"
-
- 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
Abstract
The invention provides a novel fluorescent probe Nap-NO 2 The method is used for rapid and accurate detection of the human serum albumin. The preparation method comprises the reaction of N-N-butyl-3-aldehyde-4-hydroxy-1, 8-naphthalimide and pyridinium. Fluorescent probe Nap-NO 2 The method has the advantages of red light emission, quick response, strong specificity and selectivity, low detection limit, good stability, simple detection process and the like, and has strong application value in the aspects of preparing detection products of the human serum albumin and screening or detecting medicaments related to human serum albumin IB structural domains.
Description
Technical Field
The invention relates to the field of fluorescence detection, in particular to a fluorescent probe for human serum albumin detection, and a preparation method and application thereof.
Background
As is well known, human Serum Albumin (HSA) is the most abundant protein component in human plasma (40 kg/m 3 Or 0.6 mM), has many physiological functions. Human serum albumin contains 580 amino acids, including one tryptophan and 17 tyrosines. According to X-ray crystallography investigation, human serum albumin comprises three homologous domains (I-III), each domain comprising two subdomains: the a and B subfields. Due to the interactions of the a and B domains with metabolites and drugs in vivo, the pharmacokinetic and pharmacodynamic characteristics of human serum albumin will change, which will further affect its distribution and activity on biological targets. Human serum albumin can bind a number of endogenous and exogenous compounds, such as cholesterol, fatty acids, retinol, retinoic acid, etc., facilitating their transport in the circulatory system. Human serum albumin in urine can also be used as an index for detecting chronic kidney disease. In addition, disorders in the number and structure of human serum albumin are closely related to life threatening diseases such as cancer, liver failure, etc.
Heretofore, there are many methods for detecting human serum albumin, such as electrochemical, liquid chromatography-mass spectrometry, and the like. Meanwhile, the detection method of the small molecular fluorescent probe is widely focused due to high sensitivity, good selectivity, high reaction speed and simple operation. Importantly, the "on" fluorescent probes play an important role in understanding the dynamics of biological processes. In recent years, many fluorescent probes have been reported for detecting human serum albumin, but these probes have some inherent limitations such as poor detection limits, short excitation and emission wavelengths, difficulty in distinguishing human serum albumin from bovine serum albumin, and the like.
Chinese patent document CN104341346A discloses a fluorescent probe which is a biphenyl formyl ester derivative with the C-4 hydroxyl of an N-N-butyl-4-hydroxyl-1, 8-naphthalimide matrix being substituted, is used for quantitatively determining the content of human serum albumin, has excitation wavelength of 300-500nm and emission wavelength of 410-600nm. The detection principle of the fluorescent probe is based on hydrolysis reaction of albumin pseudo esterase, the hydrolysis reaction needs to be carried out in advance before fluorescence detection, the time is 5-120 minutes, the detection process needs to synchronously detect fluorescence of a probe substrate and a hydrolysis product, the operation is complex, and the time is long; and the excitation wavelength and the emission wavelength have an overlapping area, which affects the accuracy of the result.
Therefore, developing fluorescent probes for human serum albumin detection with long emission wavelength, good selectivity, and high sensitivity remains a challenge.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a novel fluorescent dye molecular probe which is used for rapidly and accurately detecting Human Serum Albumin (HSA), can prolong the emission wavelength, has good selectivity, high sensitivity, good stability and convenient qualitative and quantitative detection, and is suitable for the detection of Human Serum Albumin (HSA).
In the structural design of the fluorescent probe, the invention adopts a carbon-carbon double bond as a tie, and combines 4-hydroxy-1, 8-naphthalimide fluorophor with pyridine salt groups capable of improving water solubility to obtain a novel fluorescent probe.
In order to achieve the object of the present invention, the present invention provides a fluorescent probe having a structure represented by the formula (I),
wherein X is halogen selected from F, cl, br or I. Preferably, X is selected from Br or I.
The invention also provides a preparation method of the fluorescent probe shown in the formula (I), which is to react a compound shown in the formula (II) with a compound shown in the formula (III) to obtain the fluorescent probe,
wherein X is halogen selected from F, cl, br or I. Preferably, X is selected from Br or I.
The fluorescent probe shown in the formula (I) has the chemical name of 4- [ (N-N-butyl-4-hydroxy-1, 8-naphthalimide) -3-vinyl]-1- (4-nitrobenzyl) pyridine-1-halonium, also designated "Nap-NO" in the present invention 2 ". Wherein when X is Br, fluorescence of formula (I)The chemical name of the probe is 4- [ (N-N-butyl-4-hydroxy-1, 8-naphthalimide) -3-vinyl]-1- (4-nitrobenzyl) pyridine-1-ium bromide, also designated "Nap-NO" in the present invention 2 -a "; when X is I, the fluorescent probe shown in the formula (I) has the chemical name of 4- [ (N-N-butyl-4-hydroxy-1, 8-naphthalimide) -3-vinyl]-1- (4-nitrobenzyl) pyridine-1-iodonium, also designated "Nap-NO" in the present invention 2 -b”。
The chemical name of the compound of formula (II) is N-N-butyl-3-aldehyde-4-hydroxy-1, 8-naphthalimide, also referred to herein as "Nap-CHO".
The chemical name of the compound of formula (III) is 4-methyl-1- (4-nitrobenzyl) pyridine-1-onium halide, also referred to as "Py-NO" in the present invention 2 ". Wherein when X is Br, the chemical name of the compound represented by formula (III) is 4-methyl-1- (4-nitrobenzyl) pyridine-1-onium bromide, which is also referred to as "Py-NO" in the present invention 2 -a "; when X is I, the chemical name of the compound shown in the formula (III) is 4-methyl-1- (4-nitrobenzyl) pyridine-1-onium iodide, and is also named as 'Py-NO' in the invention 2 -b”。
The fluorescent probe Nap-NO 2 The preparation method of (2) comprises the following steps:
preferably, the compound of formula (II) (Nap-CHO) and the compound of formula (III) (Py-NO) 2 ) The molar ratio of (2) is 1:1-1:2.
Preferably, the reaction is carried out under inert gas protection in the presence of pyridine.
Preferably, the reaction is carried out under reflux conditions.
In a further preferred embodiment, the compound Nap-CHO is prepared by reacting Nap-O with hexamethylenetetramine.
The preparation method of the compound Nap-CHO comprises the following steps:
preferably, the molar ratio of the compound Nap-O to hexamethylenetetramine is 1:1-1:3.
Preferably, the reaction is carried out in the presence of trifluoroacetic acid.
Preferably, the reaction temperature is 80-90 ℃ and the reaction time is 5-8 hours.
In a further preferred embodiment, the compound Nap-O is prepared starting from 4-bromo-1, 8-naphthalic anhydride, comprising the steps of: (a) 4-bromo-1, 8-naphthalene dicarboxylic anhydride reacts with n-butylamine to obtain a compound Nap-Br; (b) The compound Nap-Br reacts with a methoxylation reagent to obtain a compound Nap-OCH 3 The method comprises the steps of carrying out a first treatment on the surface of the (c) Nap-OCH compound 3 Removing methyl on methoxy to obtain a compound Nap-O.
The chemical name of the compound Nap-Br is N-N-butyl-4-bromo-1, 8-naphthalimide.
Nap-OCH compound 3 Is N-N-butyl-4-methoxy-1, 8-naphthalimide.
The chemical name of the compound Nap-O is N-N-butyl-4-hydroxy-1, 8-naphthalimide.
The preparation method of the compound Nap-O comprises the following steps:
preferably, the molar ratio of the 4-bromo-1, 8-naphthalic anhydride and the n-butylamine in the step (a) is 1:1-1:3, and reflux reaction is carried out.
Preferably, step (b) of the methoxylating reagent includes, but is not limited to, methanol, sodium methoxide, methyl sulfate, and the molar ratio of methoxylating reagent to compound Nap-Br is at least 1:1.
Preferably, the reagents for the demethylation of step (c) include, but are not limited to, hydroiodic acid, hydrobromic acid, demethylating reagents and the compound Nap-OCH 3 The molar ratio of (2) is at least 1:1.
Fluorescent probe Nap-NO prepared by the invention 2 The structure is confirmed by nuclear magnetic resonance hydrogen spectrum, nuclear magnetic resonance carbon spectrum and high resolution mass spectrum (HR-MS), and the structure shown in the formula (I) is proved.
Studies have shown that: nap-NO alone under 500nm excitation condition 2 The solution only has weak fluorescence emission; when N isap-NO 2 After the human serum albumin is added into the solution, the fluorescence emission intensity at 630nm is obviously enhanced, the fluorescence response time is only 5s, and the high-intensity fluorescence emission can be maintained within 60min after the response; while Nap-NO 2 Adding other substances such as bovine serum albumin, adenosine triphosphate, heparin, protamine sulfate, canavalin A, trypsin, various amino acids, etc. into the solution, and mixing the obtained solution system with independent Nap-NO 2 The fluorescence emission intensity of the solution was not significantly changed compared to that of the solution. Thus, nap-NO 2 The fluorescent probe can be used for detecting human serum albumin or preparing a detection product of the human serum albumin, and has the advantages of good fluorescence specificity and selectivity, quick response, strong signal, small interference, accurate result and good stability.
The invention also provides a reagent, test paper or kit for detecting the human serum albumin, which contains a fluorescent probe Nap-NO 2 。
The invention also provides a visual fluorescence detection method, which comprises the following steps: fluorescent probe Nap-NO 2 And the sample to be tested is fully contacted, and under the irradiation of visible light, if bright red fluorescence emission is observed, the sample to be tested contains human serum albumin.
The invention also provides a fluorescence detection method of the human serum albumin, which comprises the following steps: detecting Nap-NO containing fluorescent probe by using 450-550 nm as excitation wavelength 2 The fluorescence emission intensity of the sample mixture to be tested at 620-640 nm.
Preferably, the fluorescent probe Nap-NO 2 Is 0.2X10 g working concentration -6 mol/L～5×10 -6 mol/L。
Preferably, the quantitative detection is performed by linear correlation of human serum albumin concentration and fluorescence emission intensity; more preferably, the quantitative detection detects the fluorescence emission intensity at 630nm with 500nm as excitation wavelength.
According to fluorescent probe Nap-NO 2 A linear correlation experiment for identifying human serum albumin, wherein the concentration of the human serum albumin solution is in linear correlation with the fluorescence emission intensity at 630nm under the excitation condition of 500nm,the linear range is 0-20 mug/mL, the linear equation is y=26595.15791+20537.43171x, the fitting similarity is 0.99923, and the detection limit is 0.264 mug/mL through calculation (3 sigma/k), which shows that the fluorescent probe Nap-NO 2 The method has the advantages of linear response, low detection limit and high sensitivity, and has high application value.
In the fluorescence detection method, the sample to be detected is a water body sample or a biological sample. The biological sample includes, but is not limited to, a serum sample, a plasma sample, a whole blood sample, a urine sample, a tissue fluid sample of a human.
In the drug fluorescence response experiment, according to the influence on the fluorescent probe/human serum albumin complex solution after the drug is added, the binding site of the drug on the human serum albumin can be deduced, and the fluorescent probe Nap-NO 2 Is demonstrated to enter the IB domain of human serum albumin, rather than its usual site of drug action (IIA domain and IIIA domain). The probe can avoid the interference of the common drugs, and the characteristics also indicate that the fluorescent probe Nap-NO 2 Can be used for screening or detecting related medicaments of human serum albumin IB structural domain and has potential application value. The human serum albumin IB domain related drugs include, but are not limited to, small molecule drugs, fusion protein drugs, antibody drugs, vaccines, diagnostic reagents, and the like.
The invention synthesizes a novel fluorescence enhancement type probe Nap-NO 2 The method has the advantages of excellent specific selectivity to human serum albumin, no obvious response to other substances such as bovine serum albumin, adenosine triphosphate, trypsin, various amino acids and the like, red light emission, quick response (response time is only 5 s), good stability (stable fluorescence intensity in 60 min), low detection limit (0.264 mug/mL), small interference, high sensitivity, simple detection process and the like, and has stronger practical value in the aspect of preparing detection products of human serum albumin. Further studies speculate on Nap-NO 2 Enters human serum albumin IB structural domain, so that the method has potential application value in screening or detecting related medicines.
Drawings
FIG. 1 shows a fluorescent probe Nap-NO 2 Fluorescence selectivity plot of (2), abscissaWavelength (nm), and fluorescence intensity (a.u.) on the ordinate.
FIG. 2 shows a fluorescent probe Nap-NO 2 Response time to human serum albumin, time(s) on the abscissa and fluorescence intensity (a.u.) on the ordinate.
FIG. 3 shows a fluorescent probe Nap-NO 2 The stability study of (2) is time (min) on the abscissa and fluorescence intensity (a.u.) on the ordinate.
FIG. 4 shows a fluorescent probe Nap-NO 2 Fluorescence titration diagrams for human serum albumin were identified, with the wavelength (nm) on the abscissa and the fluorescence intensity (a.u.) on the ordinate.
FIG. 5 shows a fluorescent probe Nap-NO 2 A linear correlation diagram of Human Serum Albumin (HSA) was identified, with HSA solution concentration (. Mu.g/mL) on the abscissa and fluorescence intensity (a.u.) on the ordinate.
FIG. 6 is a fluorescent probe Nap-NO 2 And a graph of the response effect of the reference fluorescent probe Nap-C, nap-OH on human serum albumin, the abscissa indicates time (min) and the ordinate indicates fluorescence intensity (a.u.).
FIG. 7 shows a fluorescent probe Nap-NO 2 A mechanism diagram for recognizing human serum albumin.
FIG. 8 shows a fluorescent probe Nap-NO 2 The fluorescence response plot for drug is plotted on the abscissa as drug concentration (μm) and on the ordinate as normalized intensity.
FIG. 9 is Nap-NO 2 Nuclear magnetic resonance hydrogen spectrum [ ] 1 H NMR)。
FIG. 10 is Nap-NO 2 Nuclear magnetic resonance carbon spectrum of [ ] 13 C NMR)。
FIG. 11 is Nap-NO 2 High resolution mass spectrometry (HR-MS).
FIG. 12 shows the hydrogen nuclear magnetic resonance spectrum of Nap-OH 1 H NMR)。
FIG. 13 shows nuclear magnetic resonance carbon spectrum of Nap-OH 13 C NMR)。
FIG. 14 is a high resolution mass spectrum of Nap-OH (HR-MS).
FIG. 15 shows the hydrogen nuclear magnetic resonance spectrum of Nap-C 1 H NMR)。
FIG. 16 shows nuclear magnetic resonance carbon spectrum of Nap-C 13 C NMR)。
FIG. 17 is a high resolution mass spectrum of Nap-C (HR-MS).
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims of the present application.
Model of detection device: fluorescence spectrometer (Edinburgh, UK FS-5), ultraviolet-visible spectrometer (Varion Cary-300, U.S.), nuclear magnetic resonance spectrometer (Bruker advanced II (400M Hz), germany), high resolution mass spectrometer (Shimadzu LCMS-IT-TOF).
Preparation and Structure confirmation of the Compounds of example 1
The synthetic route is as follows:
1. preparation of pyridinium salts
4-methylpyridine (0.50 mL,5.05 mmol) and iodoethanol (5.05 mmol), 4-methylpyridine (5.05 mmol) and iodoethane (5.05 mmol), 4-methylpyridine (5.05 mmol) and p-nitrobenzyl bromide (5.05 mmol) were respectively added to a round-bottomed flask having a capacity of 50mL, acetonitrile was used as a solvent, heated under reflux at 70℃for 5 hours under nitrogen protection, and after the completion of the reaction, the reaction solution was distilled under reduced pressure to give the corresponding pyridine salts designated Py-OH, py-C and Py-NO 2 -a。
Referring to the method, py-NO is prepared 2 -b。
2. Fluorescent probe Nap-NO 2 Is prepared from
(1) 4-bromo-1, 8-naphthalic anhydride (2.77 g,10 mmol) was added to a 50mL round bottom flask, 20mL absolute ethanol was used as solvent, and n-butylamine (2 mL,20 mmol) was added. And (3) heating and refluxing for 5 hours in an oil bath at 80 ℃ under the protection of nitrogen, and after the reaction is completed, filtering under reduced pressure and drying to obtain N-N-butyl-4-bromo-1, 8-naphthalimide (named Nap-Br).
(2) Methanol (30 mL), N-N-butyl-4-bromo-1, 8-naphthalimide (1.60 g, 4.803 mmol) and anhydrous potassium carbonate (2.00 g,14.5 mmol) were added to a 50mL round bottom flask, heated under nitrogen at 80deg.C for 8 hours under reflux, and after the reaction was completed, the mixture was filtered under reduced pressure to give N-N-butyl-4-methoxy-1, 8-naphthalimide (designated Nap-OCH) 3 )。
(3) N-N-butyl-4-methoxy-1, 8-naphthalimide (666 mg,2.35 mmol) and 55% HI solution (8 mL) were added to a 50mL round bottom flask and heated overnight at 140℃in the absence of nitrogen. After the reaction, the mixture was slowly poured into a beaker containing 400mL of ice water, and precipitation was observed by continuous stirring. Filtering under reduced pressure, and purifying by silica gel column chromatography to obtain N-N-butyl-4-hydroxy-1, 8-naphthalimide (named Nap-O).
(4) N-N-butyl-4-hydroxy-1, 8-naphthalimide (1.29 g,4.785 mmol), hexamethylenetetramine (1.43 g,9.57 mmol) and trifluoroacetic acid (10 mL) were taken and added to a 100mL round bottom flask, the mixture was heated at 85℃for 6 hours under nitrogen protection, and after the completion of the reaction, the mixture was poured into 500mL ice water, filtered under reduced pressure, and purified by silica gel column chromatography to give N-N-butyl-3-formyl-4-hydroxy-1, 8-naphthalimide (designated Nap-CHO).
(6) N-N-butyl 3-aldehyde-4-hydroxy-1, 8-naphthalimide (100 mg,0.34 mmol) and pyridine salt Py-NO were taken 2 -a (0.34 mmol) was dissolved in ethanol (10 mL), piperidine (0.1 mL) was added and heated at 80deg.C under reflux for 8 h in an oil bath under nitrogen. After the reaction is completed, the crude product is obtained by vacuum filtration, and the fluorescent probe Nap-NO is obtained by using absolute ethyl alcohol to recrystallize for 3 times 2 -a。
(7) The method of reference (6) using a pyridinium salt Py-NO 2 -b is used as raw material to prepare the fluorescent probe Nap-NO 2 -b。
3. Preparation of reference fluorescent Probe Nap-OH and Nap-C
N-N-butyl 3-aldehyde-4-hydroxy-1, 8-naphthalimide (100 mg,0.34 mmol) and pyridine salt Py-OH (0.34 mmol) were dissolved in ethanol (10 mL), piperidine (0.1 mL) was added, and the mixture was heated under reflux in an oil bath at 80℃for 8 hours under nitrogen protection. After the reaction is completed, the crude product is obtained by vacuum filtration, and the reference fluorescent probe Nap-OH is obtained by using absolute ethyl alcohol to recrystallize for 3 times.
And replacing the Py-OH with Py-C, and obtaining the reference fluorescent probe Nap-C under the same conditions.
4. Structural confirmation of the Compounds
Through nuclear magnetic resonance hydrogen spectrum 1 H NMR nuclear magnetic resonance carbon spectrum [ ] 13 C NMR) and high resolution mass spectrometry (HR-MS) for Nap-NO 2 Structural confirmation was performed for Nap-OH and Nap-C (FIGS. 9-17).
Nap-NO 2 M/z:508.1859 (calculated: 508.1867); nap-OH, m/z:417.1804 (calculated: 417.1809); nap-C, m/z:401.1854 (calculated: 401.1860).
EXAMPLE 2 fluorescent Probe Nap-NO 2 Selectivity of (2)
Formulation with DMSO 1X 10 -3 Nap-NO in mol/L 2 A solution.
A2 mg/mL Human Serum Albumin (HSA) solution was prepared with distilled water.
A solution of 2mg/mL of other substances selected from Bovine Serum Albumin (BSA), adenosine Triphosphate (ATP), heparin (Hep), protamine sulfate (PRTM), canavanine A (Con A), trypsin (Trypsin), histidine (His), aspartic acid (Asp), isoleucine (lle), phenylalanine (Phe), methionine (Met), valine (Val), serine (Ser), cysteine (Cys), alanine (Ala), acetylcysteine (NAC), leucine (Leu), threonine (Thr), proline (Pro), tyrosine (Tyr), glutamine (Gln), tryptophan (Trp) was prepared with distilled water.
2mL of water and 2. Mu.L of the Nap-NO described above were added to each cuvette 2 After adding 20. Mu.L of each of the above human serum albumin solution and other substance solution, the probe Nap-NO was examined by a fluorescence spectrometer 2 The fluorescence emission intensity at 630nm was detected under excitation conditions of 500nm, with selectivity for human serum albumin and other substances. As a result, as shown in FIG. 1, the probe Nap-NO alone 2 The solution (Blank) has weak fluorescence emission intensity at 630nm, when human serum albumin is added, the fluorescence emission intensity at 630nm is obviously enhanced, but when other substances are added, the fluorescence emission intensity of the obtained solution system is compared with that of the solution system aloneThe fluorescence emission intensity of the probe solution was not significantly changed compared to that of the probe solution.
The experimental result shows that the fluorescent probe Nap-NO 2 Has better fluorescence specificity selectivity to human serum albumin, but has no obvious response to other substances such as bovine serum albumin, adenosine triphosphate, heparin, protamine sulfate, canavanine A, trypsin, various amino acids and the like.
EXAMPLE 3 fluorescent Probe Nap-NO 2 Response time to human serum albumin
Formulation with DMSO 1X 10 -3 Nap-NO in mol/L 2 A solution.
A2 mg/mL human serum albumin solution was prepared with distilled water.
2mL of water, 2. Mu.L of Nap-NO as described above, was added to the cuvette 2 Solution and 18. Mu.L of the above human serum albumin solution, and fluorescent probe Nap-NO was examined by a fluorescence spectrometer 2 The response time to human serum albumin, the excitation wavelength was 500nm, and the fluorescence emission intensity at 630nm was detected.
The results of fig. 2 show that: nap-NO 2 Can respond to human serum albumin within 5 seconds. This demonstrates that the fluorescent probe Nap-NO 2 Can respond to human serum albumin quickly and realize quick detection. Thus, fluorescent probe Nap-NO 2 The method is suitable for preparing detection products of human serum albumin, such as reagents, test paper or kits, and has the advantages of rapid and accurate response and strong signal.
EXAMPLE 4 fluorescent Probe Nap-NO 2 Stability of (C)
Formulation with DMSO 1X 10 -3 Nap-NO in mol/L 2 A solution.
A2 mg/mL human serum albumin solution was prepared with distilled water.
2mL of water and 2. Mu.L of Nap-NO as described above were added to a cuvette 2 The solution was kept for 60min. Detecting with a fluorescence spectrometer, wherein the excitation wavelength is 500nm, the fluorescence emission intensity at 630nm is detected, and the detection interval time is 5min.
2mL of water, 2. Mu.L of Nap-NO as described above, was added to the cuvette 2 The solution and 18. Mu.L of the human serum albumin solution were kept for 60min. Nap-NO detection by fluorescence spectrometer 2 The fluorescence response to human serum albumin, the excitation wavelength is 500nm, the fluorescence emission intensity at 630nm is detected, and the detection interval time is 5min.
The detection results are shown in fig. 3: nap-NO probe within 60min 2 Are weak fluorescent emissions. Whereas probe Nap-NO 2 The fluorescence intensity emitted by the human serum albumin was recognized to remain at a high level for 60min with little change. The above results indicate that the probe Nap-NO 2 Shows good stability.
EXAMPLE 5 fluorescent Probe Nap-NO 2 Fluorescence titration graphs and linear correlation graphs for identifying human serum albumin
Preparation of Nap-NO at 1X 10-3mol/L with DMSO 2 A solution.
A2 mg/mL human serum albumin solution was prepared with distilled water.
2mL of water and 2. Mu.L of Nap-NO as described above 2 The solutions were added to 11 clean fluorescent cuvettes, and the volumes of the human serum albumin solutions described above were gradually added to each cuvette at 0. Mu.L, 2. Mu.L, 4. Mu.L, 6. Mu.L, 8. Mu.L, 10. Mu.L, 12. Mu.L, 14. Mu.L, 16. Mu.L, 18. Mu.L, and 20. Mu.L, respectively. The fluorescence emission intensity of each sample was measured on a fluorescence spectrometer with an excitation wavelength of 500 nm.
The fluorescence probe Nap-NO is obtained by taking the fluorescence emission wavelength as the abscissa and the fluorescence emission intensity as the ordinate 2 Fluorescence titration graphs (FIG. 4) identifying human serum albumin. The results of fig. 4 show: as the concentration of the human serum albumin solution in the cuvette gradually increases to 20. Mu.g/mL, the probe Nap-NO 2 The fluorescence emission intensity of (2) increases accordingly; and, at different concentrations of human serum albumin solution, the main peak of the fluorescence emission curve is located at 630 nm.
The HSA solution concentration is taken as an abscissa and the 630nm fluorescence emission intensity is taken as an ordinate to obtain a fluorescence probe Nap-NO 2 A linear correlation diagram of human serum albumin was identified (fig. 5). FIG. 5 shows that HAS solution concentration is linearly related to fluorescence emission intensity, the linear range is 0-20 μg/mL, the linear equation is y=26595.15791+20537.43171x, the fitting similarity is 0.99923, and the fluorescence probe Nap-NO is calculated (3 sigma/k) 2 To human serumThe detection limit of the protein is 0.264 mug/mL, which shows that the probe has the advantages of linear response, low detection limit and high sensitivity, and has stronger application value.
EXAMPLE 6Nap-NO 2 Response effects of Nap-OH and Nap-C on human serum Albumin
Nap-NO of 1X 10-3mol/L was prepared with DMSO, respectively 2 Nap-OH and Nap-C.
A2 mg/mL human serum albumin solution was prepared with distilled water.
Respectively taking Nap-NO 2 2. Mu.L of each of the Nap-OH and Nap-C solutions was added to a clean fluorescent cuvette, and 2mL of water and 20. Mu.L of the human serum albumin solution described above were added, respectively. Measuring fluorescence emission intensity on a fluorescence spectrometer by taking 478nm, 472nm and 500nm as excitation wavelengths to obtain Nap-NO 2 FIG. 6 shows the effect of Nap-OH and Nap-C on human serum albumin response.
FIG. 6 shows the results of Nap-NO 2 Nap-OH and Nap-C respond to human serum albumin in the red region, but Nap-NO compared with Nap-OH and Nap-C 2 The fluorescence emission intensity of the HSA detector is the weakest, so that the interference on the HSA detection is small, and the detection result is more accurate.
Example 7 mechanism discussion
The fluorescent probe Nap-NO of the invention is discussed by using a reference fluorescent probe Nap-OH and Nap-C 2 Is a detection mechanism of (a).
Nap-OH and Nap-C as controls, which have a similar to Nap-NO 2 The same donor-pi-acceptor structure. In the research of the synthesis and structure-activity relationship of dye molecular probes, different push-pull charge groups can change the structure of the probes to adjust the characteristics of the fluorescent probes, and compared with Nap-OH with electron-pushing groups-OH and Nap-C with electron-withdrawing groups-NO 2 Nap-NO of (C) 2 Having an electron conjugated structure different from Nap-OH and Nap-C can create a Twisted Intramolecular Charge Transfer (TICT) mechanism.
FIG. 7 shows a fluorescent probe Nap-NO 2 A mechanism diagram for recognizing human serum albumin. The mechanism is discussed as follows: fluorescent probe Nap-NO 2 In a freely rotating state in solutionAfter the human serum albumin is added, the rotation of the probe is inhibited, the probe enters a distorted intramolecular charge transfer state, and strong fluorescence is stimulated to be released, so that a solution system releases a fluorescence signal, the fluorescence is obviously enhanced, and the detection of the human serum albumin is realized.
Example 8 fluorescent Probe Nap-NO 2 Fluorescent response to drugs
Formulation with DMSO 1X 10 -3 Nap-NO in mol/L 2 A solution.
An aqueous solution of 2mg/mL Human Serum Albumin (HSA) was prepared with distilled water.
Preparation of 1X 10 with distilled water -4 A mol/L heme-Hemin (binding site to HSA is located in IB domain) solution.
Preparation of 1X 10 with distilled water -4 The Warfarin (binding site to HSA is located in the IIA domain) solution in mol/L.
Preparation of 1X 10 with distilled water -4 mol/L Ibuprofen Ibuprrofen (binding site to HSA located in IIIA domain) solution.
2mL of water and 2. Mu.L of Nap-NO as described above 2 The solution was added to each clean fluorescent cuvette and 20. Mu.L of the above human serum albumin solution was added to form Nap-NO 2 HSA complex solution. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. Mu.L of the above heme solution, warfarin solution and ibuprofen solution are added to Nap-NO, respectively 2 HSA complex solution. Under the condition of excitation wavelength of 500nm, measuring fluorescence emission intensity at 630nm by using a fluorescence spectrometer, and obtaining a fluorescence probe Nap-NO by taking the concentration of the drug solution as an abscissa and the normalized intensity as an ordinate 2 Fluorescence response to drug (fig. 8).
From FIG. 8, it is observed that only heme can cause Nap-NO 2 The fluorescence of the HSA complex solution was significantly changed, whereas Nap-NO was obtained after warfarin and ibuprofen addition 2 The fluorescence change of the/HSA complex solution was not evident, confirming Nap-NO 2 The IB domain of human serum albumin is entered, rather than the very common drug action sites (IIA domain and IIIA domain). This special featureThe nature shows that the fluorescent probe Nap-NO 2 Can avoid the interference of common drugs and also shows that the fluorescent probe Nap-NO 2 Can be used for screening and detecting medicaments related to human serum albumin IB structural domain and has potential value.
Examples 2 to 8 above are in Nap-NO 2 As an example, a fluorescent probe Nap-NO was performed 2 Is a related test of (a). When the fluorescent probe Nap-NO with other coordination anions is adopted 2 When the tests as in examples 2-8 were performed, the test results were compared with Nap-NO 2 -a is consistent with the test results.
Claims (10)
1. A fluorescent probe is characterized by having a structure represented by the formula (I),
wherein X is halogen selected from F, cl, br or I.
2. The method for producing a fluorescent probe according to claim 1, wherein the fluorescent probe is obtained by reacting a compound represented by the formula (II) with a compound represented by the formula (III),
wherein X is halogen selected from F, cl, br or I.
3. The process according to claim 2, wherein the molar ratio of the compound of formula (II) to the compound of formula (III) is from 1:1 to 1:2, and the reaction is carried out under the protection of an inert gas in the presence of pyridine.
4. The use of the fluorescent probe according to claim 1 for detecting human serum albumin or for preparing a detection product of human serum albumin.
5. A reagent, test paper or kit for detecting human serum albumin, which comprises the fluorescent probe according to claim 1.
6. A visual fluorescence detection method comprising the steps of: the fluorescent probe of claim 1 is fully contacted with a sample to be tested, and under the irradiation of visible light, if obvious red fluorescent emission is observed, the sample to be tested is indicated to contain human serum albumin.
7. A fluorescence detection method of human serum albumin, which is characterized by comprising the following steps: detecting the mixture of the sample to be detected containing the fluorescent probe according to claim 1 by using 450-550 nm as excitation wavelength
Fluorescence emission intensity at 620-640 nm.
8. The method for detecting human serum albumin according to claim 7, wherein the quantitative detection is performed by linear correlation of the concentration of human serum albumin and the fluorescence emission intensity.
9. The method according to any one of claims 6 to 8, wherein the sample to be measured is a water sample or a biological sample.
10. Use of the fluorescent probe according to claim 1 for screening or detecting human serum albumin IB domain related drugs.
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