CN116425738A - Use of alpha-cyano phenyleneethylene derivative in detecting human serum albumin - Google Patents
Use of alpha-cyano phenyleneethylene derivative in detecting human serum albumin Download PDFInfo
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- CN116425738A CN116425738A CN202310687083.5A CN202310687083A CN116425738A CN 116425738 A CN116425738 A CN 116425738A CN 202310687083 A CN202310687083 A CN 202310687083A CN 116425738 A CN116425738 A CN 116425738A
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- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 77
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 77
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- 238000001514 detection method Methods 0.000 claims abstract description 31
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- 230000000007 visual effect Effects 0.000 claims abstract description 17
- RLFXJQPKMZNLMP-UHFFFAOYSA-N 2-phenylprop-2-enenitrile Chemical class N#CC(=C)C1=CC=CC=C1 RLFXJQPKMZNLMP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 125000000217 alkyl group Chemical group 0.000 claims abstract description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical group C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims description 39
- 150000001875 compounds Chemical class 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 24
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 22
- 239000011541 reaction mixture Substances 0.000 claims description 21
- 239000007864 aqueous solution Substances 0.000 claims description 19
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 18
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- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical group [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 abstract description 3
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- VEFLKXRACNJHOV-UHFFFAOYSA-N 1,3-dibromopropane Chemical compound BrCCCBr VEFLKXRACNJHOV-UHFFFAOYSA-N 0.000 description 1
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- OMIHGPLIXGGMJB-UHFFFAOYSA-N 7-oxabicyclo[4.1.0]hepta-1,3,5-triene Chemical compound C1=CC=C2OC2=C1 OMIHGPLIXGGMJB-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
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- 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
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- 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"
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- 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
- G01N21/6447—Fluorescence; Phosphorescence by visual observation
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- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides an application of an alpha-cyano phenylenevinylene derivative in detecting human serum albumin. The structural formula of the alpha-cyano phenylenevinylene derivative is as follows:
wherein R is 1 Selected from C 1‑4 Alkyl, R 2 is-O-C 3‑12 alkyl-Y, X is selected from F, cl, br, I or CN, Y is selected from pyridinium ion or ammonium ion. The alpha-cyano phenylethene derivative can be self-assembled to form a ratio fluorescent probe, and the probe has the characteristics of double fluorescence emission, high stability and the like, and can realize high sensitivity to Human Serum Albumin (HSA)Sensitivity and high selectivity detection, in particular for visual detection of HSA.
Description
Technical Field
The invention particularly relates to an alpha-cyano phenylethene derivative, a preparation method and application thereof, and comprises the application of the alpha-cyano phenylethene derivative in preparing a ratio fluorescent probe and performing on-site visual detection on Human Serum Albumin (HSA), belonging to the technical field of biological detection.
Background
Human Serum Albumin (HSA) is a biomarker closely related to human related diseases. It is the most abundant protein in blood plasma and has important function for maintaining normal physiological functions of human body. The normal concentration range of HSA in plasma is 35-55 mg/mL, and if the concentration is too low, hypoalbuminemia may be caused, reflecting malnutrition of human body, or impaired liver or kidney function, etc. The normal concentration of HSA in urine should not exceed 30 mg/L, and if the concentration is too high, it is microalbuminuria, indicating that patients with diabetes and hypertension are at risk of cardiovascular disease and kidney disease.
Among the methods currently in common clinical use for detecting HSA are the colorimetric bromocresol green (BCG) method and the enzyme-linked immunosorbent assay (ELISA). Both of these methods can give reliable results for the detection of HSA. They also have a common problem: from pretreatment of the sample to testing to data analysis, the whole procedure takes a long time (e.g., ELISA requires about 3 h), which is disadvantageous for rapid detection in the field.
Disclosure of Invention
The invention mainly aims to provide an application of an alpha-cyano phenylenevinylene derivative in detecting human serum albumin so as to overcome the defects in the prior art.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention comprises the following steps:
in one aspect, the invention provides an alpha-cyanophenyleneethylene derivative, the structure of which is shown in formula I,
wherein R is 1 Independently selected from C 1-4 Alkyl, R 2 is-O-C 3-12 alkyl-Y, X is selected from F, cl, br, I or CN, Y is selected from pyridinium ion or ammonium ion.
In one embodiment, R 1 Selected from methyl, ethyl or n-propyl. Two R 1 May be the same or different.
In one embodiment, the ammonium ions may include, but are not limited to, trimethylammonium ions, triethylammonium ions, and the like.
Another aspect of the present invention provides a method for preparing the α -cyanophenylenevinylene derivative, comprising:
by reacting a compound of formula II with Br (CH) 2 ) n Br reaction to obtain a compound shown in a formula III;
reacting a compound shown in a formula III with a compound shown in a formula IV to obtain a compound shown in a formula V;
reacting a compound represented by formula V with an organic base to obtain the alpha-cyanophenylethene derivative;
wherein R is 1 、R 2 X is the same as the above, and is not repeated here, n is any integer from 3 to 12.
In one embodiment, the method for preparing the alpha-cyanophenylenevinylene derivative comprises the steps of:
s1, in a protective atmosphere, a compound shown in a formula II and sodium hydride are mixed according to a ratio of 2.0-10.0: 1.0 to 2.5 mass ratio is dissolved in an ultra-dry polar organic solvent (such as ultra-dry tetrahydrofuran) to obtain a mixed solution 1;
s2, under the condition of stirring at normal temperature, br (CH) 2 ) n Br was added to the mixed solution 1, br (CH) 2 ) n The mass ratio of Br to the compound shown in the formula II is 1.0-2.5:1.0-1.5, and a mixed solution 2 is obtained;
s3, carrying out reflux reaction on the mixed solution 2 at the temperature of 70-105 ℃, and separating the compound shown in the formula III from the obtained reaction mixture after the reaction is finished;
s4, dissolving the compound shown in the formula III and the compound shown in the formula IV in a polar organic solvent, and adding piperidine to enable the mass ratio of the compound shown in the formula III to the compound shown in the formula IV to be 3.0-10.0: 1.0 to 6.0:1.0 to 5.0, stirring and reacting at room temperature, and separating the compound shown in the formula V from the obtained reaction mixture after the reaction is finished;
s5, carrying out reflux reaction on the compound shown in the formula V and excessive organic base at the temperature of 50-150 ℃, and separating from the obtained reaction mixture after the reaction is finished to obtain the alpha-cyano phenyleneethylene derivative.
More preferably, the mass ratio of the compound shown in the formula II in the step S1 to sodium hydride is 5.0-7.0: 1.4 to 2.2. Wherein the water content of the ultra-dry tetrahydrofuran is below 100ppm, preferably below 30 ppm.
Preferably, br (CH 2 ) n The mass ratio of Br to the compound of formula II is greater than 1:1 but less than 2.5:1.
Preferably, in step S3, the mixed solution 2 is subjected to the reflux reaction at a temperature of 75 to 85 ℃.
More preferably, the mass ratio of the compound shown in the formula III, the compound shown in the formula IV and the piperidine in the step S4 is 5.0-6.0: 3.8 to 4.6:1.7 to 3.5.
More preferably, in step S5, the compound of formula V and an excess amount of the organic base are subjected to the reflux reaction at a temperature of 100 to 110 ℃.
In one embodiment, the organic base includes, but is not limited to, pyridine, trimethylamine, triethylamine, and the like.
In one embodiment, the polar organic solvent includes, but is not limited to, methanol or ethanol, and the like.
The protective atmosphere in the present invention may be a nitrogen atmosphere, an inert atmosphere (such as an argon atmosphere), or a mixed atmosphere thereof.
In one embodiment, step S2 may specifically include: br (CH) 2 ) n Br was dissolved in ultra-dry THF and added dropwise to the mixed solution 1 under stirring at room temperature to thereby obtain theSolution 2 was mixed.
In one embodiment, step S3 may specifically include: after the reaction is finished, the obtained reaction mixture is extracted by a dichloromethane/water system, then dried and distilled under reduced pressure to obtain a crude product, and then the crude product is subjected to silica gel column chromatography, petroleum ether and ethyl acetate are used as leaching agents, and the compound shown in a formula III is obtained through separation.
In one embodiment, step S4 may specifically include: after the reaction was completed, the obtained reaction mixture was filtered and the obtained solid was washed with iced ethanol, thereby separating the compound represented by formula V.
In one embodiment, step S5 may specifically include: dissolving a compound shown in a formula V in an excessive amount of organic base to obtain a mixed solution 3, carrying out reflux reaction on the mixed solution 3 at a temperature of 100-110 ℃ for 24-30 h, carrying out reduced pressure distillation on the obtained reaction mixture after the reaction is finished to remove residual organic base, obtaining a crude product, dissolving the crude product by using a small amount of organic solvent (such as dichloromethane), then dropwise adding the crude product into a large amount of diethyl ether to precipitate a solid product, and separating to obtain the target product, namely the alpha-cyano phenyleneethylene derivative.
The light-emitting wavelength of the alpha-cyano phenylethene derivative is between 490 and 600 and nm, which can well overcome the following defects of the prior phenylethene derivative, such as: the luminescence wavelength is short, and the fluorescence signal is easy to mix with the autofluorescence of a biological system; the luminous intensity is lower, and the corresponding detection limit is lower.
Meanwhile, the alpha-cyano phenyleneethylene derivative is an amphiphilic molecule, wherein R 2 The alpha-cyano phenyleneethylene derivative is a hydrophilic group, benzothiazole and the like are hydrophobic structural units, and can self-assemble in water to form a colloid-beam-shaped aggregate by means of hydrophobic interaction and intermolecular pi-pi action, and the aggregate can generate a new fluorescence peak between the longer wavelength (550-750 nm). Furthermore, the aggregates interact specifically with Human Serum Albumin (HSA). Specifically, the aggregate is combined with human serum albumin in an aqueous phase system(HSA) is disassembled when contacted, so that the intensity of fluorescence (peak wavelength about 630 to 670 nm) generated by the aggregate is reduced, and the intensity of fluorescence (peak wavelength about 510 to 540 nm) of the alpha-cyanophenylethene derivative monomer is increased. By detecting the transition of these two fluorescent signals, detection of HSA can be achieved.
In particular, for solutions comprising HSA and α -cyanophenylenevinylene derivatives, as the HSA concentration increases, the fluorescence of the solution appears to change from red to orange to yellow, and ultimately to green in visual color. The concentration range of HSA can be judged according to the fluorescence color of the solution, so that in-situ visual detection of the HSA can be rapidly realized. Therefore, a novel HSA ratio fluorescent probe can be designed by using the alpha-cyanophenylenevinylene derivative.
The invention also provides application of the alpha-cyano phenylethene derivative in preparing a fluorescent probe, a human serum albumin detection reagent or a human serum albumin visual detection kit.
Due to the characteristics of the alpha-cyano phenylethene derivative, the fluorescent probe prepared by using the alpha-cyano phenylethene derivative has the advantages of double fluorescence emission, high stability and the like.
The present invention also provides a ratiometric fluorescent probe comprising the alpha-cyanophenylethene derivative and/or an aggregate of the alpha-cyanophenylethene derivative. The probe can be used for detecting human serum albumin and has the advantages of high selectivity, high sensitivity (about 8.3 nmol/L) and the like.
The invention also provides a reagent for detecting human serum albumin in body fluid, which comprises the alpha-cyano phenylenevinylene derivative or the ratiometric fluorescent probe.
The invention also provides a body fluid human serum albumin detection kit which comprises the alpha-cyano phenylethene derivative or the ratio fluorescent probe.
Wherein the body fluid includes, but is not limited to, plasma or urine, and the like.
By using the reagent or the kit for detecting the human serum albumin in the body fluid, the rapid and accurate detection, particularly in-situ visual detection, of the HSA in samples such as blood plasma, urine and the like can be realized, and the risk of related diseases can be accurately estimated.
The invention also provides a human serum albumin detection method, which comprises the following steps:
providing an aqueous solution of fluorescent probes comprising the ratio of fluorescent probes;
and mixing the sample to be detected with the fluorescent probe aqueous solution, and detecting the human serum albumin in the sample to be detected by observing the intensity change condition and/or the fluorescent color change condition of the fluorescent probe aqueous solution in different wavelengths before and after mixing with the sample to be detected. The detection may be qualitative or quantitative.
For example, a series of HSA standard sample solutions and fluorescent probe aqueous solutions of different concentrations may be prepared, and then the different HSA standard sample solutions may be mixed with the different fluorescent probe aqueous solutions by a method such as orthogonal test design, and the change in fluorescence intensity of the two wavelengths before and after mixing may be detected. Thus, the range of HSA concentration that can be detected by the fluorescent probe can be determined, and a standard relationship curve of HSA concentration versus the change in fluorescence intensity at both wavelengths can be established. When the sample to be detected is detected, the HSA content in the sample to be detected can be quantitatively detected according to the detected two wavelength fluorescence intensity changes and the standard relation curve.
Wherein the sample to be tested may be selected from body fluids, such as blood, urine, other biological tissue fluids, or samples of non-natural origin.
The invention also provides a visual detection method of the human serum albumin, which is non-diagnosis and treatment purpose and comprises the following steps:
providing an aqueous solution of fluorescent probes comprising the ratio of fluorescent probes;
and mixing the sample to be detected with the fluorescent probe aqueous solution, and detecting the human serum albumin in the sample to be detected at least by observing the fluorescent color change of the fluorescent probe aqueous solution before and after mixing with the sample to be detected.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The alpha-cyano phenylethene derivative has the advantages of excellent light stability, pH stability, good water solubility, longer luminous wavelength, higher luminous intensity and the like, can perform self-assembly in aqueous solution, and shows the luminescence of two forms of monomers and aggregates, namely, can realize double fluorescence emission.
(2) The alpha-cyano phenylethene derivative and the aggregate thereof can perform rapid ratio fluorescence response on HSA, have good selectivity and excellent anti-interference capability, can be used as a ratio fluorescence probe of HSA, realize in-situ visualization rapid detection on the HSA on site, and have the advantages of rapid response speed, high sensitivity, good accuracy, simplicity and convenience in operation and the like.
Drawings
FIG. 1 is a block diagram of an alpha-cyanophenylethene derivative (BCPX) of example 1 1 H NMR spectrum;
FIG. 2 is a graph of fluorescence emission spectra of BCPX in example 1 at different concentrations;
FIG. 3 is the pH stability test results of BCPX in example 1;
fig. 4 is a photo stability test result of BCPX in example 1;
fig. 5 is a test result of the selective recognition ability of BCPX to HSA in example 1;
fig. 6 is a test result of the anti-interference ability of BCPX of example 1 to different interfering substances;
FIG. 7 is a graph showing the ratio fluorescence response spectrum of BCPX in example 1 for different concentrations of HSA in aqueous solution;
FIG. 8 is a photograph of fluorescence visualization of BCPX in example 1 for different concentrations of HSA in aqueous solution;
fig. 9 is an in situ visual sensor photograph of BCPX in example 1 for different concentrations of HSA in serum;
fig. 10 is an in situ visual sensor photograph of BCPX in example 1 for different concentrations of HSA in urine.
Detailed Description
The following detailed description of the present invention is provided in connection with specific embodiments so that those skilled in the art may better understand and practice the present invention. Specific functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed embodiment. In addition, unless otherwise specified, the raw materials, detection reagents, reaction equipment and detection equipment used in the following examples are all commercially available in general, and the detection methods and the like used are also conventional in the art.
The α -cyanophenylenevinylene derivative (BCPX) in the following examples can be prepared by the following synthetic route:
wherein R is 1 Identical or different and may be selected from C 1-4 Alkyl, preferably from methyl, ethyl or n-propyl; r is R 2 is-O-C 3-12 The alkyl-Y, X is selected from F, cl, br, I or CN, Y can be selected from pyridinium ion or ammonium ion, such as trimethyl ammonium ion, triethyl ammonium ion and the like, and n is any integer from 3 to 12.
Example 1
The structural formula of an α -cyanophenylenevinylene derivative (BCPX) of this embodiment is as follows:
the synthesis method of the BCPX comprises the following specific steps:
(1) 500 mg of 4- (diethylamino) salicylaldehyde and 140 mg of NaH (60%) were dissolved in 80 mL ultra-dry THF under nitrogen protection to give a mixed solution 1, which was stirred at room temperature. Then, 1.2 g of 1, 12-dibromododecane was dissolved in 5 mL ultra-dry THF, and the solution was added dropwise to the above mixed solution 1 with a clean syringe, followed by stirring at room temperature to obtain a mixed solution 2. The mixed solution 2 was refluxed at 80℃for 3 days. After completion of the reaction, the reaction mixture was concentrated by distillation under reduced pressure. Then extracting 3 times by using dichloromethane/water (volume ratio is about 1:1), collecting an organic phase, adding a proper amount of anhydrous magnesium sulfate for drying, decompressing and distilling to obtain a crude product, and performing silica gel column chromatography (petroleum ether and ethyl acetate are used as leaching agents) on the crude product to obtain an intermediate product 1 shown in a formula III.
(2) 500. 500 mg intermediate 1, 380 mg of 2-bromobenzothiazole-2-acetonitrile was dissolved in 30 mL ethanol, 1.5. 1.5 mL piperidine was added, the reaction mixture was stirred overnight at room temperature, the obtained reaction mixture was filtered, the crude product obtained was washed with iced ethanol, and then the crude product was recrystallized twice from ethanol to obtain intermediate 2 represented by formula V.
(3) Dissolving 600 mg intermediate 2 in 30 mL pyridine to obtain a mixed solution 3, refluxing the mixed solution 3 at 100deg.C overnight, distilling under reduced pressure to remove pyridine, dissolving the crude product with small amount of dichloromethane, adding dropwise into large amount of diethyl ether for settling, centrifuging for 3 times to obtain target product BCPX, and separating by centrifugation to obtain the final product BCPX 1 The H NMR spectrum is shown in FIG. 1. 1 H NMR (400 MHz, DMSO-d 6) delta 9.07 pyridine a (d, j=5.6 Hz, 2H), 8.60 pyridine g (t, j=7.7 Hz, 1H), 8.44 α -cyanovinyl (s, 1H), 8.24 benzothiazole (d, j=9.2 Hz, 1H), 8.16 pyridine b (t, j=6.9 Hz, 2H), 8.04 benzothiazole (d, j=7.9 Hz, 1H), 7.92 diethylaminobenzene (d, j=8.0 Hz, 1H), 7.50 benzothiazole (t, j=7.5 Hz, 1H), 7.40 benzothiazole (t, j=7.5 Hz, 1H), 6.54 diethylaminobenzene (d, j=9.2 Hz, 1H), 6.23 diethylaminobenzene (s, 1H), 4.55 pyridine a-CH 2 (t, j=7.4 Hz, 2H), 4.14 phenylene ether a-CH 2 (t, j=5.4 Hz, 2H), 3.50 amino group a-CH 2 (q, j=7.0 Hz, 4H), 1.91-1.73 pyridine b-CH 2 (m, 4H), phenyl ether b-position-CH of 1.58-1.44 2 (m, 2H), 1.38 phenyl ether g position-CH 2 (s, 2H), 1.32-1.03 remaining alkyl-base region-CH 2 (m, 20H)。
The BCPX synthesized in the embodiment is dissolved in ultrapure water to form BCPX water solutions with different concentrations, and by observing the fluorescence spectrum, the BCPX can be self-assembled in water along with the increase of the concentration, so that the red shift of the fluorescence spectrum peak appears. Please refer to fig. 2, which shows the normalized fluorescence emission spectra of BCPX above (50.0 μmol/L) and below (10.0 μmol/L) CMC.
The BCPX synthesized in this example was dissolved in ultrapure water to form an aqueous BCPX solution having a concentration of about 50.0. Mu. Mol/L, and the pH of the solution was adjusted by adding hydrochloric acid, caustic soda or the like to test the stability of BCPX at various pH values, and the results are shown in FIG. 3. Meanwhile, an aqueous solution of BCPX having a concentration of about 50.0 μmol/L was left under visible light for various times to test the photostability of BCPX, and the results are shown in fig. 4.
Very surprisingly, the BCPX and its aggregates synthesized in this example have a rapid ratiometric fluorescence response to HSA, and to test whether this effect is specific, an aqueous solution of HSA was prepared at a concentration of about 50.0. Mu. Mol/L, along with a series of aqueous solutions of interfering substances, each also at a concentration of about 50.0. Mu. Mol/L. These interfering substances are, respectively, glutathione (L-serine), L-lysine (L-serine), DL-serine (DL-serine), L-glutamic acid (L-glutamic acid), glucose (glucose), ascorbic acid (ascorbic acid), lysozyme (lysozyme), lipase (lipase), gamma-Globulin (gamma-Globulin), sodium ion (Na) + ) Potassium ion (K) + ) Calcium ion (Ca) 2+ ) Carbonate (CO) 3 2− ) Sulfate radical (SO 4 2− ) Etc. And then adding BCPX to the HSA aqueous solution and the interfering substance aqueous solution respectively until the concentration is about 50.0 mu mol/L, and then testing fluorescence spectra of the BCPX respectively, wherein the results are shown in figure 5, and prove that the BCPX has good selectivity to the HSA. In addition, different interference substances are respectively added to the HSA aqueous solution, BCPX is respectively added, and then fluorescence spectra of the BCPX are respectively tested, and the results are shown in figure 6, so that the BCPX has good anti-interference capability on different interference substances when being used for detecting the HSA.
The BCPX synthesized in this example was dissolved in ultrapure water to obtain an aqueous BCPX solution having a concentration of about 400.0 mmol/L. A series of HSA standard solutions of different concentrations were additionally prepared, having concentrations of 0g/L, 0.2g/L, 0.3g/L, 0.7g/L, 1.0g/L, 1.7g/L, 3.3g/L, 5.0g/L, 6.7g/L, 13.3g/L, 26.6g/L, 53.2 g/g/L, respectively. The series of HSA standard solutions were added to the aqueous BCPX solutions, respectively, and ultrapure water was added to a final concentration of 50.0 mmol/L BCPX in each of the resulting mixed solutions, and after 2 minutes of reaction, fluorescence emission spectroscopy was performed, and fluorescence spectra and fluorescence photographs thereof were recorded, as shown in FIGS. 7 to 8. It can be seen that with BCPX of the present embodiment as a ratio fluorescent probe, visual detection of HSA can be achieved.
Then taking human serum as a matrix, adding BCPX and HSA of the embodiment, controlling the final concentration of the BCPX to be 50.0 mmol/L, and respectively controlling the final concentration of the HSA to be 0g/L, 0.2g/L, 0.3g/L, 0.7g/L, 1.0g/L, 1.7g/L, 3.3g/L, 5.0g/L, 6.7g/L, 13.3g/L, 26.6g/L and 53.2 g/L, and observing the fluorescence photographs of the human serum samples added with different concentrations of HSA (as shown in figure 9), wherein the BCPX of the embodiment can be used as a ratio fluorescent probe to realize in-situ visual detection of the HSA in the human serum.
In addition, urine is taken as a substrate, BCPX and HSA of the embodiment are added, the final concentration of the BCPX is controlled to be 50.0 mmol/L, the final concentration of the HSA is respectively 0g/L, 0.2g/L, 0.3g/L, 0.7g/L, 1.0g/L, 1.7g/L, 3.3g/L, 5.0g/L and 6.7g/L, and fluorescence photographs (shown in figure 10) of urine samples added with different concentrations of HSA are observed, so that the BCPX of the embodiment can be used as a ratio fluorescent probe to realize in-situ visual detection of the HSA in the urine.
As can be seen from fig. 7 to 10, BCPX of this example showed fluorescence change related to HSA concentration, i.e., a clear color gradient change from red to orange to yellow and finally to green, as a ratio fluorescence probe, both in water and in serum and urine, showing its excellent selectivity, interference resistance and resolution for HSA.
Example 2
The structural formula of an α -cyanophenylenevinylene derivative (BCPX) of this embodiment is as follows:
the synthesis method of the BCPX comprises the following specific steps:
(1) 500 mg of 4- (diethylamino) salicylaldehyde and 140 mg of 60% NaH were dissolved in 80 ml of ultra-dry THF under nitrogen protection to give a mixed solution 1, which was stirred at room temperature. Then, 890 mg of 1, 5-dibromopentane was dissolved in 5 ml of ultra-dry THF, and the solution was added dropwise to the above mixed solution 1 using a clean syringe, followed by stirring at room temperature to obtain a mixed solution 2. The mixed solution 2 was refluxed at 80℃for 2 days. After completion of the reaction, the reaction mixture was concentrated by distillation under reduced pressure. Then extracting 3 times by using dichloromethane/water (volume ratio is about 1:5), collecting an organic phase, adding a proper amount of anhydrous magnesium sulfate for drying, finally performing reduced pressure distillation to obtain a crude product, and performing silica gel column chromatography (petroleum ether and ethyl acetate are used as leaching agents) on the crude product to obtain an intermediate product 1 shown in a formula III.
(2) 600 mg of intermediate 1 and 460 mg of 2-iodobenzothiazole-2-acetonitrile were dissolved in 40 ml of ethanol, 1.8 ml of piperidine was added, and the obtained reaction mixture was stirred overnight at room temperature, filtered, and the obtained crude product was washed with iced ethanol, and then recrystallized twice from ethanol to obtain intermediate 2 represented by formula V.
(3) 500 mg of intermediate 2 is dissolved in 30 ml of triethylamine to obtain a mixed solution 3, the mixed solution 3 is refluxed overnight at 100 ℃, the triethylamine is removed by reduced pressure distillation from the obtained reaction mixture, the crude product is dissolved by a small amount of methylene dichloride and is added dropwise into a large amount of diethyl ether for sedimentation, the process is carried out for 3 times, and the target product BCPX is obtained after centrifugal separation.
The test on each performance of the BCPX synthesized in this example by referring to the method in example 1 shows that the BCPX in this example can also be used as a ratio fluorescent probe to realize in-situ visual detection of HSA in water, serum and urine, and also shows excellent selectivity, anti-interference performance and resolution for HSA.
Example 3
The structure of an alpha-cyanophenylenevinylene derivative (BCPX) of the embodiment is shown in the formula I, wherein R 1 Methyl, R 2 is-O-dodecyl-pyridinium ion, x=cl. The synthesis method of the BCPX comprises the following specific steps:
(1) 700 mg of 4- (dimethylamino) salicylaldehyde and 220 mg of NaH (60%) are dissolved under nitrogen in 90 ml of ultra-dry THF in a round bottom flask to give a mixed solution 1, which is stirred at ambient temperature. Then, 1200 mg of 1, 12-dibromododecane was dissolved in 5 ml of ultra-dry THF, and then added dropwise to the above mixed solution 1 by a clean syringe, followed by stirring at room temperature, to obtain a mixed solution 2. The mixed solution 2 was refluxed at 80℃for 2 days. After completion of the reaction, the reaction mixture was concentrated by distillation under reduced pressure. After 3 times of extraction with dichloromethane/water, the organic phase is collected and dried with the addition of an appropriate amount of anhydrous magnesium sulfate, and the crude product is obtained by distillation under reduced pressure. The crude product was subjected to silica gel column chromatography (using petroleum ether and ethyl acetate as eluent) to give intermediate 1.
(2) 500 mg of intermediate 1, 450 mg of 2-chlorobenzothiazole-2-acetonitrile was dissolved in 30 ml of ethanol, 1.5 ml of piperidine was added thereto, and the mixture was stirred at room temperature overnight. The obtained reaction mixture was filtered, and the obtained crude product was washed with glacial ethanol, and then recrystallized twice from ethanol to obtain intermediate 2 represented by formula V.
(3) 600 mg of intermediate 2 is dissolved in 30 ml of pyridine to obtain a mixed solution 3, the mixed solution 3 is refluxed overnight at 100 ℃, the obtained reaction mixture is distilled under reduced pressure to remove pyridine, the crude product is dissolved by a small amount of dichloromethane and is dripped into a large amount of diethyl ether for sedimentation, the process is carried out for 3 times, and the target product BCPX is obtained after centrifugal separation.
The test on each performance of the BCPX synthesized in this example by referring to the method in example 1 shows that the BCPX in this example can also be used as a ratio fluorescent probe to realize in-situ visual detection of HSA in water, serum and urine, and also shows excellent selectivity, anti-interference performance and resolution for HSA.
Example 4
The structure of an alpha-cyanophenylenevinylene derivative (BCPX) of the embodiment is shown in the formula I, wherein R 1 Methyl, R 2 is-O-propyl-trimethylammonium ion, X=Br. The synthesis method of the BCPX comprises the following specific steps:
(1) 700 mg of 4- (dimethylamino) salicylaldehyde and 220 mg of 60% NaH were dissolved in 120 ml of ultra-dry THF under nitrogen protection to give a mixed solution 1, which was stirred at room temperature. Then, 1.3 g of 1, 3-dibromopropane was dissolved in 8 ml of ultra-dry THF, and this was added dropwise to the above mixed solution 1 with a clean syringe, followed by stirring at room temperature to obtain a mixed solution 2. The mixed solution 2 was refluxed at 85 ℃ for 2 days. After completion of the reaction, the reaction mixture was concentrated by distillation under reduced pressure. Then extracting 3 times by using dichloromethane/water (volume ratio is about 1:3), collecting an organic phase, adding a proper amount of anhydrous magnesium sulfate for drying, finally performing reduced pressure distillation to obtain a crude product, and performing silica gel column chromatography (petroleum ether and ethyl acetate are used as leaching agents) on the crude product to obtain an intermediate product 1 shown in a formula III.
(2) 600 mg of intermediate 1 and 460 mg of 2-bromobenzothiazole-2-acetonitrile were dissolved in 40 ml of ethanol, 1.8 ml of piperidine was added, and the obtained reaction mixture was stirred overnight at room temperature, filtered, and the obtained crude product was washed with iced ethanol, and then recrystallized twice from ethanol to obtain intermediate 2 represented by formula V.
(3) 500 mg of intermediate 2 is dissolved in 40 ml of trimethylamine to obtain a mixed solution 3, the mixed solution 3 is refluxed for 30 hours at the temperature of 110 ℃, the obtained reaction mixture is distilled under reduced pressure to remove the trimethylamine, the crude product is dissolved by a small amount of methylene dichloride and is added dropwise into a large amount of diethyl ether for sedimentation, the process is carried out for 3 times, and the target product BCPX is obtained after centrifugal separation.
The test on each performance of the BCPX synthesized in this example by referring to the method in example 1 shows that the BCPX in this example can also be used as a ratio fluorescent probe to realize in-situ visual detection of HSA in water, serum and urine, and also shows excellent selectivity, anti-interference performance and resolution for HSA.
Example 5
An α -cyanophenylenevinylene derivative (BCPX) of this example is the same as example 3, and the synthesis method is also substantially the same as example 3, except that:
the mass ratio of 4- (dimethylamino) salicylaldehyde to NaH in step (1) is about 5:1. The mixed solution 2 was refluxed at 75℃for about 3 days.
The mass ratio of the intermediate 1, 2-chlorobenzothiazole-2-acetonitrile and piperidine in the step (2) is about 6:3.8:1.7.
the mixed solution 3 in the step (3) is refluxed at 110℃for about 24 hours.
The yield of BCPX in this example was about 3% higher than in example 3.
Example 6
An α -cyanophenylenevinylene derivative (BCPX) of this example is the same as example 3, and the synthesis method is also substantially the same as example 3, except that:
the mass ratio of 4- (dimethylamino) salicylaldehyde to NaH in step (1) is about 2.3:1.
The mass ratio of intermediate 1, 2-chlorobenzothiazole-2-acetonitrile and piperidine in step (2) is about 1.4:1.2:1.
the yield of BCPX in this example was similar to example 3.
The various aspects, embodiments, features and examples of the invention are to be considered in all respects as illustrative and not intended to limit the invention, the scope of which is defined solely by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
In addition, the inventors have conducted experiments with other materials, process operations, and process conditions as described in this specification with reference to the foregoing examples, and have all obtained desirable results.
While the invention has been described with reference to an illustrative embodiment, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (10)
1. An application of an alpha-cyano phenylenevinylene derivative in preparing a human serum albumin detection reagent, which is characterized in that: the structure of the alpha-cyano phenylenevinylene derivative is shown in a formula I,
wherein R is 1 Independently selected from C 1-4 Alkyl, R 2 is-O-C 3-12 alkyl-Y, X is selected from F, cl, br, I or CN, Y is selected from pyridinium ion, trimethylammonium ion or triethylammonium ion.
2. Use according to claim 1, characterized in that: r is R 1 Selected from methyl, ethyl or n-propyl.
3. The use according to any one of claims 1-2, wherein the process for the preparation of the α -cyanophenyleneethylene derivative comprises:
by reacting a compound of formula II with Br (CH) 2 ) n Br reaction to obtain a compound shown in a formula III;
reacting a compound shown in a formula III with a compound shown in a formula IV to obtain a compound shown in a formula V;
reacting a compound shown in a formula V with an organic base to obtain the alpha-cyano phenylenevinylene derivative, wherein the organic base is selected from pyridine, trimethylamine or triethylamine;
wherein n is any integer from 3 to 12.
4. The use according to claim 3, characterized in that the process for the preparation of the α -cyanophenylenevinylene derivatives comprises in particular:
s1, in a protective atmosphere, a compound shown in a formula II and sodium hydride are mixed according to a ratio of 2.0-10.0: 1.0 to 2.5 mass ratio is dissolved in ultra-dry tetrahydrofuran to obtain a mixed solution 1;
s2, under the condition of stirring at normal temperature, br (CH) 2 ) n Br was added to the mixed solution 1, br (CH) 2 ) n The mass ratio of Br to the compound shown in the formula II is 1.0-2.5:1.0-1.5, and a mixed solution 2 is obtained;
s3, carrying out reflux reaction on the mixed solution 2 at the temperature of 70-105 ℃, and separating the compound shown in the formula III from the obtained reaction mixture after the reaction is finished;
s4, dissolving the compound shown in the formula III and the compound shown in the formula IV in a polar organic solvent, and adding piperidine to enable the mass ratio of the compound shown in the formula III to the compound shown in the formula IV to be 3.0-10.0: 1.0 to 6.0:1.0 to 5.0, stirring and reacting at room temperature, and separating the compound shown in the formula V from the obtained reaction mixture after the reaction is finished;
s5, carrying out reflux reaction on the compound shown in the formula V and excessive organic base at the temperature of 50-150 ℃, and separating from the obtained reaction mixture after the reaction is finished to obtain the alpha-cyano phenyleneethylene derivative.
5. Use according to claim 4, characterized in that: the water content of the ultra-dry tetrahydrofuran is below 100 ppm.
6. Use according to claim 4, characterized in that: the polar organic solvent includes methanol or ethanol.
7. The application of an alpha-cyano phenylenevinylene derivative in preparing a human serum albumin visual detection kit is characterized in that: the structure of the alpha-cyano phenylenevinylene derivative is shown in a formula I,
wherein R is 1 Independently selected from C 1-4 Alkyl, R 2 is-O-C 3-12 alkyl-Y, X is selected from F, cl, br, I or CN, Y is selected from pyridinium ion, trimethylammonium ion or triethylammonium ion.
8. Use according to claim 7, characterized in that: r is R 1 Selected from methyl, ethyl or n-propyl.
9. A visual detection method of human serum albumin, which is non-diagnostic, characterized in that the detection method comprises:
providing an aqueous fluorescent probe solution comprising an alpha-cyanophenylethene derivative and water;
mixing a sample to be detected with the fluorescent probe aqueous solution, and detecting human serum albumin in the sample to be detected at least by observing the fluorescent color change of the fluorescent probe aqueous solution before and after mixing with the sample to be detected;
wherein the structure of the alpha-cyano phenyleneethylene derivative is shown in a formula I,
wherein R is 1 Independently selected from C 1-4 Alkyl, R 2 is-O-C 3-12 alkyl-Y, X is selected from F, cl, br, I or CN, Y is selected from pyridinium ion, trimethylammonium ion or triethylammonium ion.
10. The visual inspection method of human serum albumin according to claim 9, wherein: r is R 1 Selected from methyl groupsEthyl or n-propyl.
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