CN110642976A - Polymer potassium ion fluorescent probe and preparation method and application thereof - Google Patents
Polymer potassium ion fluorescent probe and preparation method and application thereof Download PDFInfo
- Publication number
- CN110642976A CN110642976A CN201910968168.4A CN201910968168A CN110642976A CN 110642976 A CN110642976 A CN 110642976A CN 201910968168 A CN201910968168 A CN 201910968168A CN 110642976 A CN110642976 A CN 110642976A
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- Prior art keywords
- potassium ion
- reaction
- fluorescent probe
- ion fluorescent
- potassium
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
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- C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
- C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/42—Introducing metal atoms or metal-containing groups
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- 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
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Abstract
The invention relates to a high polymer potassium ion fluorescent probe and a preparation method and application thereof, the probe is a ratio type high polymer potassium ion fluorescent probe based on a water-soluble high polymer framework and has a structure shown in P1, the invention obtains a completely water-soluble high polymer OEG-1 through active free radical polymerization, then a carboxyl group in the high polymer framework and a hydroxyl group in a small molecule potassium ion probe KS-23 are subjected to esterification reaction, a potassium ion sensor KS-23 is grafted onto the water-soluble high polymer framework, the ratio type potassium ion fluorescent probe P1 based on the water-soluble high polymer framework is obtained, compared with the small molecule potassium ion KS-23, the water solubility and the biocompatibility of P1 are improved, the selectivity to potassium ions is higher, the detection range of the potassium ions is between 1 and 200mM, and the quantitative analysis is expected to be carried out on the potassium ion concentration through peak value comparison, meets the requirement of quantitative detection of potassium ions in cells.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a polymer potassium ion fluorescent probe and a preparation method and application thereof.
Background
Potassium ion (K)+) Is one of the most abundant metal ions in mammalian cells, and accounts for about 4% of human body dry weight. It plays an extremely important role in numerous physiological activities, including heart beating, muscle contraction, nerve signaling, and renal excretion. K+Not only participate in maintaining the osmotic pressure and electrolyte balance inside and outside cells and regulating blood pressure, but also participate in regulating the transduction of biochemical signals of the whole nervous system. K in mammalian cell cytoplasm+The ion concentration was about 130-180mM, while the extracellular concentration was 3.5-5.0 mM. Normal serum potassium levels are maintained at 3.5-5.5mM and when ingested in excess, excess K is normally cleared by renal excretion+. When the concentration of potassium in blood is higher than 5.5mM, the hyperkalemia is generally called as hyperkalemia; at low to high concentrations (5.5-6.0mM) or at medium to high concentrations (6.1-6.9mM), nausea, fatigue, muscle weakness and sometimes arrhythmias are often caused; when blood potassium levels are higher than 7.0, cardiac arrest and even death often results. When the blood potassium concentration is below 3.5mM, it is collectively called hypokalemia. Symptoms clinically associated with hypokalemia include: weakness, paralysis, rigidity of limbs, intestinal obstruction, nausea, vomiting, etc. K+Abnormal concentrations are often an early sign of certain diseases, such as alcohol abuse, anorexia, bulimia, heart disease, diabetes, aids, cancer, and the like. Therefore, finding a highly sensitive method that can specifically study, measure or monitor the intracellular and extracellular potassium ion concentrations in real time is of great significance to the development and screening of drugs related to pathological processes and diseases.
In recent years, fluorescent potassium ion probes have been developed to some extent. The fluorescent potassium ion sensor can perform fluorescence imaging to easily obtain the time-space information of target molecules, can directly measure the concentration of potassium ions in cells in a non-invasive mode, can observe the flow and dynamic balance of potassium ions in cells, and more importantly can analyze living cells on the level of single cell and subcellular structures, is non-toxic, has no damage to cells, is easy to miniaturize and is easy to post-treat. Therefore, the fluorescent potassium ion sensor is an important way for analyzing and detecting potassium ions, and is widely concerned and loved by researchers.
Although potassium ion probes have been developed, probes that can suitably measure intracellular potassium ions (at a concentration of about 130-180mM) have been limited. Small molecules are mainly used in a few reported intracellular potassium ion probes. However, some small molecule probes are poorly water soluble and biocompatible, and the test must be performed in HEPES solution containing 0.5mM cetyltrimethylammonium bromide (CTAB, surfactant). CTAB has great cytotoxicity and cannot be used in the process of cell imaging.
CN106929008B discloses a polymer potassium ion fluorescent probe and a preparation method and application thereof, the polymer potassium ion fluorescent probe takes phenyl aza-18-crown-6-amine as an identification group and a hemicyanine dye group as a fluorescent group, has the advantages of sensitivity to the environment, good water solubility, high detection accuracy, quick response to the change of potassium ion concentration and the like, is a colorimetric and instant ratio type potassium ion detection probe, can be prepared into detection test paper, and realizes quick detection of high and low potassium ion content according to the color change of the test paper. The small molecule probe in the invention has low water solubility, and the test needs to be carried out in the presence of CTAB, and cannot be used in the cell imaging process.
CN104910894B discloses a micromolecular fluorescent probe of benzimidazole hERG potassium channel, a preparation method and application thereof. The hERG potassium ion channel gene can be used in hERG potassium ion channel and high-expression tumor cell or tissue markers thereof, high-throughput screening of hERG potassium ion channel inhibitors, new drug cardiotoxicity evaluation, probes for identifying the hERG potassium ion channel, and research on physiological, pathological and related diseases of the hERG potassium ion channel. However, the small molecular probe has poor water solubility, and the detection limit range of the probe is to be further improved.
Therefore, the development of a polymeric potassium ion fluorescent probe with high water solubility and biocompatibility, which ensures the selectivity of potassium ions required by the probe and meets the detection requirement of potassium ions in cells, is urgently needed in the art.
Disclosure of Invention
In view of the disadvantages of the prior art, an object of the present invention is to provide a polymer potassium ion fluorescent probe, and more particularly, to a ratiometric polymer potassium ion fluorescent probe, and more particularly, to a bicolor ratiometric polymer potassium ion fluorescent probe. The polymer potassium ion fluorescent probe has high water solubility and biocompatibility, a reagent with high cytotoxicity is not required in the test process, the selectivity to potassium ions is high, the response to potassium ions is between 1 and 200mM, and the requirement of potassium ion detection in cells is met.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a polymer potassium ion fluorescent probe, which has a structure shown as P1;
b is1+b2B, and b2Is not 0;
the a: b: c: d (659 + 709):240 (50-100):1, such as 660:240 (50-100):1, 670:240 (50-100):1, 680:240 (50-100):1, 690:240 (50-100):1, 700:240 (50-100):1, (659 + 709):240:60:1, (659 + 709):240:70:1, (659 + 709):240:80:1, (659 + 709):240:90:1 and the like;
n is an integer of 1 to 11, such as 1, 2,3, 4, 5, 7, 9, 10, etc.
The polymer potassium ion fluorescent probe P1 is formed by connecting a micromolecule potassium ion fluorescent probe KS-23 and a water-soluble polymer side chain, compared with the micromolecule potassium ion probe KS-23, the water solubility and the biocompatibility of P1 are obviously improved, a buffer solution containing a surfactant with high cytotoxicity is not required to be tested, and the cell imaging process can be ensured; in addition, P1 has high selectivity to potassium ions, is not interfered by other ions, responds to the potassium ions between 1 and 200mM and meets the requirement of potassium ion detection in cells;
in addition, a methacryloyloxyethyl trimethyl ammonium chloride structural unit is introduced into P1 to enhance the endocytosis capacity of the material by cells by utilizing the interaction between a positive material and a negatively charged cell, and a red fluorescence-emitting porphyrin group (Porphyrin MA,1h NMR (400MHz, Chloroform-d) δ 9.04-8.80(m, 8H), 8.54-8.23(m, 10H), 7.79(d, J ═ 6.8Hz, 9H), 6.30(d, J ═ 4.3Hz, 1H), 5.71(s, 1H), 4.74(d, J ═ 51.3Hz, 4H), 2.08(s, 3H), -2.69(s, 2H)) as an internal reference, and in actual use, the potassium ion concentration can be measured more accurately by colorimetry without adding an additional internal reference, and a ratiometric polymeric potassium ion fluorescent probe is obtained.
The arrangement of the four kinds of structural units in P1 is not limited in the present invention, and may be a random arrangement or a block arrangement.
In the fluorescent probe P1 provided by the invention, KS-23 is grafted on the carboxyl of part or all of methacrylic acid structural units, so that b exists1And b2And b is2Cannot be 0, b1The number of the carboxyl groups of all the methacrylic acid structural units may be 0 (KS-23 grafted to the carboxyl groups) or may be different from 0 (KS-23 grafted to the carboxyl groups of a part of the methacrylic acid structural units).
Preferably, the a: b1:c:d:b2＝(659-709):239:(50-100):1:1。
In the present invention, it is preferred that KS-23 is grafted onto carboxyl groups of a part of the methacrylic acid structural units, and the ratio of the methacrylic acid structural units not grafted with KS-23 to the methacrylic acid structural units grafted with KS-23 is 239:1, i.e., b1:b2Such a structure is more advantageous in increasing the selectivity of the potassium ion probe as 239: 1.
Preferably, the a: b: c: d 684:240:75: 1.
Preferably, the a: b1:c:d:b2＝684:239:75:1:1。
Preferably, the number average molecular weight of the polymeric potassium ion fluorescent probe is 4000 to 100000, such as 4200, 4300, 4400, 4500, 4600, 4700, 4800, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 and the like.
The second purpose of the invention is to provide a preparation method of the polymeric potassium ion fluorescent probe, which comprises the following steps:
(1) reacting polyethylene glycol monomethyl ether methyl methacrylate (OEG)500MA), Methacrylic Acid (MA), methacryloxyethyl trimethyl ammonium chloride (METAC) and porphyrin monomer (Porphyrin-MA) react to obtain polymer OEG-1, and the reaction formula is as follows:
(2) and (3) reacting the polymer OEG-1 with a compound KS-23 to obtain the macromolecular potassium ion fluorescent probe, wherein the reaction formula is as follows:
b is1+b2B, and b2Is not 0;
the a: b: c: d ═ (659-;
and n is an integer of 1-11.
According to the invention, the water-soluble polymer skeleton OEG-1 is obtained by a free radical polymerization method, carboxyl in OEG-1 and hydroxyl in KS-23 are subjected to esterification reaction, and KS-23 is grafted onto OEG-1 through a chemical bond to obtain P1.
Preferably, in step (1), the reaction is carried out in the presence of an initiator.
Preferably, the initiator comprises azobisisobutyronitrile and/or dibenzoyl peroxide.
Preferably, in step (1), the solvent for the reaction comprises N, N-dimethylformamide and/or tetrahydrofuran.
Preferably, in step (1), the temperature of the reaction is 60-70 ℃, such as 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, etc., preferably 65 ℃.
Preferably, the reaction time is 14-18h, such as 14.5h, 15h, 15.5h, 16h, 16.5h, 17h, 17.5h, etc., preferably 16 h.
Preferably, in the step (1), the mass ratio of the polyethylene glycol monomethyl ether methyl methacrylate, the methacrylic acid, the methacryloyloxyethyl trimethyl ammonium chloride and the porphyrin monomer is 500:30 (12.5-75):1, such as 500:30:13:1, 500:30:20:1, 500:30:25:1, 500:30:30:1, 500:30:35:1, 500:30:40:1, 500:30:45:1, 500:30:50:1, 500:30:55:1, 500:30:60:1, 500:30:70:1, and the like, preferably 500:30:25: 1.
Preferably, in step (2), the reaction is carried out in the presence of a catalyst.
Preferably, the catalyst comprises p-dimethylaminopyridine and/or N-hydroxysuccinimide.
Preferably, in step (2), the reaction is carried out in the presence of a condensing agent.
Preferably, the condensing agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and/or dicyclohexylcarbodiimide.
Preferably, in step (2), the solvent for the reaction comprises N, N-dimethylformamide and/or tetrahydrofuran.
Preferably, in step (2), the temperature of the reaction is 35-45 deg.C, such as 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C, 41 deg.C, 42 deg.C, 43 deg.C, 44 deg.C, etc., preferably 40 deg.C.
Preferably, in step (2), the reaction time is 4-8h, such as 4h, 5h, 6h, 7h, 8h, etc., preferably 6 h.
Preferably, in step (2), the mass ratio of the polymer OEG-1 to the compound KS-23 is (140-160):1, such as 142:1, 145:1, 147:1, 149:1, 150:1, 152:1, 154:1, 156:1, 158:1, etc., preferably 150: 1.
In the step (2), the preparation method of KS-23 comprises: the potassium ion probe is obtained by reacting a compound 3 with a compound 3', and the reaction formula is as follows:
preferably, the solvent for the reaction comprises any one or a combination of at least two of ethanol, toluene and benzene.
Preferably, a catalyst is added to the reaction, the catalyst comprising piperidine and/or pyridine.
Preferably, the reaction is carried out under reflux.
Preferably, the compound 3 is obtained by reacting the compound 2 with 2, 4-dimethylpyrrole, and has the following reaction formula:
preferably, the solvent for the reaction includes any one or a combination of at least two of tetrahydrofuran, dioxane, acetonitrile and dichloromethane.
Preferably, a catalyst is added to the reaction, the catalyst comprising trifluoroacetic acid and/or BF3·Et2O。
Preferably, a dehydrogenation agent is added in the reaction, and the dehydrogenation agent comprises 3-dichloro-5, 6-dicyan p-benzoquinone and/or tetrachloro p-quinone.
Preferably, an acid scavenger is added to the reaction, wherein the acid scavenger comprises triethylamine.
Preferably, the compound 2 is obtained by reacting the compound 1 with 2-bromoethanol, and the reaction formula is shown as formula III:
preferably, a catalyst is added to the reaction, the catalyst comprising potassium iodide.
Preferably, an acid-binding agent is added to the reaction, and the acid-binding agent comprises potassium carbonate.
Preferably, the solvent of the reaction comprises CH3Any one or at least two of CN, acetone and N, N-dimethylformamide.
The invention also aims to provide the application of the polymeric potassium ion fluorescent probe in potassium ion detection.
Compared with the prior art, the invention has the following beneficial effects:
compared with the micromolecule KS-23, the water solubility and biocompatibility of the polymer potassium ion fluorescent probe P1 are improved, and the test in a buffer solution containing a surfactant with high cytotoxicity is not needed, so that the cell imaging process can be ensured; moreover, P1 has high selectivity to potassium ions, is not interfered by other ions, responds to potassium ions between 1 and 200mM, and quantitatively analyzes the concentration of potassium ions by a ratio type test method, thereby meeting the requirement of quantitative detection of potassium ions in cells;
in addition, a methacryloyloxyethyl trimethyl ammonium chloride structural unit is introduced into the P1 to enhance the capacity of the material to be charged in cells by utilizing the interaction between a positive material and cells presenting negative property, a porphyrin group emitting red fluorescence is introduced to serve as an internal reference, and when the probe is actually used, the internal reference does not need to be added, so that the concentration of potassium ions can be measured more accurately by a colorimetric method, and the two-color ratio type polymer potassium ion probe is obtained.
Drawings
FIG. 1 shows the results of tests 1 with different K+Uv-vis absorption spectrum of P1 at concentration.
FIG. 2a shows the different K values in test example 1+Fluorescence emission spectrum of P1 at concentration.
FIG. 2b is the fluorescence intensity at 572nm of P1 as a function of K in test example 1+Graph of the change in concentration.
FIG. 2c is a graph showing the ratio of fluorescence intensity at 572nm and 650nm of P1 in test example 1 as a function of K+Graph of change in log concentration.
FIG. 3 is a fluorescence spectrum of P1 in the presence of different metal ions in test example 2.
Detailed Description
For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
The embodiment provides a preparation method of a polymer potassium ion fluorescent probe P1, which comprises the following specific steps:
(1) synthesis of Polymer OEG-1
Polyethylene glycol monomethyl ether methyl methacrylate (mPEG350-MMA, 1g), methacrylic acid (MAA, 60mg), methacryloyloxyethyl trimethyl ammonium chloride (METAC, 50mg), porphyrin monomer (Porphyrin-MA, 2mg) and azobisisobutyronitrile (AIBN, 20mg) were added to a 10mL Schlenk bottle and dissolved in 5mL of DMF. The mixed liquid was frozen into a solid state with liquid nitrogen. And (3) repeatedly vacuumizing, unfreezing for three times, introducing nitrogen, sealing a valve, and reacting for 16 hours in an oil bath at 65 ℃. After the reaction, the reaction solution was slowly dropped into methanol under ice bath condition to obtain a white precipitate. Suction filtration and methanol rinse 3 times to give white solid OEG-1(959.0mg) in calculated 88.0% yield;
structural characterization:1H NMR(400MHz，D2O)δ：4.10(s，2H)，3.63(d，J＝64.5Hz，40H)，3.30(s，4H)，3.17(s，2H)，1.82(s，3H)，1.26–0.56(m，7H)。
the OEG-1 polymerization degree ratio is calculated according to the feeding ratio and is about 684:240:75: 1.
OEG-1 was found to have a number average molecular weight of 4832 as measured by gel permeation chromatography (Waters 1515, USA).
(2) Synthesis of KS-23
Synthesis of Compound 2: 4-hydroxybenzaldehyde (4.8g, 0.04mol), 2-bromoethanol (4.83g, 0.06mol), KI (3.32g, 0.02mol) and K2CO3(8.29g, 0.06mol) was added to a 250mL round bottom flask, dissolved in 80mL acetonitrile and refluxed at 80 ℃ for 36 h. After cooling to room temperature, acetonitrile was removed by distillation under the reduced pressure, and the residue was taken up with CH2Cl2Extraction (80mL) was performed 3 times and washing with saturated NaCl solution (80mL) was performed 3 times. Mixing the organic phases, drying with anhydrous magnesium sulfate, filtering, concentrating, separating with silica gel column chromatography to obtain a mixture with fluidity PE (petroleum ether): EA (ethyl acetate)Ethyl acetate) ═ 1:2, yielding 2.5g of a white solid, 35.3% yield.
1H NMR(400MHz，Chloroform-d)δ9.79(s，1H)，7.76(d，J＝8.5Hz，2H)，6.95(d，J＝8.5Hz，2H)，4.17–4.04(m，2H)，4.04–3.88(m，2H)，3.32(s，1H)。
13C NMR(101MHz，CDCl3)δ191.55，163.96，132.08，129.89，114.83，69.19，61.01。
Synthesis of Compound 3: under nitrogen protection, compound 2(2.5g, 15.06mmol) and 2, 4-dimethylpyrrole (3g, 31.63mmol) were added to a 500mL two-necked flask, dissolved in 160mL tetrahydrofuran, stirred for 30min, 160. mu.L trifluoroacetic acid (TFA) was added, and the reaction solution was stirred overnight with the color changed from yellow to red. After completion, a tetrahydrofuran solution of 2, 3-dichloro-5, 6-dicyanobenzoquinone (DDQ) (3.74g, 16.57mmol) was slowly added dropwise, and after 3 hours, a triethylamine solution (60mL) was added. Stirring is continued for 2h, and BF is added dropwise3·Et2O (60 mL). After stirring for 6 hours, most of the solvent was distilled off under reduced pressure, and then dissolved in 150mL of dichloromethane, washed with dilute hydrochloric acid 2 times, washed with saturated sodium bicarbonate solution twice, washed with saturated sodium chloride solution twice, dried over anhydrous magnesium sulfate, concentrated, and separated by silica gel column chromatography, and the mobile phase PE: EA 2:1 gave 1.25g of a dark red solid with a yield of 22%.
1H NMR(400MHz，Chloroform-d)δ7.09(d，J＝8.4Hz，2H)，6.88(d，J＝8.5Hz，2H)，5.73(s，2H)，4.52(d，J＝1.1Hz，1H)，4.11–4.06(m，2H)，4.00–3.94(m，2H)，2.18(s，6H)，1.85(s，6H)。
13C NMR(101MHz，CDCl3)δ157.03，135.05，129.69，126.48，125.54，114.43，108.65，69.06，61.40，39.43，12.77，11.16。
Synthesis of Compound KS-23: compound 3(102.2mg, 0.2660mmol) and TAC-CHO (160mg, 0.2216mmol) were added to a 25mL round bottom flask, dissolved in 5mL ethanol, followed by 100. mu.L piperidine at reflux for 24 h. After completion, the reaction mixture was cooled to room temperature, the ethanol was removed by distillation under the reduced pressure, the mixture was dissolved in 20mL of dichloromethane, the mixture was washed three times with saturated brine (3X 40mL), the organic phases were combined, dried over anhydrous magnesium sulfate, concentrated, and separated by silica gel column chromatography with the flowability of DCM: MeOH: 75:1 to give 55mg of a dark blue solid with the yield of 23%. Wherein TAC-CHO is prepared by a method described in the literature (A fluorescent sensor with high selection and sensitivity for a position in water [ J ]. Journal of the American Chemical Society, 2003, 125(6): 1468-1469).
1H NMR(400MHz，Chloroform-d)δ7.53(d，J＝15.9Hz，1H)，7.26–7.01(m，8H)，6.89(d，J＝8.0Hz，2H)，6.67(d，J＝8.3Hz，2H)，6.61(s，2H)，4.34–4.21(m，2H)，4.18(t，J＝4.4Hz，2H)，4.06(q，J＝4.7Hz，8H)，3.89–3.83(m，2H)，3.70(dq，J＝29.1，8.4，7.2Hz，16H)，3.56–3.42(m，7H)，3.40–3.27(m，4H)，2.60(s，3H)，2.28(d，J＝21.7Hz，6H)，1.28(s，9H)。
Mass Spectrum (HRMS): c61H77O10N5BF2Calculating the value: 1088.57261, theoretical value: 1088.57690.
(3) synthesis of Polymer Potassium ion fluorescent Probe P1
The above white solid OEG-1300.0 mg, KS-23(2mg), dimethylaminopyridine (DMAP, 50mg) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC-HCl, 150mg) were taken and charged into a round-bottomed flask, dissolved in 5mL of DMF, and reacted at 40 ℃ overnight. After the reaction, the reaction solution was slowly dropped into methanol to obtain a white precipitate, which was filtered and washed with methanol 3 times to obtain a light blue solid. The obtained solid was dissolved in 5mL of DMF, and the solution was loaded on a dialysis membrane (3500kD), and after dialysis for 48 hours, the solution was lyophilized to give light blue solid P1.
Structural characterization:1H NMR(400MHz，D2O)δ：4.10(s，2H)，3.63(d，J＝64.5Hz，40H)，3.30(s，4H)，3.17(s，2H)，1.82(s，3H)，1.26–0.56(m，7H)。
calculating the polymerization degree ratio a to b according to the feeding ratio and the ultraviolet-visible spectrophotometry1:c:d:b2＝684:239:75:1:1。
The number average molecular weight of P1 was 4854 as determined by gel permeation chromatography (Waters 1515, USA).
Test example 1 test of sensory Properties
(1) The polymer potassium ion fluorescent probe P1 is dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, and the concentration of P1 in the stock solution is measured by an ultraviolet-visible spectrophotometry method. The appropriate stock solution was added to HEPES buffer (10mM, pH 7.4) to give a final concentration of P1 of 5. mu.M, and a working solution volume of 3 mL. Will be different from K+The working solutions with the concentrations are respectively filled in 1cm quartz dishes for testing, and K is measured+UV-visible absorption spectra (shown in FIG. 1) at concentrations ranging between 0 and 150 mM. The instrument for testing the UV-visible absorption spectrum is a UV-visible spectrophotometer (Lambda 25, Perkinelmer). Wherein mM means mmol/L.
FIG. 1 shows a difference K+As can be seen from FIG. 1, the maximum absorption peak of the internal reference porphyrin at the concentration of P1 is 419nm, and the maximum absorption of the polymeric potassium ion fluorescent probe P1 is 590 nm. With addition of 150mM K+The absorbance at 419nm is basically unchanged, the absorbance at 590nm has obvious blue shift, and the electron donating capability is weakened after the trinitrogen ether ring of P1 is combined with potassium ions, the PET effect is weakened, and the ultraviolet visible absorption has blue shift.
(2) Mixing the above different K+Working solutions with concentration (0-1000mM) are respectively filled in 1cm quartz dishes with four sides transmitting light, and fluorescence spectra (shown in figure 2 a) of potassium ion concentration in a range of 0-1000mM are measured;
quantitative analysis of fluorescence intensity at 572nm (FIG. 2 b);
linear analysis of the fluorescence intensity ratio at 572nm and 650nm and the logarithmic value of the potassium ion concentration (as shown in FIG. 2 c);
the instrument for measuring the fluorescence spectrum is a fluorescence spectrometer (FluoroMax-4, Horiba).
FIG. 2a shows a difference K+The fluorescence emission spectrum of P1 at the concentration shows that the maximum emission wavelength of internal reference porphyrin is 650nm, and the emission wavelength of polymer potassium ion fluorescent probe KS-23 is 572 nm. With the increase of the concentration of potassium ions, the fluorescence intensity at 650nm is basically unchanged, and the fluorescence intensity at 572nm is continuously enhanced;
FIG. 2b shows fluorescence intensity of P1 at 572nm as a function of K+Graph of change in concentration wherein F0Is not bound with K+Fluorescence intensity at the previous 572nm, F is the binding corresponding concentration K+Then the fluorescence intensity at 572nm, as shown in FIG. 2b, the potassium ion sensor can cause fluorescence amplification about 20 times, and it can be seen that the range of the measurable potassium ion concentration of the probe is 1-200mM, which can satisfy the measurement of the intracellular potassium ion concentration. Wherein F0To add no K+Previous fluorescence intensity at 572nm, F is the addition of the corresponding K+Fluorescence intensity at 572nm thereafter
FIG. 2c shows the ratio of fluorescence intensities (F) of P1 at 572nm and 650nm572/F650) Log [ K ] of+]Is simultaneously paired with (F)572/F650) And log [ K ]+]Performing linear fitting analysis to find that F572/F650And log [ K ]+]Linear correlation (R)20.995) and can therefore be determined from measurement F572/F650Obtaining the corresponding K+The concentration is not influenced by the concentration of potassium ions, and the method meets the requirement of quantitative analysis of potassium ions in cells.
Test example 2 Selective test
A DMSO stock solution of P1 was added to HEPES buffer (10mM, PH 7.4) to give a final concentration of P1 of 5 μ M and a working solution volume of 3 mL. Respectively filling working solution containing different metal ions in 1cm quartz dishes with four sides transparent to light for testing, and measuring different metal ions Na+(150mM)、Li+(150mM)、Mg2+(2mM)、Ca2+(2mM)、Zn2+(2mM)、Mn2+(50μM)、Cu2+(50. mu.M) and K+(10mM and 150mM) on the fluorescence intensity of P1, and the results of testing the selectivity and specificity of the polymeric potassium ion fluorescent probe are shown in FIG. 3.
FIG. 3 is a fluorescence spectrum of P1 in the presence of different metal ions, which shows that the fluorescence spectra curves of different metal ions substantially coincide with each other, and the higher the concentration of potassium ions, the higher the fluorescence intensity, which proves that the metal ions have no effect on the fluorescence intensity of the polymeric potassium ion fluorescent probe P1, and that P1 has high potassium ion selectivity.
To summarize:
example 1A completely water-soluble polymer OEG-1 was obtained by radical polymerization, and a polymer backbone containing a carboxyl group was esterified with a hydroxyl group in a small-molecule potassium ion probe KS-23 to graft KS-23 onto the water-soluble polymer backbone to obtain a polymer potassium ion fluorescent probe P1 based on the water-soluble polymer backbone. The probe is oligomeric ethylene glycol monomethyl ether methacrylate (OEG)500MA), methacrylate (MMA) and methacryloxyethyl trimethyl ammonium chloride (METAC) are taken as frameworks, porphyrin is doped as internal reference, and the water-soluble ratio type polymer potassium ion fluorescent probe P1 is obtained through active radical polymerization and esterification reaction. The P1 has high selectivity and sensitivity to potassium ions, and is not interfered by other ions, so that the strategy that the small molecular probe KS-23 is grafted on a water-soluble polymer skeleton to improve the water solubility and the sensing performance of the water-soluble polymer skeleton is feasible and effective. The novel material with high water solubility can be obtained by utilizing a high molecular technology, and the abundant fluorescence ratio type probes can be prepared, so that the research field of the probes can be enriched. The response of the P1 synthesized by the invention to potassium ions is between 1 and 200mM, and the requirement of quantitative detection of potassium ions in cells is met.
The applicant states that the present invention is illustrated by the above examples to show the detailed process equipment and process flow of the present invention, but the present invention is not limited to the above detailed process equipment and process flow, i.e. it does not mean that the present invention must rely on the above detailed process equipment and process flow to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A polymer potassium ion fluorescent probe is characterized by having a structure shown as P1;
b is1+b2B, and b2Is not 0;
the a: b: c: d ═ (659-;
and n is an integer of 1-11.
2. The polymeric potassium ion fluorescent probe according to claim 1, wherein a: b is1:c:d:b2＝(659-709):239:(50-100):1:1；
Preferably, the ratio of a to b to c to d is 684 to 240 to 75 to 1;
preferably, the a: b1:c:d:b2＝684:239:75:1:1；
Preferably, the number average molecular weight of the polymeric potassium ion fluorescent probe is 4000-100000.
3. The method for preparing the polymeric potassium ion fluorescent probe according to claim 1 or 2, wherein the method comprises the following steps:
(1) reacting polyethylene glycol monomethyl ether methyl methacrylate, methacrylic acid, methacryloyloxyethyl trimethyl ammonium chloride and a porphyrin monomer to obtain a polymer OEG-1, wherein the reaction formula is as follows:
(2) and (3) reacting the polymer OEG-1 with a compound KS-23 to obtain the macromolecular potassium ion fluorescent probe, wherein the reaction formula is as follows:
b is1+b2B, and b2Is not 0;
the a: b: c: d ═ (659-;
and n is an integer of 1-11.
4. The production method according to claim 3, wherein in the step (1), the reaction is carried out in the presence of an initiator;
preferably, the initiator comprises azobisisobutyronitrile and/or dibenzoyl peroxide;
preferably, in step (1), the solvent for the reaction comprises N, N-dimethylformamide and/or tetrahydrofuran.
5. The method according to claim 3 or 4, wherein in the step (1), the temperature of the reaction is 60 to 70 ℃, preferably 65 ℃;
preferably, the reaction time is 14-18h, preferably 16 h.
6. The production method according to any one of claims 3 to 5, wherein in the step (1), the mass ratio of the polyethylene glycol monomethyl ether methyl methacrylate, the methacrylic acid, the methacryloyloxyethyl trimethyl ammonium chloride and the porphyrin monomer is 500:30 (12.5-75):1, preferably 500:30:25: 1.
7. The production method according to any one of claims 3 to 6, wherein, in the step (2), the reaction is carried out in the presence of a catalyst;
preferably, the catalyst comprises p-dimethylaminopyridine and/or N-hydroxysuccinimide;
preferably, in step (2), the reaction is carried out in the presence of a condensing agent;
preferably, the condensing agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and/or dicyclohexylcarbodiimide;
preferably, in step (2), the solvent for the reaction comprises N, N-dimethylformamide and/or tetrahydrofuran.
8. The method according to any one of claims 3 to 7, wherein in the step (2), the temperature of the reaction is 35 to 45 ℃, preferably 40 ℃;
preferably, in step (2), the reaction time is 4-8h, preferably 6 h.
9. The production method according to any one of claims 3 to 8, wherein in the step (2), the mass ratio of the polymer OEG-1 to the compound KS-23 is (140) 160: 1, preferably 150: 1.
10. The use of the polymeric potassium ion fluorescent probe of claim 1 or 2 in potassium ion detection.
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| WO2023013175A1 (en) * | 2021-08-03 | 2023-02-09 | 国立研究開発法人物質・材料研究機構 | Cationic polymer containing porphyrin, tissue marking agent containing same, and photosensitizer |
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| WO2023013175A1 (en) * | 2021-08-03 | 2023-02-09 | 国立研究開発法人物質・材料研究機構 | Cationic polymer containing porphyrin, tissue marking agent containing same, and photosensitizer |
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