CN110642976A - Polymer potassium ion fluorescent probe and preparation method and application thereof - Google Patents

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

A kind of macromolecular potassium ion fluorescent probe and its preparation method and application

technical field

The invention relates to the technical field of biological materials, in particular to a high molecular potassium ion fluorescent probe and a preparation method and application thereof.

Background technique

Potassium ion (K ⁺ ) is one of the most abundant metal ions in mammalian cells, accounting for about 4% of the dry weight of the human body. It plays an extremely important role in numerous physiological activities, including cardiac beating, muscle contraction, nerve signaling and renal excretion. K ⁺ is not only involved in maintaining intracellular and extracellular osmotic pressure and electrolyte balance, regulating blood pressure, but also regulating the transduction of biochemical signals in the entire nervous system. The concentration of K ⁺ ions in the cytoplasm of mammalian cells is approximately 130-180 mM, while the extracellular concentration is 3.5-5.0 mM. Normal serum potassium levels are maintained between 3.5-5.5 mM, and when ingested in excess, excess K ⁺ is usually eliminated by renal excretion. When the serum potassium concentration is higher than 5.5mM, it is collectively referred to as hyperkalemia; at low and high concentrations (5.5-6.0mM) or high concentrations (6.1-6.9mM), it often causes nausea, fatigue, muscle weakness, and sometimes accompanied by Cardiac arrhythmia; when blood potassium levels are above 7.0, cardiac arrest or even death usually results. When the blood potassium concentration is less than 3.5mM, it is collectively called hypokalemia. Symptoms clinically associated with hypokalemia include weakness, paralysis, rigidity, intestinal obstruction, nausea, and vomiting. Abnormal K ⁺ concentrations are often an early sign of certain diseases, such as alcoholism, anorexia, bulimia, heart disease, diabetes, AIDS, and cancer, among others. Therefore, it is of great significance to find a highly sensitive method that can specifically study, measure or real-time monitor the intracellular and extracellular potassium ion concentration for understanding the pathological process and the development and screening of disease-related drugs.

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 spatiotemporal information of the target molecule, and can directly measure the intracellular potassium ion concentration in a non-invasive manner, and can also observe the flow and dynamic balance of intracellular potassium ions. The analysis of living cells at the level of cellular and subcellular structures is non-toxic and non-destructive to cells, easy to miniaturize, and easy to post-processing. Therefore, fluorescent potassium ion sensor is an important way to analyze and detect potassium ion, which has received extensive attention and love from scientific researchers.

Although potassium ion probes have been developed to some extent, the probes suitable for measuring intracellular potassium ions (at a concentration of about 130-180 mM) are still relatively limited. Among the few reported intracellular potassium ion probes, most of them are small molecules. However, some small molecule probes have poor water solubility and biocompatibility, and the test must be performed in HEPES solution containing 0.5 mM cetyltrimethylammonium bromide (CTAB, surfactant). CTAB is highly cytotoxic and cannot be used during cell imaging.

CN106929008B discloses a high-molecular potassium ion fluorescent probe and its preparation method and application. The high-molecular potassium ion fluorescent probe of the invention uses phenylaza-18-crown-6-amine as a recognition group, and uses hemicyanine as a recognition group. The dye group is a fluorescent group, which has the advantages of being sensitive to the environment, good water solubility, high detection accuracy, and rapid response to changes in potassium ion concentration. It is prepared as a detection test paper, and the rapid detection of potassium ion content is realized according to the color change of the test paper. The polymer potassium ion fluorescent probe of the present invention is expected to detect the potassium ion concentration in traditional Chinese medicine injections, red wine, human urine or blood, etc., and has a wide range of applications. application prospects. The small molecule probe in the invention has low water solubility, and the test needs to be carried out in the presence of CTAB, which cannot be used in the cell imaging process.

CN104910894B discloses a small molecule fluorescent probe of benzimidazole-based hERG potassium ion channel, and a preparation method and application thereof. In the hERG potassium channel and its highly expressed tumor cell or tissue markers, in the high-throughput screening of hERG potassium channel inhibitors and in the evaluation of new drugs for cardiotoxicity, and as a probe for the recognition of hERG potassium channels and in hERG Application of potassium channels in the study of physiology, pathology and related diseases. However, the small molecule probes have poor water solubility, and the detection limit of such probes needs to be further improved.

Therefore, there is an urgent need in the art to develop a polymer potassium ion fluorescent probe with high water solubility and biocompatibility, which can ensure the potassium ion selectivity required by the probe and meet the detection requirements of intracellular potassium ions.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, one of the objectives of the present invention is to provide a high molecular potassium ion fluorescent probe, especially to provide a ratio type high molecular potassium ion fluorescent probe, especially a two-color ratio type high molecular potassium fluorescent probe Ion fluorescent probes. The macromolecular potassium ion fluorescent probe has high water solubility and biocompatibility, does not need to use reagents with greater cytotoxicity in the test process, and has high selectivity to potassium ions, and the response to potassium ions is 1- 200mM, which meets the requirements of intracellular potassium ion detection.

For this purpose, the present invention adopts following technical scheme:

The present invention provides a high-molecular potassium ion fluorescent probe, and the high-molecular potassium ion fluorescent probe has the structure shown by P1;

the b ₁ +b ₂ =b, and the b ₂ is not 0;

The a:b:c:d=(659-709):240:(50-100):1, for example 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, etc.;

The n is an integer of 1-11, such as 1, 2, 3, 4, 5, 7, 9, 10, and the like.

The macromolecular potassium ion fluorescent probe P1 provided by the present invention is formed by linking the small molecule potassium ion fluorescent probe KS-23 and the water-soluble polymer side chain. Compared with the small molecule potassium ion probe KS-23, the water-soluble P1 The stability and biocompatibility are significantly improved, and there is no need to test in a buffer solution containing more cytotoxic surfactants, which can ensure the cell imaging process; and, P1 has high selectivity for potassium ions and is not affected by other ions. interference, and the response to potassium ions is between 1-200mM, which meets the requirements of intracellular potassium ion detection;

In addition, the introduction of methacryloyloxyethyltrimethylammonium chloride structural unit in P1 is to utilize the interaction between positively charged materials and negatively charged cells to enhance the ability of materials to be endocytosed by cells, and to introduce red fluorescent porphyrins group (Porphylin MA, ¹ H NMR (400 MHz, 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 internal reference, in practice When there is no need to add additional internal reference, the potassium ion concentration can be measured more accurately by colorimetric method, and a ratio-type high molecular potassium ion fluorescent probe is obtained.

The present invention does not limit the arrangement of the four structural units in P1, which may be random arrangement, block arrangement or the like.

In the fluorescent probe P1 provided by the present invention, part or all of the carboxyl groups of the methacrylic acid structural units are grafted with KS-23, so there are b ₁ and b ₂ , and b ₂ cannot be 0, and b ₁ can be 0 (all The carboxyl group of the methacrylic acid structural unit is grafted with KS-23), and may not be 0 (KS-23 is grafted on the carboxyl group of some methacrylic acid structural units).

Preferably, the a:b ₁ :c:d:b ₂ =(659-709):239:(50-100):1:1.

The carboxyl group of the preferred part of the methacrylic acid structural unit of the present invention is grafted with KS-23, and the ratio of the methacrylic acid structural unit not grafted with KS-23 to the methacrylic acid structural unit of the grafted KS-23 is 239: 1, that is, b ₁ :b ₂ =239:1, such a structure is more conducive to improving the selectivity of the potassium ion probe.

Preferably, the a:b:c:d=684:240:75:1.

Preferably, the a:b ₁ :c:d:b ₂ =684:239:75:1:1.

Preferably, the number-average molecular weight of the macromolecular potassium ion fluorescent probe is 4000-100000, such as 4200, 4300, 4400, 4500, 4600, 4700, 4800, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000 etc.

The second object of the present invention is to provide a preparation method of the macromolecular potassium ion fluorescent probe described in one of the objects, and the preparation method comprises the following steps:

(1) Make polyethylene glycol monomethyl ether methyl methacrylate (OEG ₅₀₀ MA), methacrylic acid (MA), methacryloyloxyethyltrimethylammonium chloride (METAC) and porphyrin monomers (Porphylin-MA) reaction obtains polymer OEG-1, and the reaction formula is as follows:

(2) The polymer OEG-1 is reacted with the compound KS-23 to obtain the polymer potassium ion fluorescent probe, and the reaction formula is as follows:

the b ₁ +b ₂ =b, and the b ₂ is not 0;

The a:b:c:d=(659-709):240:(50-100):1;

The n is an integer of 1-11.

In the present invention, the water-soluble polymer skeleton OEG-1 is obtained by the preparation method of free radical polymerization, the carboxyl group in OEG-1 is esterified with the hydroxyl group in KS-23, and KS-23 is grafted onto OEG-1 through chemical bond get P1.

Preferably, in step (1), the reaction is carried out in the presence of an initiator.

Preferably, the initiator includes azobisisobutyronitrile and/or dibenzoyl peroxide.

Preferably, in step (1), the solvent for the reaction includes N,N-dimethylformamide and/or tetrahydrofuran.

Preferably, in step (1), the temperature of the reaction is 60-70°C, such as 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, etc., preferably 65°C.

Preferably, the reaction time is 14-18h, such as 14.5h, 15h, 15.5h, 16h, 16.5h, 17h, 17.5h, etc., preferably 16h.

Preferably, in step (1), the mass ratio of the polyethylene glycol monomethyl ether methyl methacrylate, methacrylic acid, methacryloyloxyethyltrimethylammonium chloride and porphyrin monomer is 500:30:(12.5-75):1, e.g. 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 etc., 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 includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and/or dicyclohexylcarbodiimide.

Preferably, in step (2), the solvent for the reaction includes N,N-dimethylformamide and/or tetrahydrofuran.

Preferably, in step (2), the temperature of the reaction is 35-45°C, such as 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, etc., preferably 40°C.

Preferably, in step (2), the reaction time is 4-8h, such as 4h, 5h, 6h, 7h, 8h, etc., preferably 6h.

Preferably, in step (2), the mass ratio of the polymer OEG-1 and 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 step (2), the preparation method of KS-23 includes: using compound 3 and compound 3' to react to obtain the potassium ion probe, and the reaction formula is as follows:

Preferably, the solvent for the reaction includes any one or a combination of at least two of ethanol, toluene and benzene.

Preferably, a catalyst is added to the reaction, and the catalyst includes 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 the reaction formula is as follows:

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 in the reaction, and the catalyst includes trifluoroacetic acid and/or BF ₃ ·Et ₂ O.

Preferably, a dehydrogenating agent is added in the reaction, and the dehydrogenating agent includes, 3-dichloro-5,6-dicyano-p-benzoquinone and/or tetrachloro-p-quinone.

Preferably, an acid binding agent is added in the reaction, and the acid binding agent includes triethylamine.

Preferably, the compound 2 is obtained by reacting the compound 1 with 2-bromoethanol, and the reaction formula is shown in formula III:

Preferably, a catalyst is added to the reaction, and the catalyst includes potassium iodide.

Preferably, an acid binding agent is added in the reaction, and the acid binding agent includes potassium carbonate.

Preferably, the solvent for the reaction includes any one or a combination of at least two of CH ₃ CN, acetone and N,N-dimethylformamide.

The third object of the present invention is to provide the application of the macromolecular potassium ion fluorescent probe described in one of the objects in potassium ion detection.

Compared with the prior art, the present invention has the following beneficial effects:

The macromolecular potassium ion fluorescent probe P1 provided by the present invention is formed by linking a small molecule potassium ion fluorescent probe KS-23 and a water-soluble macromolecular side chain. Compared with the small molecule KS-23, it is water-soluble and biocompatible. The performance of P1 is improved, and there is no need to test in a buffer solution containing more cytotoxic surfactants, which can ensure the cell imaging process; in addition, P1 has high selectivity for potassium ions, is not interfered by other ions, and is sensitive to potassium ions. The response is between 1-200mM, and the potassium ion concentration is quantitatively analyzed by the ratio test method, which meets the requirements for the quantitative detection of intracellular potassium ions;

In addition, the introduction of the methacryloyloxyethyltrimethylammonium chloride structural unit in P1 is to use the positive charge material and the negative cell interaction to enhance the ability of the material to be internalized by cells, and to introduce red fluorescent porphyrin The linoline group is used as an internal reference. In actual use, there is no need to add another internal reference. The colorimetric method can be used to measure the potassium ion concentration more accurately, and a two-color ratio type macromolecular potassium ion probe is obtained.

Description of drawings

Figure 1 is the UV-Vis absorption spectrum of P1 under different K ⁺ concentrations in Test Example 1.

Figure 2a is the fluorescence emission spectrum of P1 under different K ⁺ concentrations in Test Example 1.

Fig. 2b is a graph showing the change of the fluorescence intensity of P1 at 572 nm with the concentration of K ⁺ in Test Example 1.

Figure 2c is a graph showing the change in the ratio of the fluorescence intensity of P1 at 572 nm and 650 nm with the logarithmic value of K ⁺ concentration in Test Example 1.

3 is the fluorescence spectrum of P1 in the presence of different metal ions in Test Example 2.

Detailed ways

In order to facilitate the understanding of the present invention, examples of the present invention are as follows. It should be understood by those skilled in the art that the embodiments are only for helping the understanding of the present invention, and should not be regarded as a specific limitation of the present invention.

Example 1

The present embodiment provides a method for preparing a macromolecular potassium ion fluorescent probe P1, which is specifically as follows:

(1) Synthetic polymer OEG-1

Polyethylene glycol monomethyl ether methyl methacrylate (mPEG350-MMA, 1g), methacrylic acid (MAA, 60mg), methacryloyloxyethyltrimethylammonium chloride (METAC, 50mg), porphyrin Porphylin monomer (Porphylin-MA, 2 mg) and azobisisobutyronitrile (AIBN, 20 mg) were added to a 10 mL Schlenk bottle, and 5 mL of DMF was added to dissolve. The mixed liquid was frozen into a solid state with liquid nitrogen. Repeated vacuuming, thawed three times, passed nitrogen, closed the valve, and reacted in an oil bath at 65 °C for 16 h. After the completion of the reaction, the reaction solution was slowly dropped into methanol under ice bath conditions to obtain a white precipitate. Suction filtration and rinsed with methanol 3 times to obtain a white solid OEG-1 (959.0 mg), and the calculated yield is 88.0%;

Structural characterization: ¹ H NMR (400 MHz, D ₂ O) δ: 4.10 (s, 2H), 3.63 (d, J=64.5 Hz, 40H), 3.30 (s, 4H), 3.17 (s, 2H), 1.82 ( s, 3H), 1.26–0.56 (m, 7H).

The OEG-1 degree of polymerization ratio calculated according to the feed ratio is about a:b:c:d=684:240:75:1.

The number average molecular weight of OEG-1 was 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 ₎ were combined ) was added to a 250 mL round-bottomed flask, dissolved in 80 mL of acetonitrile, and refluxed at 80 °C for 36 h. After cooling to room temperature, acetonitrile was distilled off under reduced pressure, the residue was extracted three times with CH ₂ Cl ₂ (80 mL), and washed three times with saturated NaCl solution (80 mL). The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and separated by silica gel column chromatography. The fluidity was PE (petroleum ether):EA (ethyl acetate)=1:2 to obtain 2.5 g of white solid with a yield of 35.3 g. %.

¹ H NMR (400MHz, Chloroform-d) δ 9.79 (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).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 191.55, 163.96, 132.08, 129.89, 114.83, 69.19, 61.01.

Synthesis of compound 3: under nitrogen protection, compound 2 (2.5 g, 15.06 mmol) and 2,4-dimethylpyrrole (3 g, 31.63 mmol) were added to a 500 mL two-neck flask, dissolved in 160 mL of tetrahydrofuran, and stirred for 30 min. , 160 μL of trifluoroacetic acid (TFA) was added, the color of the reaction solution changed from yellow to red, and the mixture was stirred overnight. After completion, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) (3.74 g, 16.57 mmol) tetrahydrofuran solution was slowly added dropwise, and after 3 h, triethylamine solution (60 mL) was added. After stirring continued for ₂ h, _BF3.Et2O (60 mL) was added dropwise. After stirring for 6 hours, most of the solvent was distilled off under reduced pressure, dissolved in 150 mL of dichloromethane, washed twice with dilute hydrochloric acid, twice with saturated sodium bicarbonate solution, twice with saturated sodium chloride solution, and dried over anhydrous magnesium sulfate. , concentrated, separated by silica gel column chromatography, and the mobile phase was PE:EA=2:1 to obtain 1.25 g of dark red solid with a yield of 22%.

¹ H NMR (400 MHz, Chloroform-d) δ 7.09 (d, J=8.4 Hz, 2H), 6.88 (d, J=8.5 Hz, 2H), 5.73 (s, 2H), 4.52 (d, J=1.1 Hz, 1H), 4.11–4.06 (m, 2H), 4.00–3.94 (m, 2H), 2.18 (s, 6H), 1.85 (s, 6H).

¹³ C NMR (101 MHz, CDCl ₃ ) δ 157.03, 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 (102.2 mg, 0.2660 mmol) and TAC-CHO (160 mg, 0.2216 mmol) were added to a 25 mL round-bottomed flask, 5 mL of ethanol was added to dissolve, and then 100 μL of piperidine was added, and refluxed for 24 h. After completion, it was cooled to room temperature, distilled under reduced pressure to remove ethanol, dissolved in 20 mL of dichloromethane, washed three times with saturated brine (3×40 mL), combined the organic phases, dried over anhydrous magnesium sulfate, concentrated, separated by silica gel column chromatography, and flowed. The ratio was DCM:MeOH=75:1 to give a dark blue solid 55 mg in 23% yield. Among them, TAC-CHO was prepared by the method described in the literature (A fluorescent sensor with high selectivity and sensitivity for potassium in water [J]. Journal of the American Chemical Society, 2003, 125(6): 1468-1469).

¹ H NMR (400 MHz, Chloroform-d) δ 7.53 (d, J=15.9 Hz, 1H), 7.26-7.01 (m, 8H), 6.89 (d, J=8.0 Hz, 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): Calculated for _{C61H77O10N5BF2} , _calcd : _1088.57261 , _calcd : _1088.57690 .

(3) Synthesis of macromolecular potassium ion fluorescent probe P1

Take the above white solid OEG-1 300.0mg, KS-23 (2mg), p-dimethylaminopyridine (DMAP, 50mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Salt (EDC-HCl, 150 mg) was added to a round-bottomed flask, dissolved with 5 mL of DMF, and reacted at 40°C overnight. After the completion of the reaction, the reaction solution was slowly dropped into methanol to obtain a white precipitate, which was filtered with suction and rinsed three times with methanol to obtain a light blue solid. The obtained solid was dissolved in 5 mL of DMF, and the solution was loaded into a dialysis membrane (3500KD). After dialysis for 48 h, the solution was freeze-dried to obtain a light blue solid P1.

Structural characterization: ¹ H NMR (400 MHz, D ₂ O) δ: 4.10 (s, 2H), 3.63 (d, J=64.5 Hz, 40H), 3.30 (s, 4H), 3.17 (s, 2H), 1.82 ( s, 3H), 1.26–0.56 (m, 7H).

The polymerization degree ratio a:b ₁ :c:d:b ₂ =684:239:75:1:1 was calculated according to the charging ratio and UV-Vis spectrophotometry.

The number average molecular weight of P1 was 4854 as measured by gel permeation chromatography (Waters 1515, USA).

Test Example 1 Sensing Properties Test

(1) The polymer potassium ion fluorescent probe P1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, and the concentration of P1 in the stock solution was measured by UV-Vis spectrophotometry. An appropriate amount of the stock solution was added to HEPES buffer solution (10 mM, pH=7.4) to make the final concentration of P1 5 μM, and the working solution volume was 3 mL. The working solutions of different K ⁺ concentrations were respectively placed in 1 cm quartz dishes for testing, and the UV-visible absorption spectra in the range of K ⁺ concentration of 0 and 150 mM were measured (as shown in Figure 1). The instrument for measuring the UV-Vis absorption spectrum was a UV-Vis spectrophotometer (Lambda 25, PerkinElmer). Wherein, mM refers to mmol/L.

Figure 1 is the UV-visible absorption spectrum of P1 under different K ⁺ concentrations. It can be seen from Figure 1 that the maximum absorption peak of the internal reference porphyrin is 419 nm, and the maximum absorption peak of the polymer potassium ion fluorescent probe P1 is 590 nm. With the addition of 150mM K ⁺ , the absorbance at 419nm is basically unchanged, and the absorbance at 590nm has a significant blue shift, indicating that after the combination of the triazide ring of P1 and potassium ion, the electron donating ability is weakened, and the PET effect is weakened, resulting in ultraviolet A blue shift in absorption can be seen.

(2) The working solutions of the above-mentioned different K concentrations ( ^0-1000mM ) were respectively installed in a 1cm quartz dish that was transparent on all sides, and the fluorescence spectrum in the range of 0-1000mM was measured for potassium ion concentration (as shown in Figure 2a);

Quantitative analysis of the fluorescence intensity at 572 nm (as shown in Figure 2b);

Perform linear analysis on the ratio of fluorescence intensity at 572nm and 650nm and the logarithm of potassium ion concentration, (as shown in Figure 2c);

The instrument for measuring the fluorescence spectrum was a fluorescence spectrometer (FluoroMax-4, Horiba).

Figure 2a shows the fluorescence emission spectra of P1 under different K ⁺ concentrations. The figure shows that the maximum emission wavelength of the internal reference porphyrin is 650 nm, and the emission wavelength of the macromolecular potassium ion fluorescent probe KS-23 is 572 nm. With the increase of potassium ion concentration, the fluorescence intensity at 650nm remained basically unchanged, and the fluorescence intensity at 572nm continued to increase;

Figure 2b shows the change of the fluorescence intensity of P1 at 572 nm with the concentration of K ⁺ , where F ₀ is the fluorescence intensity at 572 nm before unbound K ⁺ , and F is the fluorescence intensity at 572 nm after binding the corresponding concentration of K ⁺ , Figure 2b It is shown in the figure that the potassium ion sensor can cause a fluorescence increase of about 20 times. It can be seen from the figure that the probe can measure the potassium ion concentration in the range of 1-200mM, which can meet the measurement of intracellular potassium ion concentration. where F ₀ is the fluorescence intensity at 572 nm before adding K ⁺ , and F is the fluorescence intensity at 572 nm after adding the corresponding K ⁺

Figure 2c is a graph showing the change of the ratio of fluorescence intensity (F ₅₇₂ /F ₆₅₀ ) of P1 at 572 nm and 650 nm with log[K ⁺ ], and a linear fitting analysis was performed on (F ₅₇₂ /F ₆₅₀ ) and log[K ⁺ ] It is found that F ₅₇₂ /F ₆₅₀ is linearly related to log[K ⁺ ] (R ² =0.995), so the corresponding K ⁺ concentration can be obtained by measuring F ₅₇₂ /F ₆₅₀ , and this method is not affected by potassium ion concentration, satisfying Requirements for quantitative analysis of intracellular potassium ions.

Test Case 2 Selective Test

The DMSO stock solution of P1 was added to HEPES buffer solution (10 mM, pH=7.4) to make the final concentration of P1 5 μM and the working solution volume was 3 mL. The working solutions containing different metal ions were placed in a 1cm quartz dish with light transmission on all sides for testing, and different metal ions Na ⁺ (150mM), Li ⁺ (150mM), Mg ²⁺ (2mM), Ca ²⁺ (2mM) were measured. ), Zn ²⁺ (2mM), Mn ²⁺ (50μM), Cu ²⁺ (50μM) and K ⁺ (10mM and 150mM), the effect on the fluorescence intensity of P1 to test the selectivity of the high molecular potassium ion fluorescent probe and specificity results are shown in Figure 3.

Fig. 3 is the fluorescence spectrum of P1 in the presence of different metal ions. The figure shows that the fluorescence spectrum curves of different metal ions basically overlap. The fluorescence intensity of the fluorescent probe P1 was basically unaffected, which proved that P1 has a high degree of potassium ion selectivity.

Summarize:

Example 1 A completely water-soluble polymer OEG-1 was obtained by free radical polymerization. The polymer backbone contains carboxyl groups, which can be esterified with the hydroxyl groups in the small molecule potassium ion probe KS-23 to graft KS-23. The polymer potassium ion fluorescent probe P1 based on the water-soluble polymer backbone is obtained on the water-soluble polymer backbone. The probe is based on oligoethylene glycol monomethyl ether methacrylate (OEG ₅₀₀ MA), methacrylate (MMA), and methacryloyloxyethyltrimethylammonium chloride (METAC) as the backbone. Doping porphyrin as an internal reference, the water-soluble ratio-type high molecular potassium ion fluorescent probe P1 was obtained by living radical polymerization and esterification. The selectivity and sensitivity of P1 to potassium ions are high, and it is not interfered by other ions, which proves the strategy of grafting the small molecule probe KS-23 on the water-soluble polymer backbone in the present invention to improve its water-solubility and sensing performance. It is feasible and effective. Using polymer technology, new materials with high water solubility can be obtained, and abundant fluorescent ratiometric probes can be prepared, which will enrich the field of probe research. The P1 synthesized in the present invention responds to potassium ions in the range of 1-200 mM, which meets the requirement of quantitative detection of intracellular potassium ions.

The applicant declares that the present invention illustrates the detailed process equipment and process flow of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, that is, it does not mean that the present invention must rely on the above-mentioned detailed process equipment and process flow. Process flow can be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and disclosure scope 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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