CN110437268B

CN110437268B - Potassium ion probe and preparation method and application thereof

Info

Publication number: CN110437268B
Application number: CN201910832447.8A
Authority: CN
Inventors: 田颜清; 刘洪甜; 宁爵伟
Original assignee: Southern University of Science and Technology
Current assignee: Southern University of Science and Technology
Priority date: 2019-09-04
Filing date: 2019-09-04
Publication date: 2022-01-14
Anticipated expiration: 2039-09-04
Also published as: CN110437268A

Abstract

The potassium ion probe provided by the invention has high selectivity on potassium ions, can emit green fluorescence and can detect K⁺And the potassium ion probe contains hydroxyl, so that the potassium ion probe can be grafted with a carrier more easily, and can be grafted on the carrier together with other types of probes to realize multiple responses of the same sensing film. The invention also provides a potassium ion and dissolved oxygen dual-functional sensing film, which comprises a carrier, and a potassium ion probe, an oxygen probe and a reference probe which are fixed on the surface of the carrier in a chemical bonding mode.

Description

Potassium ion probe and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological materials, in particular to a potassium ion probe and a preparation method and application thereof, and particularly relates to a potassium ion probe and a preparation method thereof, a potassium ion and dissolved oxygen dual-functional sensing film and a preparation method thereof.

Background

Potassium ion (K)⁺) Is one of the most abundant metal ions in mammalian cells, and accounts for about 0.35% of human body dry weight. It plays an extremely important role in numerous physiological activities,including heart beat, muscle contraction, nerve signal conduction, renal excretion, etc. 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 the blood potassium concentration is higher than 7.0mM, cardiac arrest and even death is usually caused. 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.

Oxygen is one of the very important elements or parameters in the fields of environmental protection, engineering, marine production, industry and agriculture, especially in the biological field, oxygen is one of the important raw materials for cell metabolism, and is the source of energy for maintaining life activities. With the development of scientific research, the influence of oxygen concentration on cells is gradually recognized. Although oxygen is essential for the metabolism of cells, an increase in oxygen concentration also causes some toxicity. In high oxygen environments, aerobic and microaerophilic cells or tissues may initiate their own protective mechanisms to cope with the current abnormal environment. Superoxide (O) known as reactive oxygen species₂ ^-) And hydrogen peroxide (H)₂O₂) Will become a by-product of cell metabolismIntracellular accumulation, because these reactive oxygen species are more reactive than normal oxygen molecules, eventually oxidize intracellular macromolecules and thus cause apoptosis. Similarly, if the intracellular oxygen concentration is below normal, it will result in aerobic and microaerophilic cell or tissue ischemic injury and even cell death. Hypoxia of cells can also lead to a variety of diseases such as chronic vascular diseases, neurological disorders, pulmonary diseases and even cancer.

Therefore, the ability to accurately and precisely measure the oxygen content in cells is of great importance for understanding the biological viability of cells, studying the pathology of various diseases, and diagnosing and treating cancer.

It is well known that cellular respiration is a series of metabolic reactions and processes occurring in biological organs and tissues, whereby cells acquire energy such as Adenosine Triphosphate (ATP) and discharge waste materials such as carbon dioxide. Although the detection of intracellular oxygen levels has been reported, the detection of extracellular oxygen levels is still not negligible and many biological metabolic processes are still investigated by measuring extracellular oxygen levels. During cellular metabolic reactions, mitochondria are critical organelles that support aerobic respiration and gain ATP. However, cellular respiration is not dependent on mitochondria, and mitochondria are not required for yet another type of respiration, known as non-mitochondrial oxygen consumption. Studying non-mitochondrial oxygen consumption characteristics requires measuring extracellular oxygen consumption. Therefore, the ability to accurately and precisely measure the oxygen consumption of cells during respiration is very important for evaluating aerobic glycolysis rate, oxidative phosphorylation, and high-throughput drug screening, among others.

Opening and closing of potassium ion channel and K⁺Both influx and efflux at the cell membrane are affected by the concentration of Dissolved Oxygen (DO) in the body. For example, hypoxia causes myocardial K⁺Resulting in myocardial dysfunction. In addition, acute systemic hypoxia can cause arterial K⁺The concentration increases, causing peripheral circulation vasodilation. Cerebral dysfunction and pulmonary vasoconstriction also associated with hypoxia-induced K⁺The imbalance is related. Thus, K is measured simultaneously⁺Concentration ofAnd DO concentration are very important.

CN106929008B discloses a potassium ion fluorescent probe and a preparation method and application thereof, the potassium ion fluorescent probe of the invention 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, realizes quick detection of high and low potassium ion content according to the color change of the test paper, is expected to detect the potassium ion concentration in the aspects of traditional Chinese medicine injection, red wine, human urine or blood and the like, and has wide application prospect.

CN105241845B preparation and application of a single particle SPR probe for real-time monitoring of G-quadruplex formation. Based on good biocompatibility, large specific surface area and high SPR sensitivity of the noble metal nanoparticles, telomeres capable of being specifically combined with potassium ions are adopted to carry out surface modification on the noble metal nanoparticles, and the single-particle biological probe capable of detecting the potassium ions with high sensitivity and monitoring the G-quadruplex formation process in real time is simply and conveniently constructed. The probe has the function of real-time detection, and has the advantages of high detection speed, high sensitivity, wide detection range and the like. In addition, the dissociation constant K for the formation of G-quartets can be obtained by data fitting analysis_dAnd gibbs free energy Δ G, as well as two binding site morphologies during formation.

CN108333162A discloses a fluorescent oxygen probe, which comprises a polymer microsphere copolymerized with a reference probe compound and a fluorescent oxygen probe compound uniformly dispersed in the polymer microsphere copolymerized with the reference probe compound, and also provides an inventive method for preparing the fluorescent oxygen probe, preparing microspheres dispersed with a fluorescent oxygen probe compound by copolymerizing a reference probe compound with a polymerization monomer, wherein the microspheres can be used as a fluorescent oxygen probe, the fluorescent oxygen probe prepared by the invention has self-reference effect, avoids the problem that the fluorescent probe needs to be recalibrated when the detection equipment is replaced in the traditional fluorescent detection, and in addition, when the electrochemical detection device is used, the requirement on detection environment is not high, the electrochemical detection device can be used for detecting the concentration of dissolved oxygen in dynamic biological environments such as water bodies containing microorganisms, and the like, and the detection result error is less than or equal to 5 percent compared with that of the traditional electrochemical detection device.

As can be seen from the above-disclosed patents, sensors that measure the concentration of potassium ions alone or the concentration of oxygen alone have been well developed. However, no sensor capable of measuring the concentration of oxygen and potassium ions simultaneously and in real time exists at present.

Therefore, there is a need in the art to simultaneously and real-time measure oxygen and potassium ion concentrations.

Disclosure of Invention

In view of the shortcomings of the prior art, one of the objectives of the present invention is to provide a potassium ion probe, and more particularly, to provide a potassium ion probe for a potassium ion and dissolved oxygen dual-function sensing film. The potassium ion probe has high selectivity on potassium ions, emits green fluorescence and can detect K⁺And (4) concentration. And the potassium ion probe can be connected with the carrier through hydroxyl.

In order to achieve the purpose, the invention adopts the following technical scheme:

the present invention provides a potassium ion probe, wherein the potassium ion probe (KS-23) has the following structure:

the potassium ion probe provided by the invention has high selectivity on potassium ions, can emit green fluorescence, and can detect K⁺And the potassium ion probe contains hydroxyl, so that the potassium ion probe can be grafted with a carrier more easily, and can be grafted on the carrier together with other types of probes to realize multiple responses of the same sensing film.

The response mechanism of the potassium ion probe is as follows:

another object of the present invention is to provide a method for preparing the potassium ion probe according to the first object, the method comprising: 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 potassium ion and dissolved oxygen dual-function sensing film comprises a carrier, and a potassium ion probe (KS-23), an oxygen probe (OS) and a Reference Probe (RP) which are fixed on the surface of the carrier in a chemical bonding mode;

preferably, the oxygen probe (OS) has the following structure:

preferably, the Reference Probe (RP) has the following structure:

the potassium ion probe, the oxygen probe and the reference probe with specific structures are jointly bonded on the carrier, wherein the potassium ion probe responds to potassium ions and emits green fluorescence, the oxygen probe responds to dissolved oxygen and emits red fluorescence, and the reference probe does not respond to both the potassium ions and the dissolved oxygen and emits blue fluorescence. Therefore, the film has red, green and blue fluorescence, the three fluorescence colors are not interfered with each other, and the accurate simultaneous measurement of potassium ions and dissolved oxygen can be realized by using a fluorescence colorimetric method.

In addition, the three probes are fixed on the carrier in a chemical bonding mode, so that the sensing film is more stable.

In the present invention, the preparation method of the oxygen probe OS is described in the article: jianan Shi, Yifei Zhou, Jianpei Jiang, tinting Pan, Zhipeng Mei, Jianxing Wen, Cheng Yang, Zijin Wang, Yanqing Tian, European Polymer Journal, 112, 214-.

Reference probes of the present invention are synthesized by reference to the preparation methods described in the following papers or patents:

the patent: CN106947381A

The article: zhang, J.H.ZHou, H.Li, J.H.He, N.J.Li, Q.F.xu, D.Y.Chen, H.Li, J.M.Lu, The Effect of Random and Block polymerization with Pender carbide nanoparticles and nanoparticles Acceptors on Multilevel Memory, Chem Asian J.13(2018) 853-.

Preferably, the support is a quartz support modified by TMSPA, and the TMSPA has the following structure:

the fourth purpose of the present invention is to provide a method for preparing the potassium ion and dissolved oxygen dual-functional sensing thin film, which comprises the following steps:

(1') polymerizing polyethylene glycol methacrylate (PEGMA), methacrylic acid (MAA), polyethylene glycol dimethacrylate (PEGDMA), an oxygen probe and a reference probe on the surface of a carrier to obtain a carrier (F-OS-RP) covered with a hydrogel layer;

(2') grafting the potassium ion probe of one purpose on the surface of the carrier covered with the hydrogel layer to obtain the potassium ion and dissolved oxygen dual-function sensing film.

The method takes PEGMA as a hydrogel monomer and PEGDMA as a cross-linking agent to obtain stable hydrogel, an oxygen probe OS and a reference probe RP contain methacrylate ester bonds, MAA is added at the same time and can be polymerized with the used monomer, the monomer is polymerized and then integrated with a carrier to obtain a film (F-OS-RP) containing the oxygen probe, the reference probe and an active carboxyl, and then KS-23 is bonded on the carrier through the reaction of the active carboxyl and hydroxyl on KS-23, and the three probes are bonded with the carrier in a chemical bonding mode, so that the stability of the film is improved.

Preferably, the support surface contains double bonds.

Preferably, in step (1'), the initiator for the polymerization reaction comprises Azobisisobutyronitrile (AIBN).

Preferably, in step (1'), the solvent for the polymerization reaction includes N, N-Dimethylformamide (DMF).

Preferably, in step (1'), the support is a TMSPA-modified quartz support.

In a preferred embodiment of the present invention, the surface of the carrier is modified with double bond-containing and silane bond-containing TSMPA, so that the gel is stable on the surface of the carrier, and the stability of the film is further improved.

Preferably, in step (1'), the mass ratio of polyethylene glycol methacrylate, polyethylene glycol dimethacrylate, the oxygen probe, the reference probe, the initiator and the solvent is 500-1500:20-300:1-15:1-15:5-30:1000, preferably 800:50:5:5:10: 1000.

Preferably, in step (1'), the mass ratio of the methacrylic acid to the polyethylene glycol methacrylate is 10-1000: 1.

Preferably, in step (1'), the number average molecular weight of the polyethylene glycol methacrylate is 300-2000, preferably 500.

Preferably, in step (1'), the number average molecular weight of the polyethylene glycol dimethacrylate is 300-2000, preferably 750.

Preferably, in step (1'), the polymerization reaction is carried out under a nitrogen atmosphere.

Preferably, in step (1'), the polymerization temperature is 70-90 ℃, such as 72 ℃, 73 ℃, 75 ℃, 78 ℃, 80 ℃, 82 ℃, 85 ℃, 88 ℃, etc., preferably 80 ℃.

Preferably, in step (1'), the polymerization time is 0.8 to 1.5h, e.g., 0.9h, 1h, 1.2h, 1.3h, 1.4h, etc., preferably 1 h.

Preferably, step (1') comprises in particular: mixing polyethylene glycol methacrylate, methacrylic acid, polyethylene glycol dimethacrylate, an oxygen probe, a reference probe, an initiator and a solvent, taking the mixed solution to be arranged on the surface of a TMSPA modified quartz carrier, and polymerizing for 0.8-1.5h at the temperature of 70-90 ℃ to obtain the carrier covered with the hydrogel layer.

Preferably, in step (2'), a coupling agent and/or a catalyst is added.

Preferably, the coupling agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

Preferably, the catalyst comprises 4-dimethylaminopyridine.

Preferably, in the step (2'), the mass ratio of the potassium ion probe, the coupling agent and the catalyst is 2:1000: 200.

Preferably, in step (2'), the reaction solvent for grafting comprises N, N-dimethylformamide.

Preferably, in step (2'), the reaction time for the grafting is 10 to 15h, such as 11h, 12h, 13h, 14h, etc., preferably 12 h.

Preferably, in step (2'), the reaction of grafting is carried out at room temperature.

Preferably, step (2') specifically comprises the steps of: and (3) placing the carrier covered with the hydrogel layer in a mixed solution of a potassium ion probe, a coupling agent and a catalyst, and carrying out grafting reaction for 10-15h at room temperature to obtain the potassium ion and dissolved oxygen dual-function sensing film.

The fifth purpose of the present invention is to provide an application of the three-purpose dual-function sensing film for potassium ions and dissolved oxygen in potassium ion detection and dissolved oxygen detection.

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

(1) the potassium ion probe provided by the invention has high selectivity on potassium ions, can emit green fluorescence, and can detect K⁺The concentration and the hydroxyl contained in the potassium ion probe are easier to graft with a carrier, and the potassium ion probe and other types of probes are grafted on the carrier together, so that multiple responses of the same sensing film are realized;

(2) the potassium ion probe, the oxygen probe and the reference probe with specific structures are jointly bonded on the carrier, wherein the potassium ion probe responds to potassium ions and emits green fluorescence, the oxygen probe responds to dissolved oxygen and emits red fluorescence, and the reference probe does not respond to both the potassium ions and the dissolved oxygen and emits blue fluorescence. Therefore, the film has red, green and blue fluorescence, the three fluorescence colors are not interfered with each other, and the accurate simultaneous measurement of potassium ions and dissolved oxygen can be realized by using a fluorescence colorimetric method;

(3) the method takes PEGMA as a hydrogel monomer and PEGDMA as a cross-linking agent to obtain stable hydrogel, the oxygen probe OS and the reference probe RP contain methacrylate bonds and can be polymerized with the used monomer, the monomer is polymerized and then integrated with a carrier, MAA is added to enable KS-23 containing hydroxyl to be connected into a colloid, and the three probes are combined with the carrier in a chemical bonding mode, so that the stability of the film is improved.

Drawings

FIG. 1 is a schematic diagram of a method for preparing a potassium ion and dissolved oxygen dual-functional sensing film in example 1 of the present invention.

FIG. 2a is a graph showing the response of the reference probe to oxygen in test example 1.

FIG. 2b is a graph showing the response of the reference probe to potassium ions in test example 1.

FIG. 3a is a graph showing the response of the film to potassium ions under excitation at 405 nm in test example 1.

FIG. 3b is a graph showing the response of the film to oxygen under excitation at 405 nm for test example 1.

FIG. 4a is a graph showing the relationship between the oxygen concentration in test example 1 and the fluorescence ratio of the reference probe to the oxygen probe.

FIG. 4b is a graph showing the relationship between the concentration of potassium ions and the fluorescence ratio of potassium ion probe to reference probe in test example 1.

FIG. 5a is a graph comparing the effect of the film in test example 2 on the concentration of potassium and sodium ions on the fluorescence intensity.

FIG. 5b is a graph showing the comparison of the fluorescence intensity of the film in test example 2 for potassium ions and different metal ions,

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.

Synthesis example 1

This synthesis example provides a method for synthesizing a potassium ion probe KS-23, specifically as follows:

(1) 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 with 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. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and subjected to silica gel column chromatography with a flowability of PE (petroleum ether): EA (ethyl acetate): 1:2 to obtain 2.5g of a white solid with a yield of 35.3%.

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。

(2) Synthesis of Compound 3: under the protection of nitrogen, 2(2.5g, 15.06mmol) and 2, 4-dimethylpyrrole (3g, 31.63mmol) were added into a 500mL two-necked flask, dissolved in 160mL of tetrahydrofuran, stirred for 30min, added with 160. mu.L of trifluoroacetic acid, and the reaction solution was stirred overnight with the color changed from yellow to red. After completion, a solution of DDQ (3.74g, 16.57mmol) in tetrahydrofuran was slowly added dropwise and after 3h, a solution of triethylamine (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。

(3) 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%.

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.

synthesis example 2

This synthesis example provides a method for synthesizing a reference probe RP, which specifically comprises:

(1) synthesis of Compound 5

Compound 4(5g, 25.2mmol), 2-aminoethanol (15.4g, 252mmol) and 80ml of ethanol were refluxed in a 150ml round-bottomed flask for 10 hours and then cooled. Filtering out precipitated solid and recrystallizing with ethanol to obtain pure product. Yield: 88.5 percent.

(2) Synthesis of RP

Compound 5(1.50g, 5mmol), Et₃N (1.01g, 10mmol) and 80mL CH₂Cl₂After mixing, the mixture was cooled in an ice bath. Methacryloyl chloride (1.04g, 10mmol) was then added. After 6 hours at room temperature, concentrate in vacuo. Purifying the rest solid with silica gel column, and using petroleum ether/ethyl acetate (5:1) as developing agent to obtain pure solid. The yield was 89%.

¹H NMR(400MHz，Chloroform-d)δ8.63(dd，J＝7.3，1.1Hz，2H)，8.25(dd，J＝8.3，1.1Hz，2H)，7.78(dd，J＝8.2，7.3Hz，2H)，6.07(dt，J＝1.8，1.0Hz，1H)，5.53(p，J＝1.6Hz，1H)，4.59(ddd，J＝6.0，5.1，1.1Hz，2H)，4.52(ddd，J＝6.0，5.0，1.1Hz，2H)，1.89(t，J＝1.3Hz，3H)。

The NMR instrument used for characterization of the synthesis examples was Bruker Avance III and the Mass Spectrometer was Thermo LTQ Orbitrap XL Mass Spectrometer.

Example 1

The embodiment provides a potassium ion and dissolved oxygen dual-functional sensing film, and the preparation method thereof is as follows:

(1') mixing polyethylene glycol methacrylate, methacrylic acid, polyethylene glycol dimethacrylate, an oxygen probe, a reference probe, an initiator and a solvent, wherein the mass ratio of the polyethylene glycol methacrylate, the polyethylene glycol dimethacrylate, the oxygen probe, the reference probe, the initiator and the solvent is 800:50:5:5:10:1000, placing 30 microliter of mixed solution on a TMSPA modified quartz carrier, covering the quartz carrier with a cover glass, and placing the quartz carrier in an 80-DEG oven for polymerization for 1 hour. Taking out the glass cover, and removing the glass cover to obtain a film covered with a gel layer;

(2 ') the film obtained in step (1') was placed in 1mL of DMF solution containing 0.2mg of KS-23, 100mg of EDC and 20mg of DMAP to conduct grafting of a potassium ion probe. The reaction was carried out at room temperature after 12 hours. And taking out the film, washing the film by using DMF (dimethyl formamide) and deionized water to obtain the potassium ion and dissolved oxygen dual-functional sensing film, and performing performance test.

The method for preparing the potassium ion and dissolved oxygen dual-functional sensing film in example 1 is shown in fig. 1.

Test example 1

The potassium ion and dissolved oxygen dual-function sensing film (referred to as film for short) of example 1 was placed in a quartz watch glass, and 3ml of water was added. The concentration of oxygen is controlled by introducing mixed gas of nitrogen and oxygen with different oxygen partial pressures into the liquid. The concentration of potassium ions was controlled by adding varying concentrations of KCl to the liquid. The quartz dishes were tested in a PerkinElemer LS55 fluorescence tester (PerkinElemer, Waltham, MA, USA).

FIGS. 2a and 2b show the response of the reference probe to oxygen and potassium ions in the film, respectively, at an excitation wavelength of 350 nm. Since the probe does not contain groups responsive to potassium ions and oxygen, it can be clearly seen that the reference probe does not respond to both potassium ions and oxygen, demonstrating that the probe is a good reference.

FIG. 3a shows the response of the film to potassium ions under excitation at 405 nm, the concentration gradient of potassium ions is 0mM, 20mM, 40mM, 60mM, 80mM, 100mM, 120mM, 150mM, 200mM, 300mM, and it can be seen that the fluorescence at 574 nm increases with the increase of potassium ion concentration, while the fluorescence intensity from the oxygen probe at 670nm is not affected by the potassium ion concentration. FIG. 3b shows the response of the film to oxygen under 405 nm excitation, and it can be seen that the potassium probe does not respond to oxygen. Only the peak at 670nm of the oxygen probe increases as the concentration of oxygen decreases. Thus, a three-color bifunctional probe film is realized.

FIG. 4a shows the oxygen concentration and the fluorescence ratio (F) of the reference probe to the oxygen probe₃₈₀/F₆₇₀) FIG. 4b shows the potassium ion concentration and the fluorescence ratio of potassium ion probe to reference probe (F)₅₇₄/F₃₈₀) Wherein the fluorescence intensity of 380nm is used for the fluorescence of the reference probe, the fluorescence intensity of 670nm is used for the fluorescence of the oxygen probe, and the fluorescence intensity of 574 nm is used for the fluorescence of the potassium ion probe.

From FIG. 4a, it can be seen that the oxygen concentration (controlled by a 21-1-15-0-500-KMG3 model mass flow controller (Alicat Scientific, Tucson, AZ, USA)) and fluorescence intensity ratio obeyed the Stern-Volmer linear equation, indicating that oxygen concentration from anoxic (0mg/L) to saturated oxygen (oxygen solubility 8.6mg/L at normal atmospheric pressure with 21% oxygen in water at 23 degrees Celsius and saturated oxygen solubility 40.9mg/L at 100% oxygen partial pressure) can be measured well. Wherein the oxygen concentration refers to the concentration of dissolved oxygen.

From FIG. 4b, it can be seen that the probe can detect potassium ions from potassium ion-free solution to 100mM potassium solution with higher sensitivity, and the fluorescence intensity in this range is more sensitive to the change of the potassium ion concentration. Although the sensitivity decreased above 100mM, detection was still possible. Therefore, the material can realize the detection of potassium ion-free solution to solution containing 250mM potassium ions. Can be applied to the detection of potassium ions in blood (generally 3.5-5.5mM), can also realize the detection of potassium ions in urine (generally 25-125mM), and has certain application prospect.

Test example 2

A quartz plate (size 12.7X 1mm) with the sensor film of example 1 was placed diagonally in a 10X 10mm cuvette. Incident light was directed at an angle of 45 degrees to the quartz plate, 3mL of a buffer solution (Tris-HCl) having a pH of 7.4 was added to the cuvette, and then a 3M KCl solution was gradually added dropwise to increase the concentration from 0 to 300mM, and each titration was waited for 1min, and then data was collected.

FIG. 5a shows a graph comparing the effect of the film on the concentration of potassium and sodium ions on the fluorescence intensity (F)₀Is the fluorescence intensity before no ions are added, F is the fluorescence intensity at different ion concentrations), figure 5b gives a graph comparing the fluorescence intensity of potassium ions with different physiologically relevant ions. It can be seen that sodium ions and other ions hardly affect the fluorescence of the film. Indicating that the sensing film has high selectivity to potassium ions.

(2) The oxygen and potassium ion concentrations in an aqueous solution containing about 150mM potassium ions were measured using 2 films (all obtained by the method of example 1), and the measured data were compared with the values obtained by electrode measurement, as shown in Table 1, and an error of less than 10% was found, indicating that there is a certain application prospect.

TABLE 1

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

1. The potassium ion and dissolved oxygen dual-function sensing film is characterized by comprising a carrier, and a potassium ion probe, an oxygen probe and a reference probe which are fixed on the surface of the carrier in a chemical bonding mode;

the potassium ion probe has the following structure:

the oxygen probe has the following structure:

the reference probe has the following structure:

2. the potassium ion and dissolved oxygen dual-function sensing film of claim 1, wherein the support is a TMSPA modified quartz support, and the TMSPA has the following structure:

3. a method for preparing the potassium ion and dissolved oxygen dual-function sensing film according to claim 1 or 2, wherein the method comprises the following steps:

(1') polymerizing polyethylene glycol methacrylate, methacrylic acid, polyethylene glycol dimethacrylate, an oxygen probe and a reference probe on the surface of a carrier to obtain the carrier covered with a hydrogel layer;

(2') grafting a potassium ion probe on the surface of the carrier covered with the hydrogel layer to obtain the potassium ion and dissolved oxygen dual-function sensing film.

4. The method according to claim 3, wherein the surface of the carrier contains a double bond.

5. The process according to claim 3, wherein in the step (1'), the initiator for the polymerization reaction is azobisisobutyronitrile.

6. The method according to claim 3, wherein in the step (1'), the solvent for the polymerization reaction is N, N-dimethylformamide.

7. The method according to claim 3, wherein in the step (1'), the support is a TMSPA-modified quartz support.

8. The method according to claim 3, wherein in the step (1'), the mass ratio of the polyethylene glycol methacrylate, the polyethylene glycol dimethacrylate, the oxygen probe, the reference probe, the initiator and the solvent is 500-1500:20-300:1-15:1-15:5-30: 1000.

9. The method according to claim 8, wherein in the step (1'), the mass ratio of the polyethylene glycol methacrylate, the polyethylene glycol dimethacrylate, the oxygen probe, the reference probe, the initiator and the solvent is 800:50:5:5:10: 1000.

10. The production method according to claim 3, wherein in the step (1'), the mass ratio of the methacrylic acid to the polyethylene glycol methacrylate is 10-1000: 1.

11. The method as claimed in claim 3, wherein the number average molecular weight of the polyethylene glycol methacrylate in step (1') is 300-2000.

12. The production method according to claim 11, wherein in the step (1'), the number average molecular weight of the polyethylene glycol methacrylate is 500.

13. The preparation method as claimed in claim 3, wherein in the step (1'), the number average molecular weight of the polyethylene glycol dimethacrylate is 300-2000.

14. The method according to claim 13, wherein in the step (1'), the number average molecular weight of the polyethylene glycol dimethacrylate is 750.

15. The production method according to claim 3, wherein in the step (1'), the polymerization reaction is carried out under a nitrogen atmosphere.

16. The production method according to claim 3, wherein in the step (1'), the temperature of the polymerization reaction is 70 to 90 ℃.

17. The production method according to claim 16, wherein in the step (1'), the temperature of the polymerization reaction is 80 ℃.

18. The process according to claim 3, wherein in the step (1'), the polymerization reaction time is 0.8 to 1.5 hours.

19. The method according to claim 18, wherein in the step (1'), the polymerization reaction time is 1 hour.

20. The process according to claim 3, wherein step (1') comprises in particular: mixing polyethylene glycol methacrylate, methacrylic acid, polyethylene glycol dimethacrylate, an oxygen probe, a reference probe, an initiator and a solvent, taking the mixed solution to be arranged on the surface of a TMSPA modified quartz carrier, and polymerizing for 0.8-1.5h at the temperature of 70-90 ℃ to obtain the carrier covered with the hydrogel layer.

21. The process according to claim 3, wherein in the step (2'), a coupling agent and/or a catalyst is added.

22. The method of claim 21, wherein the coupling agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

23. The method of claim 21, wherein the catalyst is 4-dimethylaminopyridine.

24. The production method according to claim 3, wherein in the step (2'), the mass ratio of the potassium ion probe, the coupling agent and the catalyst is 2:1000: 200.

25. The method according to claim 3, wherein in the step (2'), the reaction solvent for grafting is N, N-dimethylformamide.

26. The method according to claim 3, wherein in the step (2'), the reaction time for grafting is 10 to 15 hours.

27. The method according to claim 26, wherein in the step (2'), the reaction time for grafting is 12 hours.

28. The method according to claim 3, wherein in the step (2'), the reaction of grafting is carried out at room temperature.

29. The method according to claim 3, wherein the step (2') comprises the steps of: and (3) placing the carrier covered with the hydrogel layer in a mixed solution of a potassium ion probe, a coupling agent and a catalyst, and carrying out grafting reaction for 10-15h at room temperature to obtain the potassium ion and dissolved oxygen dual-function sensing film.

30. Use of the potassium ion and dissolved oxygen bifunctional sensing membrane of claim 1 or 2 for potassium ion detection and dissolved oxygen detection.