Cu < 2 + > ion fluorescence chemical sensor as well as synthesis method and application thereof
By introducing polyether groups and isothiocyanate fluorescein into the polyvinylamine structure, a Cu2+ ion fluorescent chemical sensor was formed, solving the technical problem of existing technologies' difficulty in penetrating cells. This enabled highly sensitive detection of intracellular copper content, accurate detection of intracellular copper, highly sensitive detection of intracellular copper content, highly sensitive detection of intracellular copper ions, and highly sensitive detection of intracellular copper. This also solved the technical problem of existing fluorescent sensors' difficulty in penetrating cells, achieving highly sensitive detection of intracellular copper and highly efficient detection of intracellular copper ions.
Patent Information
- Application Number
- CN202511310258.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-15
- Publication Date
- 2025-11-11
AI Technical Summary
Existing fluorescent probes have difficulty penetrating cells, which limits their application in the field of biofluorescent probes, and they also lack the ability to detect changes in intracellular copper ions with high sensitivity.
Using a polyvinylamine structure as the backbone, polyether groups and isothiocyanate fluorescein are introduced. The polyether segments are modified with benzenesulfonyl chloride and linked to isothiocyanate fluorescein to form a Cu2+ ion fluorescent chemical sensor with high water solubility and biocompatibility.
It achieves highly sensitive detection of changes in intracellular Cu2+ ions, exhibits good biocompatibility and optical response, and is suitable for cell imaging and disease diagnosis in biomedical research.
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Figure CN120923797A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of analytical chemistry materials and relates to a Cu 2+ Ion fluorescence chemical sensors, their synthesis methods, and applications. Background Technology
[0002] Copper, as an important trace element, plays a crucial role in numerous physiological processes within cells, including but not limited to mitochondrial respiration, antioxidant defense, and neurotransmitter synthesis. However, imbalances in intracellular copper levels are closely associated with various diseases, such as Wilson's disease and Alzheimer's disease. Therefore, developing a fluorescent chemical sensor capable of accurately detecting intracellular copper levels is of paramount importance.
[0003] This study focuses on a novel fluorescent chemical sensor for detecting intracellular copper levels. Based on a specific chemical structure design, the core principle of this sensor is the unique chemical reaction between copper ions and specific functional groups in the sensor molecule, triggering detectable signal changes such as alterations in fluorescence intensity and shifts in the absorption spectrum. The effectiveness of the fluorescent chemical sensor was validated through a series of cell experiments at the cellular level. Introducing the sensor into different cell types successfully enabled the monitoring of intracellular copper levels. Experimental results show that the sensor exhibits good cell compatibility and minimal interference with normal cellular physiological functions. Furthermore, it demonstrates high selectivity and sensitivity to copper ions, accurately recognizing and binding them in the complex intracellular environment, exhibiting a good linear response over a wide range of copper ion concentrations, with a detection limit as low as the nanomolar level.
[0004] Furthermore, this fluorescent chemical sensor possesses excellent optical properties, such as a strong fluorescence emission peak and good photostability, making the detection process more convenient, efficient, and accurate. By detecting changes in copper content in cells under different physiological and pathological states, the potential application value of this fluorescent chemical sensor in the field of biomedical research has been further confirmed. It is expected to provide powerful tools and technical support for in-depth research into the pathogenesis of diseases related to copper ions and apoptosis, as well as for the early diagnosis of these diseases, thus advancing research in related fields. Summary of the Invention
[0005] The purpose of this invention is to overcome the above-mentioned defects of the prior art and provide a highly sensitive Cu with good biocompatibility and specific optical response. 2+ Ion fluorescence chemical sensor. The Cu of this invention... 2+The ion fluorescence chemical sensor uses a polyethyleneamine structure as its backbone, and introduces polyether groups and fluorescein isothiocyanate isomer (FITC). The polyethyleneamine backbone contains nitrogen atoms, which are easily protonated and used as bonding groups. FITC acts as a fluorophore, and has the advantages of good water solubility, high quantum yield, and sensitive fluorescence response.
[0006] To achieve the objectives of this invention, the technical solution adopted is: highly sensitive Cu 2+ An ion fluorescence chemical sensor comprises a polyethyleneamine (PVAm) molecular framework, a polyether highly water-soluble unit, a hydrogen ion bonding unit, and a signal transmission unit, an isothiocyanate fluorophore; wherein, the Cu... 2+ The ion fluorescence chemical sensor is shown in formula (I):
[0007]
[0008] Where n, a, and b are all integers greater than 1, and nab > 1; Y is a polyether of polyethylene glycol-polypropylene glycol-polyethylene glycol copolymer (molecular weight 500-18000), and R is a fluorescent group. Further, R is fluorescein isothiocyanate (FITC). The structural formula (II) is as follows:
[0009]
[0010] This invention also provides a highly sensitive Cu with specific optical response. 2+ The method for preparing an ion-fluorescence chemical sensor is based on a polyvinylamine molecular backbone, with the introduction of a polyether segment and a fluorophore, thereby giving it high water solubility in aqueous solution and sensitivity to Cu. 2+ The ions exhibit a specific fluorescence response. Specifically, using polyvinylamine molecules as the molecular backbone, polyether and fluorescein groups are introduced onto its amino groups, respectively. The amino groups on the polyvinylamine molecule serve as bonding sites, the polyether improves water solubility and biocompatibility, and the fluorescein group serves as a signal unit, thus synthesizing Cu. 2+ Ion fluorescence chemical sensor. Its reaction route is as follows:
[0011] This invention is the first to propose linking isothiocyanate fluorescein with polyethyleneamine molecules to obtain Cu. 2+ Ion fluorescence chemical sensor.
[0012] Furthermore, benzenesulfonyl chloride (BsCl) is used in the reaction, and the reaction is carried out at room temperature.
[0013] This invention Cu 2+ Ion fluorescence chemical sensors can detect Cu in cells 2+The ion changes trigger a response. In the prior art, the application of isothiocyanate fluorescein in the field of biofluorescent probes is limited because it cannot penetrate cells. This invention, Cu... 2+ Ion fluorescence chemical sensors can detect tiny Cu particles within cells through cell imaging experiments. 2+ The invention responds to changes in ions. Through cell imaging experiments, the Cu of this invention... 2+ The fluorescent chemical sensor can enter cells after being co-cultured with cells in serum-free medium to perform fluorescence imaging, thus providing a new application for isothiocyanate fluorescein.
[0014] This invention provides a highly sensitive Cu with specific optical response. 2+ The preparation method of the ion fluorescence chemical sensor has the advantages of mild reaction conditions, simple operation, and high yield. In particular, the purification process only requires dialysis, avoiding the use of time-consuming and labor-intensive column chromatography purification methods.
[0015] The Cu provided by this invention 2+ The ion fluorescence chemical sensor has good biocompatibility, low cytotoxicity, and is suitable for use in biological detection.
[0016] The specific synthesis method is as follows:
[0017] (1) Synthesis of polyether-modified polyethyleneamine derivatives
[0018] Step 1: The polyether (Y) was pre-dried in a 60℃ oven under vacuum for 24 hours and then set aside. The polyether was dissolved in dry anhydrous dichloromethane (DCM), followed by the addition of a certain amount of anhydrous pyridine (Py) and benzenesulfonyl chloride (BsCl). The mixture was stirred at room temperature for 3 hours under nitrogen protection. After the reaction was complete, the mixture was extracted with 3 mol / L hydrochloric acid and washed with saturated NaHCO3 solution. After extraction, the organic phase was dissolved in tetrahydrofuran, recrystallized in ice-cold diethyl ether solvent, and then dried under vacuum to obtain the benzenesulfonated polyether intermediate (Y-OBs). The specific reaction formula is as follows:
[0019]
[0020] Step 2: Polyvinylamine is dissolved in deionized water to obtain a polyvinylamine solution. The benzenesulfonated polyether (Y-OBs) intermediate obtained in Step 1 is dissolved in deionized water to obtain an active benzenesulfonated intermediate solution. The polyvinylamine solution and the active benzenesulfonated intermediate solution are mixed and reacted at 60°C for 12–24 h. After the reaction is complete, the liquid is placed in a 2000 Da dialysis bag and purified by dialyzing in water. After dialysis purification, the liquid is concentrated by rotary evaporation and freeze-dried to obtain a white solid polyether-polyvinylamine copolymer. The specific reaction formula is as follows:
[0021]
[0022] (2) Cu based on polyether-polyvinylamine copolymer 2+ Synthesis of ion fluorescence chemical sensor molecules
[0023] A certain amount of fluorescein isothiocyanate (FITC) was weighed and dissolved in a 0.1M Na₂CO₃-NaHCO₃ buffer solution (pH approximately 9-10). First, a polyether-polyethyleneamine copolymer solution (10 mg / ml, dissolved in DEPC water) was mixed with the FITC solution (2 mg / ml, dissolved in Na₂CO₃-NaHCO₃ buffer solution). The mixture was reacted at room temperature in the dark for 3 hours. Then, the reaction solution was dialyzed through a 2000 kDa dialysis bag to remove unreacted FITC (alternating dialyzing with Na₂CO₃-NaHCO₃ buffer and distilled water). Finally, the mixture was freeze-dried to obtain an orange-red solid, which is Cu. 2+ Ion chemical sensor. The specific reaction formula is as follows:
[0024]
[0025] The present invention also provides Cu 2+ Ion fluorescence chemical sensor for detecting Cu in solution 2+ Applications of Cu ions. In the pH range of 4.00-12.00, Cu... 2+ Ion fluorescence chemical sensors are unaffected by pH value and common metal ions such as Li. + Na + Mg 2+ Al 3 + K + Ca 2+ Cr 3+ Mn 2+ Fe 3+ Fe 2+ Zn 2+ Ti 4+ Cs + Ag + 、Sr 2+ Ba 2+ Pb 2+ and anion Cl - ,Br - I - CO3 2- HCO3 - H2PO4 - SO4 2- SO3 2- and OAC - Interference. Cu 2+ Ions can react with Cu 2+In ion-fluorescence chemical sensors, the formation of selective complexes alters the fluorescence properties of the signal unit, causing energy transfer and a decrease in fluorescence (quenching), thus achieving selective sensing of Cu. 2+ The role of ions.
[0026] Cu 2+ Ion fluorescence chemical sensor for Cu in solution 2+ The ion concentration sensing study was conducted in deionized water. Cu was prepared... 2+ An ion fluorescence chemical sensor was used to analyze the fluorescence emission spectra of a series of solutions with a fixed concentration (0.1 g / mL) but different pH values. After standing for 0.5 hours, the fluorescence emission spectra of the solutions at different pH values were measured. The obtained data were then plotted and analyzed using Origin Pro 2022 to observe the changes in the spectra. This invention also provides the Cu... 2+ Ion fluorescence chemical sensors for biological cell imaging and Cu in intracellular environments 2+ Applications of ion detection. Fluorescein isothiocyanate, as an organic dye, cannot penetrate cells, limiting its application in the field of bioluminescent probes. This invention provides the application of fluorescein isothiocyanate in the preparation of Cu... 2+ Applications in ion fluorescence chemical sensors. This invention uses Cu... 2+ Ion fluorescence chemical sensors can detect Cu in cells 2+ The ion changes elicit a response. This invention combines isothiocyanate fluorescein with polyether-modified polyethyleneamine to obtain Cu. 2+ Ion fluorescence chemical sensor. This invention's pH fluorescence chemical sensor links isothiocyanate fluorescein to polyethyleneamine via the formation of a thiourea group, enabling co-culture with cells to detect intracellular Cu. 2+ Ion changes. The CCK8 experiment showed that Cu... 2+ The ion-fluorescence chemical sensor exhibits low cytotoxicity. This invention holds significant importance in bioanalysis and pharmaceutical biology. Cell imaging experiments have demonstrated that the Cu of this invention… 2+ The ion-fluorescent chemical sensor can enter cells after co-culturing with cells in serum-free medium, enabling fluorescence imaging of Cu in the cells. 2+ Applications in detection. This invention innovatively proposes a new application approach for isothiocyanate fluorescein.
[0027] This invention Cu 2+ The signal unit of the ion fluorescence chemical sensor, fluorescein isothiocyanate, possesses advantages such as good water solubility, good biocompatibility, and easily observable fluorescence. Fluoroscein isothiocyanate can be applied to cell imaging and Cu... 2+ Ion detection provides a new application for isothiocyanate fluorescein. Attached Figure Description
[0028] Figure 1 Cu of the present invention 2+ Infrared spectra of molecules in an ion fluorescence chemical sensor.
[0029] Figure 2 Cu of the present invention 2+ The proton NMR spectrum of an ion fluorescence chemical sensor molecule.
[0030] Figure 3 Cu of the present invention 2+ Determined spectra of fluorescence excitation, emission wavelengths, and ultraviolet absorption of an ion fluorescence chemical sensor; in the figure: a is the ultraviolet spectrum, b is the emission spectrum, and c is the excitation spectrum.
[0031] Figure 4 Cu of the present invention 2+ Fluorescence lifetime spectrum of an ion fluorescence chemical sensor.
[0032] Figure 5 Cu of the present invention 2+ A graph showing the relationship between the fluorescence intensity at the maximum emission peak of an ion fluorescence chemical sensor and the variation of different metal ions.
[0033] Figure 6 Cu of the present invention 2+ A graph showing the relationship between the fluorescence intensity at the maximum emission peak of an ion fluorescence chemical sensor and different anions.
[0034] Figure 7 Cu of the present invention 2+ Ion fluorescence chemical sensors in different Cu 2+ Fluorescence spectrum under ion concentration conditions.
[0035] Figure 8 Cu of the present invention 2+ The fluorescence intensity of the ion fluorescence chemical sensor at the maximum emission peak is related to Cu 2+ A graph showing the relationship between changes in ion concentration.
[0036] Figure 9 Cu of the present invention 2+ Graphs showing the detection of CCK8 toxicity in HeLa cells at different concentrations using an ion fluorescence chemical sensor.
[0037] Figure 10 Cu of the present invention 2+ Graphs showing the detection of CCK8 toxicity in B16F1 cells at different concentrations using an ion fluorescence chemical sensor.
[0038] Figure 11 Cu of the present invention 2+ Ion fluorescence chemical sensor and HeLa cells, Cu 2+ Ion fluorescence chemical sensor and intracellular Cu 2+Fluorescence microscopy images of HeLa cells after co-culturing in the intracellular environment.
[0039] Figure 12 Cu of the present invention 2+ Ion fluorescence chemical sensor and B16F1 cells, Cu 2+ Ion fluorescence chemical sensor and intracellular Cu 2+ Fluorescence microscopy image of B16F1 cells after co-culture in the intracellular environment. Detailed Implementation
[0040] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.
[0041] Example 1: Cu containing isothiocyanate fluorescein 2+ Synthesis of fluorescent chemical sensors:
[0042] (1) Synthesis of polyether-modified polyethyleneamine derivative (L44-PVAm):
[0043] Step 1: Polyether L44 (Maclean Chemicals Co., Ltd.) was dried under vacuum at 60°C for 24 hours and then set aside for use. 10.0 g (1.6 mmol) of polyether L44 was added to a 250 mL round-bottom flask, followed by 40 mL of anhydrous pyridine and 11 mL of benzenesulfonyl chloride. The reaction was carried out at room temperature under nitrogen for 3 hours. After the reaction was complete, the mixture was extracted with 3 mol / L hydrochloric acid, then washed with saturated NaHCO3 solution, and finally washed with saturated NaCl solution. After washing, the mixture was dried over anhydrous magnesium sulfate and filtered to obtain a white solid. The collected white solid was dissolved in tetrahydrofuran and recrystallized three times in ice-cold diethyl ether. The resulting product was then dried under vacuum to obtain a white solid benzenesulfonated L44 intermediate with a yield of 80%.
[0044] The structural formula of the benzenesulfonated L44 intermediate, the product of the first step above, is as follows:
[0045]
[0046] Step 2: Dissolve 0.05 g of polyvinylamine (PVAm) prepared in our laboratory with a number average molecular weight of 2500–12000 in 10 mL of deionized water to obtain a polyvinylamine solution. Simultaneously, weigh 0.05 g of the benzenesulfonated L44 intermediate obtained in Step 1 and dissolve it in 5 mL of deionized water to obtain an active benzenesulfonated intermediate solution. Mix the two liquids and react at 60 °C for 24 h. After the reaction is complete, place the liquid in a 2000 kDa dialysis bag and dialyze it in water for purification. Change the liquid every 6-8 h and dialyze for 72 h. After dialysis purification, concentrate the liquid by rotary evaporation and freeze-dry for 24 h to obtain a white solid L44-PVAm with a yield of 76%.
[0047] The structural formula of the product L44-PVAm from the second step is as follows:
[0048]
[0049] (2) Cu based on the second-step product L44-PVAm 2+ Synthesis of ion fluorescence chemical sensor molecules
[0050] Weigh 0.005 g of fluorescein isothiocyanate (FITC) and dissolve it in 1 mL of 0.1 M Na2CO3-NaHCO3 (pH approximately 9-10) buffer solution to obtain FITC solution. First, mix the L44-PVAm (10 mg / mL, dissolved in DEPC water) obtained in step (1) with the above FITC solution (5 mg / mL, dissolved in 1 mL of 0.1 M Na2CO3-NaHCO3 buffer solution) and react at room temperature in the dark for 3 h. Then dialyze the reaction solution with a 2000 kDa dialysis bag for 24 h, and dialyze alternately with Na2CO3-NaHCO3 buffer and distilled water for a total of 6 dialyzes to remove unreacted FITC. Finally, freeze-dry to obtain an orange-red solid, which is Cu. 2+ Ion chemical sensor, yield 86%. Figure 1 Cu of the present invention 2+ Infrared spectra of molecules in an ion fluorescence chemical sensor. Figure 2 Cu of the present invention 2+ The proton NMR spectrum of an ion fluorescence chemical sensor molecule.
[0051] The Cu prepared in this embodiment 2+ Structure of ion chemical sensor:
[0052]
[0053] Where n, a, and b are all integers greater than 1, and nab>1; R is fluorescein isothiocyanate (FITC).
[0054] Example 2: Cu2+ Ion chemical sensor in solution Cu 2+ Applications of ion detection
[0055] Prepare a solution with a concentration of 1 mg / mL. -1 Cu prepared in Example 1 2+ The ion chemical sensor solution was allowed to stand for 0.5 hours, and its fluorescence and ultraviolet spectra were measured. Figure 3 The spectrum shown indicates the fluorescence excitation and emission wavelengths of the pH fluorescence chemical sensor of this invention. The excitation wavelength of the molecule is determined to be 494 nm, and the emission wavelength is 517 nm. The fluorescence lifetime spectrum was measured to determine the Cu... 2+ The fluorescence lifetime of the ion chemical sensor is 30 ns (see...) Figure 4 ).
[0056] To investigate Cu 2+ The ion chemical sensor solution's immunity to potential ions, and its resistance to polymers and metal ions (Li). + Na + Mg 2+ Al 3+ K + Ca 2+ Cr 3+ Mn 2+ Fe 3+ Fe 2+ Cu 2+ Zn 2+ Ti 4+ Cs + Ag + 、Sr 2+ Ba 2+ Pb 2+ and anion Cl - ,Br - I - CO3 2- HCO3 - SO4 2- SO3 2- H2PO4 - and OAC - The reaction was monitored. A solution containing ions (10M, 0.1mL) was added to 10mL of the L44-PVAm-FITC polymer. After standing for 1 hour, its fluorescence emission spectrum was measured. Figure 5 and Figure 6 As shown, these potential metal ion interfering substances affect the Cu of this invention. 2+ The maximum emission intensity of the ion chemical sensor solution is not significantly affected, and the effect on Cu is not significant. 2+ The probe exhibits significant selectivity for Cu ions, indicating that it is sensitive to Cu. 2+The ion exhibits outstanding selectivity compared to other ions. In particular, the decrease in fluorescence intensity at an excitation wavelength of 520 nm under UV radiation (Ex = 395 nm) is significantly different from that of other metal ions.
[0057] According to Cu 2+ The concentration range of the ions is 0–70 μM, for Cu 2+ The ion chemical sensor performed ratiometric fluorescence detection on Cu. 2+ Ion chemical sensor and Cu 2+ The fluorescence intensity of Cu after the interaction of ions at 520 nm varies. 2+ The change in ion concentration shows a decreasing trend. Figure 7 and Figure 8 The above results indicate that Cu 2+ Ion chemical sensors are potential ratiometric probes that can be used to detect Cu in complex biological samples. 2+ Highly selective detection by a chemical sensor. This experimental study demonstrates the detection of Cu. 2+ The design of the ion fluorescence chemical sensor is both reasonable and practically significant.
[0058] Example 3: Cu 2+ Ion fluorescence chemical sensors for detecting Cu in biological cells 2+ Applications of ion concentration in cytotoxicity (CCK8) assays
[0059] Collect cells in the logarithmic growth phase. Digest adherent cells with 0.1 wt% trypsin, then resuspend them in fresh culture medium to adjust the cell suspension concentration. Seed the cells in 100 μL wells of a 96-well plate to achieve a cell density of 2 × 10⁶ cells / well. 5 Each experimental and control group had 5 replicates. The culture was incubated overnight at 37°C with 5 wt% CO2. The culture medium was then discarded, and different concentrations (0 μg / mL–20 μg / mL) of Cu were added. 2+ The ion fluorescence chemiluminescence sensor culture medium (100 μL) was incubated for 24 hours. 10 μL of CCK8 solution was added to each well, and after shaking for 5 minutes, the cells were incubated for another hour at 37°C with 5 wt% CO2. The optical density of each well was then measured at 570 nm. The values of Cu at different concentrations were calculated. 2+ Cell viability under the action of an ion-fluorescence chemiluminescence sensor. Cell viability = (mean absorbance of experimental group / mean absorbance of blank control) × 100%. Figure 9 Cu 2+ An ion-fluorescent chemical sensor was co-cultured with HeLa cells for 24 hours. Figure 10 Cu 2+ The ion fluorescence chemical sensor was co-cultured with B16F1 cells for 24 hours. Figure 9 and Figure 10This invention represents Cu 2+ Ion fluorescence chemical sensor cell toxicity studies showed low levels; cell viability exceeded 95%, i.e., Cu 2+ Ion fluorescence chemical sensors exhibit excellent biocompatibility and low cytotoxicity, making them a promising candidate for applications in the field of biomaterials.
[0060] Different Cu 2+ Cell imaging experiments at ion concentrations
[0061] Collect cells in the logarithmic growth phase. Digest adherent cells with 0.1 wt% trypsin and then resuspend them in fresh culture medium (serum-free). Adjust the cell suspension concentration and seed the cells into 24-well plates covered with coverslips, 1000 μL per well, to achieve a cell concentration of 2 × 10⁻⁶ cells / well. 5 After incubating at 37°C for 12 hours in a 5 wt% CO2 environment, the old culture medium solution in each well was discarded, and then a solution containing 10 μg / mL Cu was added. 2+ The culture medium for the ion-fluorescent chemical sensor was co-cultured with cells for 12 hours. The culture medium was aspirated, the cells were washed three times with PBS, and then PBS was added again. The cells were then observed and photographed under a fluorescence microscope. Figure 11 and Figure 12 This invention represents HeLa cells, B16F1, and Cu. 2+ Fluorescence image of an ion fluorescence chemical sensor after 12 hours of co-culturing.
[0062] After imaging with a fluorescence inverted microscope, carefully aspirate the old PBS and add a solution containing 10 μg / mL Cu. 2+ The cells were co-cultured with the ion culture medium for 12 hours. The culture medium was aspirated, the cells were washed three times with PBS, and then fresh complete culture medium was added to culture the cells for 24 hours. After complete culture, the cells were washed three times with PBS and fixed with 4 wt% paraformaldehyde solution for 20 minutes. The cells were then observed and photographed under a fluorescence microscope.
[0063] Cu 2+ After co-culturing cells, the ion-fluorescent chemical sensor, as demonstrated by fluorescence inverted microscopy imaging, showed the presence of normal Cu in the cells. 2+ At certain ion concentrations, intracellular fluorescence intensity is stronger; when using high concentrations of Cu, the fluorescence intensity is higher. 2+ After co-culturing cells with ions, intracellular fluorescence quenched, which is the result of the Cu synthesized in this invention. 2+ The ion-fluorescent chemical sensor has good biocompatibility and can detect intracellular Cu. 2+ The concentration of ions is detected.
[0064] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.
Claims
1. A Cu 2+ An ion fluorescence chemical sensor, characterized in that, It comprises a polyethyleneamine molecular backbone, polyether, and isothiocyanate fluorescein fluorophore; the Cu 2+ The structural formula of the ion fluorescence chemical sensor is as follows: (I) Structure: ； Wherein, n, a, and b are all integers greater than 1, and nab>1; Y is a polyether of polyethylene glycol-polypropylene glycol-polyethylene glycol copolymer, wherein the molecular weight of the polyether is 500-18000, and R is fluorescein isothiocyanate.
2. The Cu according to claim 1 2+ A method for preparing an ion fluorescence chemical sensor, characterized in that, A pH fluorescent chemical sensor was synthesized by introducing polyether and isothiocyanate fluorescein groups onto the amino groups of polyvinylamine as the molecular backbone, using the amino groups on the polyvinylamine as the bonding sites and the fluorescein groups as the signal units. Its reaction route is shown in equation (II): 。 3. The Cu as described in claim 2 2+ A method for preparing an ion fluorescence chemical sensor, characterized in that, The reaction uses benzenesulfonyl chloride (BsCl) and is carried out at room temperature.
4. The Cu as described in claim 2 2+ A method for preparing an ion fluorescence chemical sensor, characterized in that, The specific steps are as follows: (1) Synthesis of polyether-modified polyethyleneamine derivatives: Step 1: The polyether was dried under vacuum at 60℃ for 24 h and then set aside for use. 5.0-15.0 g of polyether was added to a 250 mL round-bottom flask, followed by 20-60 mL of anhydrous pyridine and 5-15 mL of benzenesulfonyl chloride. The mixture was reacted at room temperature under nitrogen atmosphere for 3 h. After the reaction was completed, the mixture was first extracted with 3 mol / L hydrochloric acid, then washed with saturated NaHCO3 solution, and finally washed with saturated NaCl solution. After washing, the mixture was dried with anhydrous magnesium sulfate and filtered to obtain a white solid. The collected white solid was dissolved in tetrahydrofuran and recrystallized three times in ice-cold diethyl ether. The solid was then dried under vacuum to obtain a white solid benzenesulfonated polyether intermediate. Step 2: Polyvinylamine is dissolved in deionized water to obtain a polyvinylamine solution. The benzenesulfonated polyether intermediate obtained in Step 1 is dissolved in deionized water to obtain an active benzenesulfonated polyether intermediate solution. The polyvinylamine solution and the active benzenesulfonated polyether intermediate solution are mixed and reacted at 60°C for 12-24 h. After the reaction is completed, the liquid is placed in a 2000 Da dialysis bag and purified by dialysis in water. After dialysis purification, the liquid is concentrated by rotary evaporation and freeze-dried to obtain a white solid polyether-polyvinylamine copolymer. (2) Cu based on the second-step product, polyether-polyvinylamine copolymer 2+ Synthesis of ion fluorescence chemical sensor molecules Weigh 0.005~0.015 g of fluorescein isothiocyanate (FITC) and dissolve it in 1~3 mL of 0.1 M Na2CO3-NaHCO3 buffer solution to obtain FITC solution. Mix the polyether-polyethyleneamine copolymer obtained in step (1) with the above FITC solution and react at room temperature in the dark for 2~5 h. Then dialyze the reaction solution with a 2000 Da dialysis bag for 24 h, and dialyze alternately with Na2CO3-NaHCO3 buffer and distilled water. Finally, obtain an orange-red solid by freeze drying, which is Cu. 2+ Ion chemical sensor.
5. The Cu according to claim 1 2+ The Cu obtained by the preparation method according to any one of claims 2-4 is an ion fluorescence chemical sensor. 2+ Ion fluorescence chemical sensor for detecting Cu in solution 2+ Applications in ions.
6. The application as described in claim 5, characterized in that, Within the pH range of 4.00 to 12.00, the Cu 2+ Ion fluorescence chemical sensors can quantitatively detect Cu in solution. 2+ Ion concentration, unaffected by metal ions Li + Na + Mg 2+ Al 3+ K + Ca 2+ Cr 3 + Mn 2+ Fe 3+ Fe 2+ Zn 2+ Ti 4+ Cs + Ag + 、Sr 2+ Ba 2+ Pb 2+ and anion Cl - ,Br - I - CO3 2- HCO3 - H2PO4 - SO4 2- SO3 2- and OAC - Interference.
7. The Cu according to claim 1 2+ Fluorescent chemical sensor or Cu prepared by any of the preparation methods described in claims 2-4 2+ Ion fluorescence chemical sensors for cell imaging and Cu in intracellular environments 2+ Applications in detection.
8. The Cu according to claim 1 2+ Fluorescent chemical sensor or Cu prepared by any of the preparation methods described in claims 2-4 2+ Ion fluorescence chemical sensor in cells Cu 2+ Applications in detection.