CN113604213B - Nano fluorescent probe and preparation method and application thereof - Google Patents
Nano fluorescent probe and preparation method and application thereof Download PDFInfo
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
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Abstract
The invention provides a nano fluorescent probe which is a composite nano particle, wherein the composite nano particle mainly comprises C, N, O and Cl four elements to form a nanocrystal, and the nanocrystal comprises an-OH functional group, -NH 2 functional group and a C=C functional group. The preparation method comprises the following steps: s10, dissolving o-phenylenediamine, catecholamine and ferric chloride in a solvent to prepare a first solution, and adjusting the pH value to 1-6 by using an acidic reagent; step S20, placing the first solution in a reaction kettle for heating reaction to form a second solution; step S30, adding a surfactant into the second solution, and placing the mixture in a stirring container for stirring reaction to form a third solution; and S40, purifying the third solution, and drying to form composite nano particle solid powder to obtain the nano fluorescent probe. The fluorescent nano probe provided by the invention has different color responses to different pH values, and has good chemical stability and biocompatibility.
Description
Technical Field
The invention belongs to the technical field of nano biological sensing, and particularly relates to a nano fluorescent probe and a preparation method and application thereof.
Background
Fluorescence-based microscopy is one of the most widely used techniques for visualizing biological samples, as it allows non-invasive real-time monitoring of cells with high spatial-temporal resolution. Various fluorescent probes have been developed for detecting biologically important ions/substances or micro-environmental fluctuations in cells. In these techniques, pH sensitive fluorescent probes have been used to visualize intracellular compartments with acidic or basic pH values. Various physiological and pathological processes can cause changes in tissue and intracellular pH, including cell proliferation, apoptosis, receptor-mediated signal transduction, ion transport, muscle contraction, inflammation, and tumor growth, and the pH microenvironment can regulate the structure and function of all biologically active macromolecules. Abnormalities in cells and tissues are closely related to changes in pH, such as diseases like cancer and senile dementia. In addition, the intracellular pH profile is different, and the characteristic pH environment in each organelle ensures that the organelle functions normally. For example, lysosomes and endosomes are acidic pH (4.7-6.5), cytoplasm and nucleus are neutral pH (7.2-7.4), and mitochondrial pH is about 8. Therefore, monitoring the change in cell pH with an effective method is of great importance to better understand cells and to provide critical assistance for early disease diagnosis.
The fluorescent probe is an effective method for monitoring the pH value change of cells, and the existing fluorescent probe for detecting the pH value of cells has the defects of complex synthesis, poor light stability, poor water solubility, poor biocompatibility and the like, so that the application of the fluorescent probe in organisms is greatly limited. Chinese patent application (CN 111138639A) discloses a pH responsive conjugated polymer nanoparticle, a preparation method and application thereof, and the pH responsive conjugated polymer nanoparticle aqueous solution prepared by using a polythiophene derivative as a main raw material has the characteristics of good biocompatibility, high fluorescence intensity, low toxicity and the like, can be used for accurately measuring weak acidic pH value in the aqueous solution, and can also be used for sensitive detection and fluorescence imaging of tumor cell microenvironment response. However, the response interval of the nano particles prepared by the method to pH is smaller (5-8), a larger range of pH detection cannot be achieved, and the used raw materials are organic small molecular materials and have certain toxicity.
Disclosure of Invention
In view of the defects existing in the prior art, the invention provides a nano fluorescent probe, and a preparation method and application thereof, so as to solve the problem that the response interval of the existing nano particle fluorescent probe to pH is smaller.
In order to solve the problems, the invention adopts the following technical scheme:
In one aspect of the present invention, a nano fluorescent probe is provided, and the nano fluorescent probe is a composite nanoparticle, and the composite nanoparticle is a nanocrystal composed of four elements, namely C, N, O and Cl, and includes an-OH functional group, -NH 2 functional group, and a c=c functional group.
Specifically, the particle size of the composite nano particles is 10 nm-100 nm.
Another aspect of the present invention is to provide a method for preparing the nano fluorescent probe as described above, comprising:
s10, dissolving o-phenylenediamine, catecholamine and ferric chloride in a solvent to prepare a first solution, and adjusting the pH value to 1-6 by using an acidic reagent;
Step S20, placing the first solution in a reaction kettle for heating reaction to form a second solution;
step S30, adding a surfactant into the second solution, and placing the mixture in a stirring container for stirring reaction to form a third solution;
and S40, purifying the third solution, and drying to form composite nano particle solid powder to obtain the nano fluorescent probe.
Specifically, in the step S10, the first solution formed by the preparation is prepared, wherein the concentration of o-phenylenediamine is 1 g/L-10 g/L, the concentration of catecholamine is 1 g/L-10 g/L, and the concentration of ferric chloride is 0.5 g/L-5 g/L.
Specifically, in the step S10, the catecholamine is selected from any one of dopamine, epinephrine and norepinephrine, the solvent is selected from any one of deionized water, ethanol, methanol and dimethyl sulfoxide, and the acidic reagent is concentrated hydrochloric acid.
Specifically, in the step S20, the reaction temperature of the heating reaction is 70-200 ℃ and the reaction time is 6-36 h.
Specifically, in the third solution formed in the step S30, the concentration of the surfactant is 0.1g/L to 2g/L, and the surfactant is selected from any one of four-arm polyethylene glycol amino group, polyethylene glycol-400 or polyethylene glycol-800.
Specifically, in the step S30, the stirring reaction is performed in a magnetic stirrer, and the stirring time is 6-36 hours.
Specifically, the step S40 specifically includes: placing the third solution in a dialysis container for dialysis for more than 3 days, and changing the dialysate at least 3 times during the dialysis; and after the dialysis is finished, the solution in the dialysis container is taken for freeze drying to obtain composite nano particle solid powder, so that the nano fluorescent probe is formed.
The invention also provides application of the nano fluorescent probe in preparing a cell fluorescent imaging material.
The fluorescent nano probe provided by the embodiment of the invention has different color responses to different pH values, the response interval of the pH value is 2-10, and the response interval of the pH value is larger; the preparation method of the fluorescent nano probe provided by the embodiment of the invention has the advantages of simple process and green and environment-friendly raw materials, and the prepared fluorescent nano probe has good biocompatibility.
Drawings
FIG. 1 is a process flow diagram of a method of preparing a fluorescent nanoprobe in an embodiment of the invention;
FIG. 2 is a transmission electron microscope image of the nano fluorescent probe obtained by the preparation of example 1;
FIG. 3 is a high resolution transmission electron microscope image of the nano-fluorescent probe prepared in example 1;
FIG. 4 is a Fourier infrared spectrum of the nano-fluorescent probe prepared in example 1;
FIG. 5 is an X-ray photoelectron spectrum of the nano fluorescent probe obtained in example 1;
FIG. 6 is a photograph showing the color of the nano-fluorescent probe of example 2 under different pH conditions;
FIG. 7 is a graph showing fluorescence spectra of the nano-fluorescent probe of example 3 under different pH conditions;
FIG. 8 is a plot of fluorescence intensity for a nano-fluorescent probe at 720nm wavelength at different pH values in example 3;
FIG. 9 is a graph showing the linear relationship between the fluorescence intensity and pH of the nano-fluorescent probe at 720nm in example 3;
FIG. 10 is a plot of fluorescence intensity for cyclic detection of pH change for the nano-fluorescent probe of example 4;
FIG. 11 is a graph of fluorescence kinetics of nano-fluorescent probe to pH detection in example 5;
FIG. 12 is a graph showing the results of toxicity test of the nano-fluorescent probe of example 6 on 293T cells;
FIG. 13 is a graph showing the results of toxicity test of the fluorogenic probe of example 6 on 4T1 cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the embodiments of the present invention will be given with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described in accordance with the drawings are merely exemplary and the invention is not limited to these embodiments.
It should be noted that, in order to avoid obscuring the present invention due to unnecessary details, only structures and/or processing steps closely related to aspects of the present invention are shown in the drawings, and other details not greatly related to the present invention are omitted.
The embodiment of the invention firstly provides a nano fluorescent probe which is a composite nano particle, wherein the composite nano particle mainly comprises C, N, O and Cl four elements to form a nanocrystal, and the nanocrystal comprises an-OH functional group, -NH 2 functional group and a C=C functional group.
Wherein the particle size of the composite nano particles is 10 nm-100 nm.
The embodiment of the invention provides a preparation method of a nano fluorescent probe, referring to fig. 1, the preparation method comprises the following steps:
Step S10, o-phenylenediamine (o-PD), catecholamine (CA) and ferric chloride (FeCl 3) are dissolved in a solvent to form a first solution, and the pH value of the first solution is adjusted to 1-6 by using an acidic reagent.
In a preferred scheme, the concentration of the o-phenylenediamine in the first solution is 1-10 g/L, the concentration of catecholamine is 1-10 g/L, and the concentration of ferric chloride is 0.5-5 g/L.
In a preferred embodiment, the catecholamine is selected from any one of Dopamine (DA), epinephrine (Ad) or Norepinephrine (NA), the solvent is selected from any one of deionized water, ethanol, methanol or dimethyl sulfoxide, and the acidic reagent is concentrated hydrochloric acid.
And step S20, placing the first solution in a reaction kettle for heating reaction to form a second solution.
In a preferred scheme, the reaction temperature of the heating reaction is 70-200 ℃ and the reaction time is 6-36 h.
And step S30, adding a surfactant into the second solution, and placing the mixture in a stirring container for stirring reaction to form a third solution.
In a preferred embodiment, the concentration of the surfactant in the third solution formed in the step S30 is 0.1g/L to 2g/L, and the surfactant is any one of four-ARM polyethylene glycol amino (4 ARM-PEG-NH 2), polyethylene glycol-400 or polyethylene glycol-800.
In a preferred scheme, the stirring reaction is carried out in a magnetic stirrer, and the stirring time is 6-36 h.
And S40, purifying the third solution, and drying to form composite nano particle solid powder to obtain the nano fluorescent probe.
In a preferred embodiment, the step S40 specifically includes: placing the third solution in a dialysis container (e.g., a dialysis bag) for dialysis for more than 3 days, and changing the dialysate at least 3 times (preferably 3-5 times) during the dialysis; and after the dialysis is finished, the solution in the dialysis container is taken for freeze drying to obtain composite nano particle solid powder, so that the nano fluorescent probe is formed.
The embodiment of the invention also provides application of the nano fluorescent probe in preparing a cell fluorescent imaging material.
Example 1:
the embodiment provides a nano fluorescent probe and a preparation method thereof, and the nano fluorescent probe comprises the following specific steps:
(1) 0.10g of o-phenylenediamine, 0.10g of dopamine and 0.05g of ferric chloride are weighed and dissolved in 15mL of deionized water to form a mixed solution, the mixed solution is placed in a beaker, and the pH of the mixed solution is adjusted to 3.0 by using concentrated hydrochloric acid (1M). It should be noted that in other embodiments, dopamine may be replaced by epinephrine or norepinephrine.
(2) Pouring the mixed solution into a clean reaction kettle liner with the volume of 50mL, placing the liner into a reaction kettle, screwing a cover, placing into an oven, and reacting for 12h at 180 ℃. And after the reaction is finished, taking out the reaction kettle, naturally cooling to room temperature, opening the cover, and pouring reactants in the liner into the beaker.
(3) 15Mg of four-arm polyethylene glycol amino group is added to 15mL of the reaction product, and the mixture is placed on a magnetic stirrer for vigorous stirring for 24 hours. It should be noted that in other embodiments, the four-arm polyethylene glycol amino group may be replaced with polyethylene glycol-400 or polyethylene glycol-800.
(4) And (3) placing the reactants in a dialysis bag, dialyzing for 3 days by using deionized water, changing water for more than 3 times, taking out dialyzate after the dialyzate is completely dialyzed, placing the dialyzate in a 50mL centrifuge tube, and freeze-drying to obtain solid powder, namely the composite nano particles, thereby obtaining the nano fluorescent probe.
Fig. 2 is a Transmission Electron Microscope (TEM) of the nano fluorescent probe prepared in this example, and fig. 3 is a High Resolution Transmission Electron Microscope (HRTEM) of the nano fluorescent probe prepared in this example. As can be seen from fig. 2 and 3, the size of the nano fluorescent probe (composite nanoparticle) prepared in this example is about 20nm, and the nano fluorescent probe has an obvious lattice structure and a lattice spacing of 0.21nm.
Fig. 4 is a fourier infrared spectrum of the nano fluorescent probe prepared in this example, and it can be seen from the figure that the nano fluorescent probe contains-OH functional group, -NH 2 functional group, and c=c functional group.
Fig. 5 is an X-ray photoelectron spectrum of the nano fluorescent probe prepared in this embodiment, and it can be seen from the figure that the nano fluorescent probe mainly contains C, N, O, cl elements, and the contents of the C, N, O, cl elements are respectively: 70.47%, 10.01%, 15.78% and 3.74%.
Example 2
(1) The nano fluorescent probe solid powder prepared in the example 1 is prepared into a probe solution with the concentration of 1mg/mL by deionized water for standby.
(2) Tris-HCl buffer solutions of different pH (pH 2,3,4, 5, 6, 6.5, 7, 8,9 and 10 respectively) were prepared at concentrations of 10 mM.
(3) At room temperature, 0.5mL of Tris-HCl buffer solution with each pH value is placed in a 96-well plate, 10 mu L of the probe solution in the step (1) is respectively added, shaking is carried out fully, and the color change of the solution in each well of the 96-well plate is observed.
Fig. 6 is a photograph illustration of the colors of the nano fluorescent probe under different pH values in the present embodiment, and as can be seen from fig. 6, the nano fluorescent probe provided by the present invention can show different macroscopic colors under different pH values, has high response sensitivity, the response interval of pH is 2-10, and has a larger response interval of pH. Specifically, in this embodiment: at pH values of 2, 3, 4, 5, 6 and 6.5, the nano fluorescent probe solution is yellow, the yellow color at pH value of 2 is dark yellow, the color gradually becomes lighter along with the increase of the pH value, and the yellow color at pH value of 6.5 is light and approaches to transparency; at pH 7, the nano-fluorescent probe solutions all appear bluish; at pH 8, 9 and 10, the nanofluorescent probe solutions all appear brown, the brown at pH 8 is dark brown, and the color gradually becomes lighter as the pH increases.
Example 3
(1) The nano fluorescent probe solid powder prepared in the example 1 is prepared into a probe solution with the concentration of 1mg/mL by deionized water for standby.
(2) Tris-HCl buffer solutions of different pH (pH 2,3, 4, 5, 6, 7, 8, 9 and 10 respectively) were prepared at concentrations of 10 mM.
(3) At room temperature, 0.5mL of Tris-HCl buffer solution with each pH value is placed in a 1mL centrifuge tube, 10 mu L of the probe solution in the step (1) is respectively added, shaking is carried out fully, the fluorescence intensity of the reaction solution in each centrifuge tube is measured by a fluorescence spectrophotometer, and a fluorescence intensity graph is drawn.
FIG. 7 is a graph showing fluorescence spectra of the nano-fluorescent probe at different pH values in the present example. FIG. 8 is a plot of fluorescence intensity of the nano-fluorescent probe of the present example at 720nm wavelength under different pH conditions. As can be seen from fig. 7 and 8, the fluorescence intensity of the nano fluorescent probe provided by the invention decreases with the increase of pH, and has high response sensitivity and a larger response interval of pH.
FIG. 9 is a graph showing the linear relationship between the fluorescence intensity and pH of the nano-fluorescent probe in this example at 720nm wavelength. As can be seen from FIG. 9, the fluorescence of the nano fluorescent probe provided by the invention has a good linear relationship with the pH between 3 and 7, so that the quantitative detection of the pH can be realized.
Example 4
(1) The nano fluorescent probe solid powder prepared in the example 1 is prepared into a probe solution with the concentration of 1mg/mL by deionized water for standby.
(2) A10 mM Tris-HCl buffer solution with pH of 2 was prepared, 0.5mL was placed in a 1mL centrifuge tube, 10. Mu.L of the probe solution described in step (1) was added, and the mixture was shaken well with sufficient shaking, and the fluorescence intensity of the reaction solution was recorded in the manner described in example 3.
(3) And (3) regulating the pH value of the reaction solution in the step (2) to 6 by using a high-concentration Tris solution, sufficiently shaking and shaking uniformly, and recording the fluorescence intensity.
(4) The pH of the reaction solution is regulated to 2 by using a high-concentration HCl solution, the reaction solution is fully vibrated and shaken uniformly, and the fluorescence intensity of the reaction solution is recorded.
(5) Repeating the steps (3) and (4) 5 times.
FIG. 10 is a plot of fluorescence intensity for cyclic detection of pH change for the nano-fluorescent probe in this example. As can be seen from fig. 10, the nano fluorescent probe provided by the invention has stable cycle detection performance between pH 2 and pH 6.
Example 5
(1) The nano fluorescent probe solid powder prepared in the example 1 is prepared into a probe solution with the concentration of 1mg/mL by deionized water for standby.
(2) Tris-HCl buffer solutions with pH 2, 4 and 6 were prepared at a concentration of 10mM, respectively.
(3) At room temperature, 0.5mL of Tris-HCl buffer solution with each pH value is placed in a 1mL centrifuge tube, 10 mu L of the probe solution in the step (1) is respectively added, shaking is carried out fully, and the fluorescence intensity of the reaction solution is measured.
(4) And under different time periods, measuring and recording the fluorescence intensity of each reaction liquid, monitoring for 2 hours, and drawing a fluorescence dynamics graph.
Fig. 11 is a fluorescence kinetics diagram of the nano fluorescent probe for pH detection in this example, and as can be understood from fig. 11, the fluorescent probe of the present invention has good fluorescence stability at different pH, and the fluorescence intensity remains stable for 2h, and has good chemical stability.
Example 6
(1) The nano fluorescent probe solid powder prepared in the example 1 is prepared into probe solutions with the concentration of 0mg/mL, 0.25mg/mL, 0.5mg/mL, 0.75mg/mL, 1mg/mL, 1.5mg/mL and 3mg/mL by deionized water for standby.
(2) Human kidney epithelial cells (293T) and breast cancer cells (4T 1) were cultured in 96-well plates at 37℃under 5% CO 2, respectively.
(3) After the cells proliferate to 70% -80%, adding probe solutions with different concentrations (0/0.25/0.75/0.5/1/1.5/3 mg/mL) in the step (1) into each well of the 96-well plate, incubating for 24 hours together, and then performing cytotoxicity test (MTT test) to detect the cytotoxicity of the nano fluorescent probe.
FIG. 12 is a graph showing the toxicity test results of the nano-fluorescent probe of the present example on 293T cells, and FIG. 13 is a graph showing the toxicity test results of the nano-fluorescent probe of the present example on 4T1 cells. As can be seen from fig. 12 and 13, the nano fluorescent probe provided by the invention has small toxicity to cells and excellent biocompatibility.
In summary, the fluorescent nano probe provided by the embodiment of the invention has different color responses to different pH values, has very high response sensitivity, has a response interval of pH value of 2-10, and has a response interval of larger pH value; the preparation method of the fluorescent nano probe provided by the embodiment of the invention has the advantages of simple process, green and environment-friendly raw materials and the like, and the prepared fluorescent nano probe has good chemical stability and biocompatibility.
The foregoing is merely illustrative of the embodiments of this application and it will be appreciated by those skilled in the art that variations and modifications may be made without departing from the principles of the application, and it is intended to cover all modifications and variations as fall within the scope of the application.
Claims (10)
1. The preparation method of the nano fluorescent probe is characterized by comprising the following steps:
s10, dissolving o-phenylenediamine, catecholamine and ferric chloride in a solvent to prepare a first solution, and adjusting the pH value to 1-6 by using an acidic reagent;
Step S20, placing the first solution in a reaction kettle for heating reaction to form a second solution;
Step S30, adding a surfactant into the second solution, and placing the mixture in a stirring container for stirring reaction to form a third solution; the surfactant is selected from any one of four-arm polyethylene glycol amino, polyethylene glycol-400 or polyethylene glycol-800;
and S40, purifying the third solution, and drying to form composite nano particle solid powder to obtain the nano fluorescent probe.
2. The method for preparing a nano-fluorescent probe according to claim 1, wherein in the first solution prepared in the step S10, the concentration of o-phenylenediamine is 1 g/L-10 g/L, the concentration of catecholamine is 1 g/L-10 g/L, and the concentration of ferric chloride is 0.5 g/L-5 g/L.
3. The method for preparing a nano-fluorescent probe according to claim 2, wherein in the step S10, the catecholamine is selected from any one of dopamine, epinephrine and norepinephrine, the solvent is selected from any one of deionized water, ethanol, methanol and dimethyl sulfoxide, and the acidic reagent is concentrated hydrochloric acid.
4. The method of preparing a nano-fluorescent probe according to claim 1, wherein in the step S20, the reaction temperature of the heating reaction is 70 ℃ to 200 ℃ and the reaction time is 6h to 36h.
5. The method of preparing a nano-fluorescent probe according to claim 1, wherein the concentration of the surfactant in the third solution formed in the step S30 is 0.1g/L to 2g/L.
6. The method of preparing a nano-fluorescent probe according to claim 5, wherein in the step S30, the stirring reaction is performed in a magnetic stirrer for a period of time ranging from 6h to 36h.
7. The method for preparing a nano-fluorescent probe according to claim 1, wherein the step S40 specifically comprises: placing the third solution in a dialysis container for dialysis for more than 3 days, and changing the dialysate at least 3 times during the dialysis; and after the dialysis is finished, the solution in the dialysis container is taken for freeze drying to obtain composite nano particle solid powder, so that the nano fluorescent probe is formed.
8. A nano fluorescent probe prepared by the preparation method according to any one of claims 1 to 7, wherein the nano fluorescent probe is a composite nanoparticle, and the composite nanoparticle is a nanocrystal mainly composed of C, N, O and Cl, and comprises an-OH functional group, -NH 2 functional group and a c=c functional group.
9. The nano-fluorescent probe of claim 8, wherein the particle size of the composite nano-particles is 10nm to 100nm.
10. Use of a nano-fluorescent probe according to claim 8 or 9 for the preparation of a cell fluorescent imaging material.
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