CN113337278B - Silicon quantum dot-based fluorescent probe for high-selectivity detection of hydroxyl radicals and preparation method and application thereof - Google Patents
Silicon quantum dot-based fluorescent probe for high-selectivity detection of hydroxyl radicals and preparation method and application thereof Download PDFInfo
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- ethylenediamine
- trimethoxysilyl
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 48
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 229910052710 silicon Inorganic materials 0.000 title claims abstract description 25
- 239000010703 silicon Substances 0.000 title claims abstract description 25
- 238000001514 detection method Methods 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000002096 quantum dot Substances 0.000 title abstract description 7
- 239000000243 solution Substances 0.000 claims abstract description 18
- PHQOGHDTIVQXHL-UHFFFAOYSA-N n'-(3-trimethoxysilylpropyl)ethane-1,2-diamine Chemical compound CO[Si](OC)(OC)CCCNCCN PHQOGHDTIVQXHL-UHFFFAOYSA-N 0.000 claims abstract description 17
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- 229940071125 manganese acetate Drugs 0.000 claims abstract description 11
- UOGMEBQRZBEZQT-UHFFFAOYSA-L manganese(2+);diacetate Chemical compound [Mn+2].CC([O-])=O.CC([O-])=O UOGMEBQRZBEZQT-UHFFFAOYSA-L 0.000 claims abstract description 11
- 239000002019 doping agent Substances 0.000 claims abstract description 8
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention discloses a silicon quantum dot-based fluorescent probe for high-selectivity detection of hydroxyl radicals and a preparation method and application thereof, and belongs to the technical field of biological detection. Respectively dissolving manganese acetate and disodium ethylene diamine tetraacetate in water to obtain manganese acetate solution and disodium ethylene diamine tetraacetate solution; and (2) dropwise adding the manganese acetate solution into the ethylene diamine tetraacetic acid disodium salt solution, uniformly mixing, adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine or adding N- [3- (trimethoxysilyl) propyl ] ethylenediamine and a dopant for expanding the emission wavelength range of the fluorescent probe, and carrying out hydrothermal reaction to obtain the fluorescent probe. The fluorescent probe can be selectively combined with hydroxyl radicals under physiological conditions, the fluorescence intensity is obviously weakened, and organisms, other common substances and biological media in the environment do not interfere with the organisms, and the fluorescent probe can dynamically monitor the change of the concentration of the hydroxyl radicals in cells.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a silicon quantum dot-based fluorescent probe for detecting hydroxyl radicals (OH) with high selectivity, a preparation method of the fluorescent probe and application of the fluorescent probe in detecting hydroxyl radicals in cells.
Background
Hydroxyl radicals (. OH) are highly reactive, have very short half-lives (. about.1 ns), are highly susceptible to oxidation, and are considered to be the most aggressive radicals. Its concentration is closely related to many physiological and pathological processes, including ischemia-reperfusion injury, cancer, neurodegenerative disease, and the like. So far, many methods for detecting. OH have been established, such as high performance liquid chromatography, electrochemical methods, and chemiluminescence methods. Among these methods, the fluorescence method is attracting attention because of its simplicity in operation, low cost, and capability of providing distribution and content information in real time, and is an ideal tool for detection and imaging of OH in biological samples. The fluorescent nano material silicon quantum dots (SiQDs) have the advantages of high light stability, good biocompatibility, good water solubility, low toxicity, surface modification and the like, and have the potential to become an ideal fluorescent detection probe. The detection and imaging of OH in a living body are easily interfered by other active oxygen substances and metal ions, so that a fluorescent probe for detecting OH with high selectivity is designed and synthesized, is used for detecting the OH content in cells in situ in real time, and has very important guiding significance for researching the signal transduction pathway and the physiological and pathological regulation of OH in the living body.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a fluorescent probe for detecting hydroxyl free radicals (OH) with high selectivity based on silicon quantum dots, and a preparation method and application thereof, so as to realize real-time monitoring of the content of OH in cells.
The silicon quantum dot fluorescent probe synthesized by using the disodium ethylenediamine tetraacetate as the main raw material shows good selectivity on OH, and is free from other active oxygen substances such as hydrogen peroxide (H) during detection2O2) Hypochlorite (ClO)-) Singlet oxygen (a)1O2) Peroxy radical (O)2 -) Peroxynitrite (ONOO)-) And tert-butyl hydroperoxide (TBHP). Carboxyl and amino on the surface of the silicon quantum dots, p-Mn2+The fluorescent probe has complexing ability, can mask the interference of other metal ions, and shows extremely high selectivity to OH.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a fluorescent probe for detecting hydroxyl radicals at high selectivity based on silicon quantum dots is disclosed, the synthetic process of the fluorescent probe is shown in figure 1, and the method specifically comprises the following steps: respectively dissolving manganese acetate and disodium ethylene diamine tetraacetate in water to obtain manganese acetate solution and disodium ethylene diamine tetraacetate solution; and dropwise adding a manganese acetate solution into an ethylene diamine tetraacetic acid disodium salt solution, uniformly mixing, adding silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine or adding silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine and a dopant for expanding the emission wavelength range of the fluorescent probe to carry out hydrothermal reaction, and obtaining a reaction solution containing the silicon quantum dot-based fluorescent probe for detecting hydroxyl radicals at high selectivity. The dopant is aniline or phenol substances, such as phenylenediamine, catechol or 3-aminophenol, and the like, and can extend the wavelength of the fluorescent probe to a green light region.
In the preparation method, the molar ratio of N- [3- (trimethoxysilyl) propyl ] ethylenediamine, ethylene diamine tetraacetic acid disodium salt and manganese acetate is preferably 7:7: 1-14: 7:1, and more preferably 14:7: 1. If aniline or phenol such as p-phenylenediamine, catechol or 3-aminophenol is added as a dopant, the molar ratio of the dopant to N- [3- (trimethoxysilyl) propyl ] ethylenediamine is preferably 1:690 to 33: 69.
In the preparation method, the hydrothermal reaction condition is preferably 150-200 ℃ for 6-12 hours.
Further, the preparation method also comprises the following steps: and dialyzing and purifying the obtained reaction solution, and freeze-drying to obtain fluorescent probe powder.
The main raw material for preparing the fluorescent probe is ethylene diamine tetraacetic acid disodium salt, the doped metal ions are divalent manganese, and the synthetic process is environment-friendly, non-toxic and non-corrosive.
A fluorescent probe for detecting hydroxyl radicals with high selectivity based on silicon quantum dots is obtained through the preparation method.
The carboxyl group and the adjacent amino group on the fluorescent probe for detecting the hydroxyl free radical with high selectivity based on the silicon quantum dot have complexing ability on metal ions, can mask the interference of the metal ions, and have strong fluorescence. Under physiological conditions, the fluorescent probe can be selectively combined with hydroxyl radicals, the fluorescence intensity is obviously weakened, and organisms, other substances and biological media which are common in the environment do not form interference. Based on the above, the invention provides the fluorescent probe with the application in hydroxyl radical detection for non-diagnosis or treatment purposes, such as detecting the content of hydroxyl radicals in cells and dynamically monitoring the change of the concentration of the hydroxyl radicals in the cells.
The invention has the following advantages and beneficial effects:
(1) according to the invention, the ethylene diamine tetraacetic acid disodium salt is firstly used as a main raw material to synthesize the silicon quantum dots, and the obtained fluorescent probe shows excellent selectivity on hydroxyl radicals, and is the best in the currently reported OH fluorescent probes based on carbon dots/silicon dots.
(2) According to the invention, metal ion bivalent manganese is doped in the silicon quantum dots firstly, so that the fluorescent probe is free from interference of other metal ions during measurement.
(3) The OH fluorescent probe provided by the invention can realize real-time in-situ detection of OH in cells.
(4) The fluorescence intensity of the OH fluorescent probe provided by the invention is obviously weakened before and after the OH fluorescent probe is combined with OH.
(5) The preparation method of the OH fluorescent probe provided by the invention only needs one step and is simple and feasible.
Drawings
FIG. 1 is a schematic diagram showing a synthetic scheme of the fluorescent probe of the present invention and its reaction with hydroxyl radical (. OH).
FIG. 2 is a fluorescence spectrum of probe (I) and its reaction product with hydroxyl radical.
FIG. 3 is a graph showing the results of selective measurement of different substances by the probe (I). In the figure, blank represents no substance added, I represents the fluorescence intensity of the sample to be measured, I0The fluorescence intensity of the single probe (I) is shown.
FIG. 4 is a graph showing fluorescence intensity of probe (I) for real-time detection of hydroxyl radical content. In the figure, F represents the fluorescence intensity of the sample after 30 minutes of the hydroxyl radical addition reaction, F0Represents the fluorescence intensity of the probe (I) alone.
FIG. 5 is a fluorescence diagram of real-time in situ detection of hydroxyl radical content in cells by probe (I). In the figure, (a) probe, (b) probe + 0.1% DMSO, (c) probe + LPS, (d) probe + PMA; DMSO represents dimethyl sulfoxide, LPS represents lipopolysaccharide derived from Escherichia coli, and PMA represents phorbol-12-myristate-13-acetate.
FIG. 6 is a graph showing the results of selectivity measurement of silicon dots (I) for different substances. In the figure, blank represents no substance added, I represents the fluorescence intensity of the sample to be measured, I0The fluorescence intensity of the probe (I) alone is shown.
FIG. 7 is a graph showing the results of selectivity measurement of silicon dots (II) for different substances. In the figure, blank represents no substance added, I represents the fluorescence intensity of the sample to be measured, I0The fluorescence intensity of the probe (I) alone is shown.
FIG. 8 is a graph showing the results of selectivity measurement of silicon dots (III) for different substances. In the figure, blank represents no substance added, I represents the fluorescence intensity of the sample to be measured, I0The fluorescence intensity of the probe (I) alone is shown.
Detailed Description
The invention will now be further described with reference to the following examples and figures, which are intended to illustrate the invention without in any way limiting it.
Example 1
Synthesis of hydroxyl radical fluorescent probe for blue light:
(1) 1.335g of ethylenediaminetetraacetic acid disodium salt dihydrate (3.5. mu. mol) was dissolved in 10mL of ultrapure water, 2mL of an aqueous solution containing 0.122g of manganese acetate tetrahydrate (0.5. mu. mol) was added dropwise at room temperature, and after mixing uniformly, 1.5mL of silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine (7. mu. mol) was added to obtain a clear and transparent mixed solution.
(2) And transferring the solution into a polytetrafluoroethylene reaction kettle, reacting in a constant-temperature air-blowing drying oven at 160 ℃ for 12 hours, cooling to room temperature, dialyzing and purifying in ultrapure water for 6 hours by using a 500Da dialysis bag, and freeze-drying to obtain golden yellow powder, thus obtaining the fluorescent probe for high-selectivity detection of blue light (Em 433 nm).
Example 2
Synthesis of hydroxyl radical fluorescent probe for blue light:
(1) 1.335g of ethylenediaminetetraacetic acid disodium salt dihydrate (3.5. mu. mol) was dissolved in 10mL of ultrapure water, 2mL of an aqueous solution containing 0.122g of manganese acetate tetrahydrate (0.5. mu. mol) was added dropwise at room temperature, and after mixing well, 0.0108g of p-phenylenediamine (0.1. mu. mol) and 1.5mL of silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine (7. mu. mol) were added to obtain a clear and transparent mixed solution.
(2) And transferring the solution into a polytetrafluoroethylene reaction kettle, reacting in a constant-temperature air-blowing drying oven at 160 ℃ for 12 hours, cooling to room temperature, dialyzing and purifying in ultrapure water for 6 hours by using a 500Da dialysis bag, and freeze-drying to obtain golden yellow powder, thus obtaining the fluorescent probe for high-selectivity detection of OH with green light (Em 522 nm).
Example 3
Synthesis of hydroxyl radical fluorescent probe of green light:
(1) 1.335g of ethylenediaminetetraacetic acid disodium salt dihydrate (3.5. mu. mol) was dissolved in 10mL of ultrapure water, 2mL of an aqueous solution containing 0.122g of manganese acetate tetrahydrate (0.5. mu. mol) was added dropwise at room temperature, and after mixing uniformly, 0.0110g of catechol (0.1. mu. mol) and 1.5mL of silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine (7. mu. mol) were added to obtain a clear and transparent mixed solution.
(2) And transferring the solution into a polytetrafluoroethylene reaction kettle, reacting in a constant-temperature air-blowing drying oven at 160 ℃ for 12 hours, cooling to room temperature, dialyzing and purifying in ultrapure water for 6 hours by using a 500Da dialysis bag, and freeze-drying to obtain yellow powder, thus obtaining the fluorescent probe for high-selectivity detection of OH with green light (Em 515 nm).
Example 4
Synthesis of hydroxyl radical fluorescent probe of green light:
(1) 1.335g of ethylenediaminetetraacetic acid disodium salt dihydrate (3.5. mu. mol) was dissolved in 10mL of ultrapure water, 2mL of an aqueous solution containing 0.122g of manganese acetate tetrahydrate (0.5. mu. mol) was added dropwise at room temperature, and after mixing well, 0.0109g of 3-aminophenol (0.1. mu. mol) and 1.5mL of silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine (7. mu. mol) were added to obtain a clear and transparent mixed solution.
(2) And transferring the solution into a polytetrafluoroethylene reaction kettle, reacting for 12 hours at 160 ℃ in a constant-temperature air-blowing drying oven, cooling to room temperature, dialyzing and purifying for 6 hours in ultrapure water by using a 500Da dialysis bag, and freeze-drying to obtain yellow powder, thus obtaining the fluorescent probe for high-selectivity detection of green light (Em 522 nm).
Example 5
The properties of the fluorescent probe for hydroxyl radicals obtained in example 3 (hereinafter referred to as probe (I)) and the reaction product thereof with hydroxyl radicals were measured:
(1) spectral properties
After 50. mu.g/mL of the probe (I) was reacted with 100. mu.M. OH in a water bath at 37 ℃ for 30 minutes using 0.1M phosphate buffer (pH7.4) as a solvent, the ultraviolet-visible absorption spectrum and the fluorescence spectrum of the probe (I) and the reaction product thereof with. OH were measured. The results are shown in Table 1.
TABLE 1 spectroscopic Properties of Probe (I) and its reaction product with hydroxyl radical (. OH)
Experimental results show that the fluorescence of the probe (I) is strong, and the excitation wavelength and the emission wavelength are respectively 424nm and 515 nm; the fluorescence intensity of the derivative product obtained after the probe (I) reacts with OH is greatly weakened. The fluorescence spectrum of probe (I) and its reaction product with. OH is shown in FIG. 2.
(2) Selectivity of Probe (I) for measurement of hydroxyl radical
Probes (I) and OH and various active oxygen species (H) were examined2O2、ClO-、1O2、O2 -、ONOO-TBHP), active Nitrogen (NO)2 -) Inorganic salt (F)-、Cl-、Br-) Inorganic acid (NO)3 -、SO4 2-、CO3 -、HCO3 -) Sulfhydryl compounds (GSH, Cys, Hcy), and reducing substances (DA, AA, H)2S), glucose and metal ions (Cd)2+、K+、Na+、Mn2+、Ca2+、Zn2+、Mg2+、Cu2+、Ag+、Hg2 +、Fe2+、Fe3+、Al3+) The reaction conditions of (1). The reactions were all carried out in 0.1M phosphate buffer (pH7.4) at 37 ℃ for 40 minutes with the concentration of probe (I) being 50. mu.g/mL, H2O2、ClO-、1O2、O2 -、ONOO-、TBHP、NO3 -、NO2 -、F-、Cl-、Br-、SO4 2-、CO3 -、HCO3 -、F-、Cl-、Br-、HCO3 -、GSH、Cys、Hcy、DA、AA、H2S, glucose, Cd2+、K+、Na+、Mn2+、Ca2+、Zn2+、Mg2+、Cu2+、Ag+、Hg2+、Fe2+、Fe3+、Al3+All concentrations of (2) were 100. mu.M. The measurement result shows that the fluorescence intensity of the probe (I) is slightly changed after reacting with the active substances, and the fluorescence intensity of the derivative product after reacting with the OH is greatly weakened, which shows that the probe (I) has excellent selectivity on the OH measurement, and is shown in figure 3.
Example 6: the probe (I) is used for detecting the content of hydroxyl free radicals in real time
The probe (I) was added to a 0.1M phosphate buffer solution (pH7.4) at a concentration of 50. mu.g/mL, and 0.8, 2, 4, 10, 20, 30, 40, 50. mu.M probe-discovering OH solution was added thereto, and the reaction was carried out in a water bath at 37 ℃ for 30 minutes, and then the fluorescence intensity was measured. The fluorescence intensity gradually decreased with increasing OH concentration, as shown in FIG. 4.
Example 7: the probe (I) is used for detecting the content of hydroxyl free radicals in cells in situ in real time
The human cervical cancer cells HeLa cells are taken as research objects, and the reaction conditions of the probe (I) on OH are examined in detail, wherein the reaction conditions comprise probe toxicity, concentration, incubation time and the like. It was found that the probe (I) has a high quenching efficiency for. OH at 37 ℃ and pH7.4, and that the survival rate of the HeLa cells was maintained at 93% or more after incubating the HeLa cells for 24 hours with the probe (I) at concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, and 150. mu.g/mL in the MTT assay. Therefore, the probe (I) is suitable for imaging experiments of HeLa cells. The results showed that the cells exhibited bright green fluorescence with probe (I) at a concentration of 50. mu.g/mL for 4 hours of incubation (FIG. 5 a); FIG. 5b shows a stronger fluorescence than FIG. 5a after addition of OH scavenger (0.1% DMSO); fluorescence of the green channel was quenched after addition of 500ng/mL of the. OH stimulator PMA (phorbol-12-myristate-13-acetate) (FIG. 5c) and 10. mu.g/mL of LPS (lipopolysaccharide from E.coli) (FIG. 5 d).
Examples 5, 6 and 7 were repeated using the probes prepared in the other examples, and results similar to those of the probe (I) were obtained.
Example 8
Silicon dots (I) synthesized by using ethylene diamine tetraacetic acid disodium salt dihydrate and N- [3- (trimethoxysilyl) propyl ] ethylenediamine as raw materials; silicon dots (II) synthesized by taking ethylene diamine tetraacetic acid disodium salt dihydrate, manganese acetate tetrahydrate and N- [3- (trimethoxysilyl) propyl ] ethylenediamine as raw materials; silicon dots (III) synthesized using manganese acetate tetrahydrate and N- [3- (trimethoxysilyl) propyl ] ethylenediamine as raw materials were synthesized by the method described in example 1. Among the three, silicon spot (II) showed good selectivity for hydroxyl radical (fig. 7), the fluorescence of silicon spot (I) was interfered by metal ions (fig. 6), and silicon spot (III) did not respond to hydroxyl radical (fig. 8), and the measurement method was the same as example 5.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Claims (6)
1. A preparation method of a fluorescent probe for detecting hydroxyl radicals with high selectivity based on silicon quantum dots is characterized by comprising the following steps: the method comprises the following steps: respectively dissolving manganese acetate and disodium ethylene diamine tetraacetate in water to obtain manganese acetate solution and disodium ethylene diamine tetraacetate solution; dropwise adding a manganese acetate solution into an ethylene diamine tetraacetic acid disodium salt solution, uniformly mixing, adding silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine or adding silicon source N- [3- (trimethoxysilyl) propyl ] ethylenediamine and a dopant for expanding the emission wavelength range of a fluorescent probe, and carrying out hydrothermal reaction to obtain a reaction solution containing the fluorescent probe for detecting hydroxyl radicals at high selectivity based on silicon quantum dots;
the dopant is phenylenediamine, catechol or 3-aminophenol;
the molar ratio of N- [3- (trimethoxysilyl) propyl ] ethylenediamine, ethylenediaminetetraacetic acid disodium salt and manganese acetate is 7:7: 1-14: 7: 1; the molar ratio of the dopant to the N- [3- (trimethoxysilyl) propyl ] ethylenediamine is 1: 690-33: 69;
the hydrothermal reaction is carried out for 6-12 hours at the temperature of 150 ℃ and 200 ℃.
2. The method for preparing a fluorescent probe according to claim 1, characterized in that: further comprising the steps of: the obtained reaction solution is dialyzed, purified and freeze-dried.
3. A fluorescent probe for detecting hydroxyl radicals with high selectivity based on silicon quantum dots is characterized in that: obtained by the production method according to claim 1 or 2.
4. Use of the fluorescent probe of claim 3 for the detection of hydroxyl radicals for non-diagnostic or therapeutic purposes.
5. Use according to claim 4, characterized in that: the application is to detect the content of hydroxyl free radicals in cells.
6. Use according to claim 4, characterized in that: the application is dynamic monitoring of changes in intracellular hydroxyl radical concentration.
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| CN107149685A (en) * | 2017-04-17 | 2017-09-12 | 南开大学 | A kind of water-soluble Mn2+The preparation method of the silicon nano of functionalization |
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| CN105694871A (en) * | 2016-03-24 | 2016-06-22 | 武汉大学 | Preparation method of water-soluble green fluorescent silicon quantum dot |
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