CN111303877A

CN111303877A - Quantum dot fluorescent probe in cell and human blood Hg2+Application in visual detection

Info

Publication number: CN111303877A
Application number: CN202010150121.XA
Authority: CN
Inventors: 陆阳清; 王凯; 陈泽楷; 张鑫洋
Original assignee: Hangzhou Zhongdao Photoelectric Technology Co ltd
Current assignee: Hangzhou Zhongdao Photoelectric Technology Co ltd
Priority date: 2020-03-06
Filing date: 2020-03-06
Publication date: 2020-06-19

Abstract

The invention belongs to the technical field of fluorescence detection of quantum dot fluorescent probes, and particularly relates to application of a quantum dot fluorescent probe in visual detection of Hg2+ in cells and human blood, wherein the quantum dot fluorescent probe comprises a silicon dioxide microsphere, 10Ca/6Tb Bi2O2S nanocrystalline and aminated fluorescent carbon quantum dots, a layer of 10Ca/6Tb Bi2O2S nanocrystalline is modified after the surface of the silicon dioxide microsphere is aminated, and the aminated fluorescent carbon quantum dots are wrapped and modified on the outer layer of the 10Ca/6Tb Bi2O2S nanocrystalline. The quantum dot fluorescent probe adopts a SiO2@10Ca/6Tb: Bi2O2S @ CDS fluorescent probe, has dual-emission fluorescence, can be used for a ratio fluorescence analysis method, does not depend on expensive instruments during in vitro analysis, can be used for completely quantitative or semi-quantitative analysis by naked eyes, can detect the fluorescence ratio intensity under two emission wavelengths or excitation wavelengths, can well correct errors brought by detection environment, and improves the signal intensity.

Description

Quantum dot fluorescent probe in cell and human blood Hg2+Application in visual detection

Technical Field

The invention belongs to the technical field of fluorescence detection of quantum dot fluorescent probes, and particularly relates to a quantum dot fluorescent probe in cells and human blood Hg²⁺Application in visual detection.

Background

One of the metal elements with strong toxicity is Hg0 and Hg²⁺And the organic mercury exists in three forms, wherein the toxicity of the organic mercury is the greatest. Hg is a mercury vapor²⁺After being absorbed, the medicine can enter the whole body and various tissues and organs through the blood circulation system, which can cause central nerve abnormality and irreversible permanent damage to internal organs such as liver, kidney and the like, which can cause symptoms such as inappetence, laziness and the like. Detection of Hg with fluorescent probes²⁺The probe with high sensitivity and high resolution mainly studied at present comprises carbon quantum dots, MOF or organic matter systems, and the optical performance of the systems is unstable, for example, the luminous efficiency of the organic matter is reduced along with the prolonging of the irradiation time.

At present, mercury ion detection methods are mainly divided into two categories, one category is a detection method depending on a large-scale analytical instrument, and the detection methods mainly comprise atomic absorption photometry (AAS), Atomic Fluorescence Spectrometry (AFS), inductively coupled plasma mass spectrometry (ICP-MS), resonance Rayleigh Scattering Spectrometry (RSS), an electrochemical method, a gas chromatography, a liquid chromatography, a mass spectrometry, various instrument combination technical methods and the like, wherein professional and complicated pretreatment processes such as enrichment and separation of a sample are required, so that the detection methods have certain potential safety hazards to an operator, and the instrument is expensive to operate and high in maintenance cost and detection cost. The other is a chemical sensor detection method which has the outstanding advantages of sensitivity, simplicity, short response time, high analysis speed, high selectivity, real-time detection, biological application and the like which are different from the detection method of a large-scale analytical instrument.

Quantum dots (Quantum dot QDs), also known as nanocrystals or artificial atoms, are quasi-zero-dimensional (quasi-zero-dimensional) nanoparticles composed of group II-VI or group III-V elements. Quantum dots are typically below 100 nanometers (nm) in size in all three dimensions. Electrons and holes in the quantum dots have discrete energy level structures and can emit fluorescence after being excited due to quantum confinement effect (quantumproficient effect). The electronic states such as the energy gap width, the size of exciton binding energy and the energy blue shift of excitons can be conveniently adjusted by controlling the shape, the structure and the size of the quantum dots. The phenomenon of blue shift occurs in the light absorption spectrum of the quantum dots as the size of the quantum dots is gradually reduced. The phenomenon of spectral blue shift is more pronounced the smaller the size, the so-called quantum size effect. Because quantum dots can absorb all photons above their band gap energy and emit light with a wavelength (i.e., color) that is size dependent, a range of labels with different emission wavelengths (i.e., colors) can be formed with the same type of quantum dots of different sizes.

The Chinese patent application (application number) discloses that the nucleation energy barrier of the bismuth oxysulfide nanocrystalline is reduced by adding alkaline earth calcium ions in the process of preparing the nanocrystalline, so that the growth of the bismuth oxysulfide nanocrystalline is promoted, and the surface of the nanocrystalline is negatively charged due to the coating of oleic acid. Prepared Ca/Tb Bi₂O₂The S nanocrystalline material emits bright green fluorescence under the irradiation of an ultraviolet lamp with the wavelength of 254nm, and the quantum efficiency is 40.2%. Tb in this system³⁺The 4f-5d transition with the lower 5d energy level position of the ion is positioned between the ultraviolet region and Hg²⁺The absorption peak position of ions in the ultraviolet region is close to that of the nano-crystalline surface through electrostatic adsorption of Hg²⁺Rear Tb of ion³⁺Electrons at the 5d level of the ion are passed through Hg²⁺Radiationless relaxation of ions to the ground state reduces Tb³⁺Ion(s)⁵D₄Electron population of energy level resulting in Tb³⁺The luminous intensity of the ions is reduced, and the reduced amplitude is very obviously and very suitable for Hg²⁺And (4) detecting ions.

In recent years, the detection of ions by chemical sensing has been receiving more and more attention in the fields of medical diagnosis, biotechnology, analytical chemistry, and the like. In various biochemical sensors, the fluorescence probe method not only provides an application method of in vitro determination, but also provides in vivo imaging research due to the advantages of higher sensitivity, easy operation, less spectral interference and the like. The outstanding fluorescence performance and good biocompatibility are the most outstanding characteristics of the quantum dot fluorescent probe and are one of the reasons why the quantum dot fluorescent probe is widely concerned. In recent years, researchers haveVarious application possibilities of the quantum dot fluorescent probe in the biological field are tried, including cell imaging, biochemical sensing, medicine carrying and the like. The present application has studied mainly on modified SiO₂@10Ca/6Tb:Bi₂O₂The fluorescent probe with the S @ CDS structure is applied to cell imaging and ion detection in cells as the fluorescent probe. For SiO by cell biology means₂@10Ca/6Tb:Bi₂O₂The cytotoxicity of the fluorescent probe with the S @ CDS structure is evaluated, and then SiO is observed by using a fluorescence microscope₂@10Ca/6Tb:Bi₂O₂Imaging effect of fluorescent probe with S @ CDS structure in cells and external addition of Hg²⁺Influence on cell imaging effect, in order to realize Hg in cells by quantum dot fluorescent probe²⁺The detection of (2) lays the foundation.

Disclosure of Invention

It is an object of the present application to provide quantum dot fluorescent probes in cells of Hg²⁺The application in visual detection is that the quantum dot fluorescent probe comprises silicon dioxide microspheres and 10Ca/6Tb: Bi₂O₂S nano-crystal and aminated fluorescent carbon quantum dot, wherein a layer of 10Ca/6Tb Bi is modified after the surface of the silicon dioxide microsphere is aminated₂O₂S nanocrystal of 10Ca/6Tb Bi₂O₂The outer layer of the S nanocrystal is also wrapped and modified with aminated fluorescent carbon quantum dots. The quantum dot fluorescent probe adopts SiO₂@10Ca/6Tb:Bi₂O₂The S @ CDS fluorescent probe has dual-emission fluorescence, can be used for a ratio fluorescence analysis method, does not depend on expensive instruments when used for in vitro analysis, can be used for completely quantitative or semi-quantitative analysis by naked eyes, can detect the fluorescence ratio intensity under two emission wavelengths or excitation wavelengths, can well correct errors brought by detection environments, and improves the signal intensity.

Preferably, the silica microspheres, 10Ca/6Tb: Bi₂O₂The molar ratio of the S nanocrystal to the aminated fluorescent carbon quantum dot is 1:5-10: 10-30; preferably 1:6-8: 12-25.

Preferably, the preparation method of the quantum dot fluorescent probe comprises the following steps:

1) synthesis of silica microspheres

First, 80.0mL of ethanol, 4.850mL of water, and 3.6mL of 25% aqueous ammonia were sequentially added to a 250.0mL flask, which was heated to 55 ℃ with vigorous stirring. Then a mixed solution of 3.1mL Tetraethoxysilane (TEOS) and 8.0mL ethanol was rapidly added to the flask; keeping the temperature of the solution at 55 ℃, reacting for 5 hours to prepare silicon dioxide microspheres with the particle size of about 40 +/-5 nm, and taking the prepared microspheres as seeds for continuous growth of the microspheres;

adding more than 10.0mL of seed solution of the silica microspheres prepared by reaction, 70.0mL of ethanol, 13.0mL of water and 7.5mL of 25% ammonia water into a 250.0mL flask, mixing and stirring, dropwise adding prepared mixed solution of 1.0mL of tetraethoxysilane and 10.0mL of ethanol, and keeping continuous stirring reaction for more than 5 hours to prepare the silica microspheres with the diameter of about 220 cm and 5 nm;

2) synthesis and purification of carbon amide quantum dots

Adding 1.O mL of 3-Aminopropyltriethoxysilane (APTES) and 9.0mL of glycerol into a tetrafluoroethylene container, introducing high-purity argon for 5min to remove dissolved oxygen in the solution, then placing the solution into a reaction kettle at 200 ℃, completing the reaction after half an hour, and taking out a light yellow transparent solution in the tetrafluoroethylene container when the container is slightly cooled; injecting the light yellow solution into a dialysis bag of 1000Da, dialyzing and purifying with ultrapure water, replacing the ultrapure water every 6 hours, dialyzing for at least 12 hours, and finally obtaining the light yellow solution in the dialysis bag, namely the prepared carbon amide quantum dot solution;

3) amination of silicon dioxide microsphere and surface modified quantum dot thereof

Re-dissolving 20.0mg of silicon dioxide microspheres in a freshly prepared 2.0mL of anhydrous methanol solution, ultrasonically dispersing the 3-aminopropyltriethoxysilane in the freshly prepared anhydrous methanol solution at a concentration of 10%, and then stirring at room temperature for reaction for 2 hours; after the reaction, the aminated silica spheres were washed repeatedly three times with methanol solution and water in this order and dispersed in 10Ca/6Tb: Bi of 2, OmL₂O₂Mixing and stirring the mixture of S nanocrystal (5mg/mL) and 2.0mL EDC (20mg/mL) at 4 ℃ for 12h, washing the mixture by water centrifugation for 3 times after the reaction is finished, and removing the uncouplingWashing the nano-crystals to obtain 10Ca/6Tb: Bi₂O₂S nanocrystalline-modified silicon spheres;

4) mixing 10Ca/6Tb Bi₂O₂And dissolving the S nanocrystal modified silicon spheres in 2.0mL of EDC (20mg/mL) solution, adding 1.0mL of prepared aminated fluorescent carbon quantum dots, stirring and reacting at room temperature for 12h, centrifuging and washing the solution for three times after the reaction is finished, removing redundant carbon quantum dots, and dispersing the solution in 2.0mL of water to obtain the double-emission ratio type fluorescent probe.

In another aspect, the present application additionally provides a cell Hg²⁺The visual detection kit comprises a quantum dot fluorescent probe, wherein the quantum dot fluorescent probe comprises silicon dioxide microspheres and 10Ca/6Tb: Bi₂O₂S nano-crystal and aminated fluorescent carbon quantum dot, wherein a layer of 10Ca/6Tb Bi is modified after the surface of the silicon dioxide microsphere is aminated₂O₂S nanocrystal of 10Ca/6Tb Bi₂O₂The outer layer of the S nanocrystal is also wrapped and modified with aminated fluorescent carbon quantum dots.

On the other hand, the application also provides the application of the quantum dot fluorescent probe in preparing human blood Hg²⁺The quantum dot fluorescent probe comprises silicon dioxide microspheres and 10Ca/6Tb: Bi₂O₂S nano-crystal and aminated fluorescent carbon quantum dot, wherein a layer of 10Ca/6Tb Bi is modified after the surface of the silicon dioxide microsphere is aminated₂O₂S nanocrystal of 10Ca/6Tb Bi₂O₂The outer layer of the S nanocrystal is also wrapped and modified with aminated fluorescent carbon quantum dots.

On the other hand, the application also provides human blood Hg²⁺The visual detection kit comprises a quantum dot fluorescent probe, wherein the quantum dot fluorescent probe comprises silicon dioxide microspheres and 10Ca/6Tb: Bi₂O₂S nano-crystal and aminated fluorescent carbon quantum dot, wherein a layer of 10Ca/6Tb Bi is modified after the surface of the silicon dioxide microsphere is aminated₂O₂S nanocrystal of 10Ca/6Tb Bi₂O₂The outer layer of the S nanocrystal is also wrapped and modified with aminated fluorescent carbon quantum dots.

By adopting the technical scheme, the quantum dot fluorescent probe adopts SiO₂@10Ca/6Tb:Bi₂O₂The S @ CDS fluorescent probe has dual-emission fluorescence, can be used for a ratio fluorescence analysis method, does not depend on expensive instruments when used for in vitro analysis, can be used for completely quantitative or semi-quantitative analysis by naked eyes, can detect the fluorescence ratio intensity under two emission wavelengths or excitation wavelengths, can well correct errors brought by detection environments, and improves the signal intensity.

Drawings

FIG. 1: in example 1 of this patent, 10Ca/6Tb Bi₂O₂X-ray diffraction pattern of S nanocrystal.

FIG. 2: in example 1 of this patent, 10Ca/6Tb Bi₂O₂Transmission electron microscopy of S nanocrystals.

FIG. 3: in example 1 of this patent, 10Ca/6Tb Bi₂O₂And (3) a down-conversion luminescence spectrum of the S nanocrystal.

FIG. 4: in example 1 of this patent, 10Ca/6Tb Bi₂O₂Fluorescence intensity and Hg of S nanocrystal²⁺Ion concentration dependence.

FIG. 5: comparative example 6Tb Bi₂O₃X-ray diffraction pattern of the nanocrystals.

FIG. 6: comparative example 6Tb Bi₂O₃Fluorescence intensity and Hg of nanocrystals²⁺Ion concentration dependence.

FIG. 7: (A) NaCl solutions of different concentrations; (B) the pH value; (C) the influence of the illumination time and the standing time (D) on the fluorescence intensity of the carbon quantum dot solution.

FIG. 8: (A) influence of different metal ions on fluorescence intensity of the carbon quantum dots; (B) hg in the presence of other metal ions²⁺The measurement of (1).

FIG. 9: example 2SiO of this patent₂@10Ca/6Tb:Bi₂O₂S @ CDS scanning electron micrograph.

FIG. 10: example 2SiO of this patent₂@10Ca/6Tb:Bi₂O₂S (A, B) and SiO₂@10Ca/6Tb:Bi₂O₂S@CDS(C，D) High resolution transmission electron microscopy.

FIG. 11: example 2SiO of this patent₂@10Ca/6Tb:Bi₂O₂S @ CDS fluorescent probe for Hg²⁺Sequence 1 was 550nm and sequence 2 was 425 nm.

FIG. 12: different concentrations of 4SiO₂@10Ca/6Tb:Bi₂O₂And (3) analyzing the cytotoxicity of the S @ CDS fluorescent probe after 24h incubation with HeLa.

FIG. 13: cytotoxicity assays 24h after incubation of different concentrations of si02@ QDs @ CDs with L929 cells.

FIG. 14: hg addition to L929 cells²⁺Pre (A1, B1, C1, D1) and addition of Hg²⁺Confocal laser imaging of the rear (a2, B2, C2, D2), (a1, a2) is a blue channel, (B1, B2) is a red channel, (D1, D2) is a superposition of the three, and (Cl, C2) is bright field imaging with a scale of 20 μm.

Detailed Description

Example 110Ca/6Tb Bi₂O₂Preparation of S nanocrystal

(1) Adding 0.84 mmol of bismuth acetate, 0.06 mmol of terbium acetylacetonate, 0.1 mmol of calcium acetate and 5ml of oleic acid into a 50ml three-neck bottle at room temperature, heating to 110 ℃, and preserving the temperature for 1 hour;

(2) after the solution in the step (1) is cooled to below 50 ℃, adding 10 mmol of sulfur powder and 20 ml of oleylamine, vacuumizing the three-necked bottle by using a mechanical pump for about 10 minutes, then heating to 120 ℃, keeping the temperature for 30 minutes, then rapidly heating to 310 ℃ under the protection of argon and keeping the temperature for 1 hour;

(3) and (3) cooling the solution in the step (2) to room temperature, adding ethanol, centrifuging to obtain a precipitate, and adding ethanol: washing the product with a mixture of cyclohexane in a ratio of 3:1, and drying at 40 ℃ to obtain the final product.

The powder X-ray diffraction analysis and the transmission electron microscope observation analysis show that: the product was in the form of petals (FIG. 2) with an orthorhombic system (FIG. 1) size of about 30 nm. The sample under 254nm ultraviolet lamp irradiation emits bright green light emission spectrum containing 493nm, 550nm, 594nm and 628nm⁵D₄→⁷F₆，⁵D₄→⁷F₅，⁵D₄→⁷F₄And⁵D₄→⁷F₃(iii) transition (FIG. 3). Dispersing the nanocrystals in cyclohexane and then in solution with different concentrations of mercuric chloride Tb³⁺The luminous intensity of the ion is dependent on Hg²⁺The ion concentration increases and gradually decreases (fig. 4). This is mainly due to Tb³⁺Electrons at the 5d level of the ion are passed through Hg²⁺Radiationless relaxation to the ground state reduces Tb³⁺Ion(s)⁵D₄Electron population of energy level resulting in Tb³⁺The luminous intensity of the ions decreases. When Hg is contained³⁺Tb with an ion concentration of only 1. mu. mol/l³⁺The reduction amplitude of the ion luminous intensity is more than 90 percent, which indicates that the system has high sensitivity.

Comparative example 1

(1) Adding 0.94 mmol of bismuth acetate and 0.06 mmol of terbium acetylacetonate into 5ml of oleic acid at room temperature, heating to 110 ℃ in a 50ml three-neck bottle, and keeping the temperature for 1 hour;

Powder X-ray diffraction analysis showed: the product is 6Tb: Bi₂O₃Phase (fig. 5). The sample under 254nm ultraviolet lamp irradiation has weak green light emission spectrum including 493nm, 550nm, 594nm and 628nm⁵D₄→⁷F₆，⁵D₄→⁷F₅，⁵D₄→⁷F₄And⁵D₄→⁷F₃similar to fig. 3. Dispersing the nanocrystals in cyclohexane and then in solution with different concentrations of mercuric chloride Tb³⁺The luminous intensity of the ions remained substantially unchanged (fig. 6). This is achieved byIndicates Ca²⁺The ion doping can promote Bi₂O₂Growth of S nanocrystals with Bi content of 6Tb₂O₃Tb in the system³⁺Electrons on the 5d level of the ion are free of Hg²⁺Influence of ions.

Test example 110Ca/6Tb Bi₂O₂Stability of S nanocrystals under different conditions

The results of the stability test of the carbon quantum dots in different NaCl solutions are shown in FIG. 7A, and the fluorescence intensity of the carbon quantum dots is independent of the concentration of the NaCl solution (up to 1 mol/L). When the pH value of the solution is changed within 3-11, the fluorescence intensity of the carbon quantum dots is slightly changed, which shows that the fluorescence intensity of the carbon quantum dots does not change along with the pH value (fig. 7B). Further, the carbon quantum dot solution was irradiated with xenon lamp (500W) for 7h, and the fluorescence intensity of the carbon quantum dot was almost unchanged (FIG. 7C). The fluorescence intensity of the carbon quantum dots was stable even after 3 months of standing at room temperature (FIG. 7D). The experimental results show that the carbon quantum dot has better stability.

Test example 210Ca/6Tb Bi₂O₂S nanocrystalline pair Hg²⁺Selectivity of (2)

As shown in FIG. 8A, 50. mu. mol/L Cd were obtained under the same experimental conditions²⁺，Zn²⁺，Fe²⁺，Mn²⁺，Ba²⁺，Cu²⁺，Co²⁺，Ca²⁺，K⁺，Fe³⁺，Mg²⁺，Pb²⁺，Ni²⁺And Na⁺And the like have almost no influence on the fluorescence intensity of the carbon quantum dots. Pb in the presence of other metal ions (concentration of 1. mu. mol/L)²⁺And Fe³⁺For Hg²⁺The assay was weakly affected, while the remaining ions were almost undisturbed (FIG. 8B). These results indicate that this carbon quantum dot acts as Hg²⁺The sensor has better selectivity. At the same time, different mercury salts (Hg (NO) were investigated₃)₂，HgCl₂，NaClO₃、Hg(ClO₄)，(CH₃COO)₂Hg) on the fluorescence intensity of the carbon quantum dot solution. Experiments have shown that all mercury salts cause essentially the same degree of quenching. Control experiments were performed with sodium salts having the same anion as the mercury salts and all sodium salts were found to have no effect on solution fluorescence. This indicates that the carbon quantum dots are only pairedHg²⁺There is a fluorescent response.

Example 2SiO₂@10Ca/6Tb:Bi₂O₂S @ CDS Synthesis

1) Synthesis of silica microspheres

2) synthesis and purification of carbon amide quantum dots

Adding 1, OmL 3-Aminopropyltriethoxysilane (APTES) and 9.0mL of glycerol into a tetrafluoroethylene container, introducing high-purity argon for 5min to remove dissolved oxygen in the solution, then placing into a reaction kettle at 200 ℃, completing the reaction after half an hour, and taking out a light yellow transparent solution in the tetrafluoroethylene container when the container is slightly cooled; injecting the light yellow solution into a dialysis bag of 1000Da, dialyzing and purifying with ultrapure water, replacing the ultrapure water every 6 hours, dialyzing for at least 12 hours, and finally obtaining the light yellow solution in the dialysis bag, namely the prepared carbon amide quantum dot solution;

Re-dissolving 20.0mg of silicon dioxide microspheres in a freshly prepared 2.0mL of anhydrous methanol solution, ultrasonically dispersing the 3-aminopropyltriethoxysilane in the freshly prepared anhydrous methanol solution at a concentration of 10%, and then stirring at room temperature for reaction for 2 hours; after the reaction, the aminated silica spheres were repeatedly washed with methanol solution and water in this orderThree times of the mixture are dispersed in 10Ca/6Tb B i of 2, OmL₂O₂Mixing and stirring the S nanocrystal (5mg/mL) and 2.0mL of EDC (20mg/mL) for 12h at 4 ℃, centrifugally washing for 3 times after the reaction is finished, and washing away the uncoupled nanocrystal to obtain 10Ca/6Tb: Bi₂O₂S nanocrystalline-modified silicon spheres;

4) mixing 10Ca/6Tb Bi₂O₂Dissolving S nanocrystal modified silicon spheres in 2.0mL of EDC (20mg/mL) solution, adding 1.0mL of prepared aminated fluorescent carbon quantum dots, stirring and reacting at room temperature for 12h, centrifugally washing the solution for three times after the reaction is finished, removing redundant carbon quantum dots, and dispersing the solution in 2.0mL of water to obtain the double-emission ratio type SiO₂@10Ca/6Tb:Bi₂O₂S @ CDS fluorescent probe.

SiO₂@10Ca/6Tb:Bi₂O₂The S @ CDS fluorescent probe is analyzed and characterized by a transmission electron microscope image and a scanning electron microscope. FIG. 9 is a scanning electron micrograph showing that SiO is observed when the resolution is gradually increased from 15000 to 35000₂@10Ca/6Tb:Bi₂O₂The S @ CDS probe has uniform particle size distribution and good sphericity and dispersity, the diameter of the silicon sphere is about 250nm, and the particle size of the microsphere finally reaches about 265nm after quantum dots and carbon dots with particle sizes of about 4.5nm and 9.5nm are decorated according to a high-resolution electron microscope picture 10. As can be clearly seen from a and B in fig. 10, black dots are uniformly distributed on the surface of the silicon sphere, which are quantum dots on the surface modification of the silicon sphere; after the carbon dots are wrapped on the outermost layer of the microsphere, as can be seen from C and D in FIG. 10, the surface of the microsphere is of a paste structure, and the black dots in the previous step are almost completely wrapped inside, that is, the carbon dots are tightly wrapped on the SiO₂@10Ca/6Tb:Bi₂O₂And (5) the surface of the S microsphere.

Test example 3SiO₂@10Ca/6Tb:Bi₂O₂S @ CDS fluorescent probe for Hg²⁺Analysis and detection of

As shown in fig. 11, with Hg²⁺The fluorescence intensity of the quantum dots at the characteristic peak of 550nm gradually decreases while the fluorescence intensity of the carbon dots at the characteristic peak of 425nm hardly changesAnd the color change under the ultraviolet light shown in the figure is the color change process from red to blue of the system directly visible to the naked eye. When Hg is contained²⁺When the concentration is more than 1 mu mol/L, the fluorescence of the quantum dots at the 550nm characteristic peak is completely quenched, and the corresponding fluorescence ratio can not be changed any more. As can be seen from the quenching trend of quantum dot fluorescence, in the range of O-1 mu mol/L, the ratio of the fluorescence intensity at the characteristic peak of 550nm to the fluorescence intensity at the characteristic peak of 425nm, namely the change of F550/F425 to Hg²⁺The concentration is in a very good linear relationship. Due to Hg²⁺Can quench corresponding fluorescence in a short time after being added into the system, and the sensitive quenching mode is used for realizing the rapid detection of Hg²⁺Is very advantageous.

Test example 4SiO₂@10Ca/6Tb:Bi₂O₂Application of S @ CDS fluorescent probe in L929 cell fluorescence imaging research

First, experimental material

Mouse fibroblasts (L929), provided by cells of the chinese academy of sciences; DMEM high-sugar culture medium, trypsin solution with the mass fraction of 0.25%, fetal bovine serum, penicillin and streptomycin, provided by Hangzhou Jinuo biological medicine technology limited company; phosphate (PBS) buffer solution pH 7, provided by Hangzhou ilex bioengineering materials, Inc.; thiazole blue (MTT), available from blue sky biotechnology limited; dimethyl sulfoxide (DMSO), available from national pharmaceutical group chemical agents, Inc.; lead chloride, available from national drug group chemical reagents, ltd.

According to the method, a normal mouse fibroblast L929 cell is selected as a research object to evaluate the biotoxicity and the imaging effect of the BETA-cyclodextrin modified 10Ca/6Eu: Bi2O2S nanocrystal. The L929 fibroblast has clear and easily-distinguished outline, is mostly in a protruded spindle-shaped or star-shaped flat structure, and has vigorous viability.

Firstly, recovering the frozen cells, transferring the recovered cells into a cell culture bottle, and placing the cell culture bottle in CO at 37 DEG C₂The incubator of (2) for cultivation. The next day, the culture medium was changed with fresh medium, and the cells were continuously cultured until a certain number of cells were proliferated, digested, and planted in a 24-well plate. The preparation of the culture medium for L929 cell culture is as follows: 90% MEM in 100mL of the culture mediumMedium + 10% fresh fetal bovine serum FBS +1mL diabody (penicillin, streptomycin).

Second, MTT cytotoxicity assay

The application adopts an MTT colorimetric method, selects an L929 cell as an experimental cell, takes 24h as a survey point, and surveys the influence of a Si02@ QDs @ CDs probe on the cell growth.

L929 cells in flasks were trypsinized and dispersed in DMEM medium containing 10% calf serum and 1% antibiotics. After adjusting the cell suspension concentration, 100. mu.L of the suspension was added to each well of a 96-well cell culture plate, and the plate was incubated at 5% C02 and 37 ℃ until the cells adhered to the wall. Ultrafiltering, centrifuging and purifying the 4SiO₂@10Ca/6Tb:Bi₂O₂S @ CDS fluorescent probe was lyophilized and weighed, dissolved in DMEM medium to obtain stock solutions of different mass concentrations, and 100g of the stock solution was added to each well to give final concentrations of Si02@ QDs @ CDs of O.05, 0.1, 0.2, 0.4, 0.8, 1.6mg/mL, each concentration being repeated six times. After 24 hours of incubation, 10g of LM TT solution was added to each well, incubated for 4 hours and washed with PBS. 150g of LDMSO was added to each well, and the absorbance at 490nm was measured on a microplate reader after 10min of shaking. Cell survival (%) — absorbance value (treatment)/absorbance value (control) X100%.

Another 96-well plate is taken, the other steps are repeated by using Si02@ QDs with final concentrations of 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6mg/mL, and a group of cytotoxicity tests of Si02@ QDs are carried out for comparison analysis.

Tri, 4SiO₂@10Ca/6Tb:Bi₂O₂S @ CDS fluorescent probe for L929 cell imaging

The L929 cell strain is inoculated in a six-hole culture plate and cultured in an incubator with 5% C02 and 37 ℃. The Si02@ QDs @ CDs probe was added for co-incubation in the logarithmic growth phase of the cells. 1, 3, 8 and 18h were selected as observation time points. A time plan was made, 2.3 washes with sterile PBS, 90% glycerol mounting, and Si02@ QDs @ was observed with the aid of confocal microscope Zeiss510META, binding of CDs probe to L929 cells. One group is Hg-containing²⁺The L929 cell of (1), and a comparative experiment is designed. This demonstrated that Si02@ QDs @ CDs act on L929 intracellular Hg²⁺Specificity of detection.

Fourth, results and analysis

4.1SiO₂@10Ca/6Tb:Bi₂O₂Cytotoxicity assay of S @ CDS fluorescent probes

As shown in FIGS. 12 and 13, SiO was compared₂@10Ca/6Tb:Bi₂O₂The S @ CDS fluorescent probe respectively influences the survival rate of L929 cells before and after wrapping the aminated carbon point. First, quantum dots of different concentrations were added to the cell culture dish for SiO₂@10Ca/6Tb:Bi₂O₂For S microspheres, when the concentration of quantum dots in cell sap is lower than O.05mg/mL, the cell survival rate is higher than 75%, but when the concentration of quantum dots in cell sap is greater than or equal to 0.05mg/mL, the cell survival rate is obviously reduced.

From FIG. 13, SiO after wrapping the carbon dots is observed₂@10Ca/6Tb:Bi₂O₂The cell survival rate of the S @ CDS fluorescent probe is kept to be higher than 85% in the concentration range of 0.05mg/mL.1.6m g/mL, so that the cell compatibility of the probe can be improved by wrapping a dense carbon dot material on the surface of the microsphere, and the L929 cell imaging using the probe with low biological toxicity does not cause great influence on the growth of cells.

4.2 cellular imaging analysis

In order to evaluate the application prospect of the designed probe in the fluorescent labeling of a biological system, the probe is directly used for Hg in L929 cells²⁺The fluorescence detection of (3). As can be observed from FIG. 14, with SiO₂@10Ca/6Tb:Bi₂O₂The L929 cells incubated by the S @ CDS fluorescent probe show clear green fluorescence, and the dazzling green fluorescence is enough to cover the blue fluorescence of the carbon dots; when the right concentration of Hg is added²⁺Then, the quantum dot as the signal label in the dual-emission fluorescent probe is desirably subjected to fluorescence quenching reaction, and at this time, the blue fluorescence of the carbon dot is gradually highlighted and easily captured by the naked eye. Due to the presence of osmotic pressure difference of fluorescent probe inside and outside the cell membrane, and SiO₂@10Ca/6Tb:Bi₂O₂The biological property of the S @ CDS fluorescent probe is low, and the L929 cell takes up enough to completely realize the intracellular Hg²⁺And sensitively detected fluorescence imaging ofProvides a new research idea for realizing the detection of the probe on the metal ions in the living body.

The application realizes the SiO₂@10Ca/6Tb:Bi₂O₂High-sensitivity and high-selectivity detection of Hg by S @ CDS fluorescent probe in cell system²⁺The cell imaging technique of (1). This work is not only intracellular Hg²⁺The quantitative analysis provides a new idea, and has important significance for preparing high-performance fluorescent probes and promoting the application of the fluorescent probes in life science.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention, including any reference to the above-mentioned embodiments. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Quantum dot fluorescent probe in cell Hg²⁺The application in visual detection is that the quantum dot fluorescent probe comprises silicon dioxide microspheres and 10Ca/6Tb: Bi₂O₂S nano-crystal and aminated fluorescent carbon quantum dot, wherein a layer of 10Ca/6Tb Bi is modified after the surface of the silicon dioxide microsphere is aminated₂O₂S nanocrystal of 10Ca/6Tb Bi₂O₂The outer layer of the S nanocrystal is also wrapped and modified with aminated fluorescent carbon quantum dots.

2. Use according to claim 1, characterized in that the silica microspheres, 10Ca/6Tb: Bi₂O₂The molar ratio of the S nanocrystal to the aminated fluorescent carbon quantum dot is 1:5-10: 10-30; preferably 1:6-8: 12-25.

3. The use of claim 1 or 2, wherein the quantum dot fluorescent probe is prepared by a method comprising the steps of:

1) synthesis of silica microspheres

First, 80.0mL of ethanol, 4.850mL of water, and 3.6mL of 25% ammonia water were sequentially added to a 250.0mL flask, heated to 55 ℃ with vigorous stirring, and then a mixed solution of 3.1mL of Tetraethoxysilane (TEOS) and 8.0mL of ethanol was rapidly added to the flask; keeping the temperature of the solution at 55 ℃, reacting for 5 hours to prepare silicon dioxide microspheres with the particle size of about 40 +/-5 nm, and taking the prepared microspheres as seeds for continuous growth of the microspheres;

2) synthesis and purification of carbon amide quantum dots

Re-dissolving 20.0mg of silicon dioxide microspheres in a freshly prepared 2.0mL of anhydrous methanol solution, ultrasonically dispersing the 3-aminopropyltriethoxysilane in the freshly prepared anhydrous methanol solution at a concentration of 10%, and then stirring at room temperature for reaction for 2 hours; after the reaction, the aminated silica spheres were washed repeatedly three times with methanol solution and water in this order and dispersed in 10Ca/6Tb: Bi of 2, OmL₂O₂A mixture of S nanocrystals (5mg/mL) and 2.0mL of EDC (20mg/mL) was brought to 4 deg.CMixing and stirring for 12h, centrifugally washing for 3 times after the reaction is finished, and washing away the uncoupled nanocrystals to obtain 10Ca/6Tb: Bi₂O₂S nanocrystalline-modified silicon spheres;

4. Cell Hg²⁺The visual detection kit comprises a quantum dot fluorescent probe, wherein the quantum dot fluorescent probe comprises silicon dioxide microspheres and 10Ca/6Tb: Bi₂O₂S nano-crystal and aminated fluorescent carbon quantum dot, wherein a layer of 10Ca/6Tb Bi is modified after the surface of the silicon dioxide microsphere is aminated₂O₂S nanocrystal of 10Ca/6Tb Bi₂O₂The outer layer of the S nanocrystal is also wrapped and modified with aminated fluorescent carbon quantum dots.

5. The kit of claim 4, wherein the silica microspheres and 10Ca/6Tb: Bi₂O₂The molar ratio of the S nanocrystal to the aminated fluorescent carbon quantum dot is 1:5-10: 10-30; preferably 1:6-8:12-25, and the quantum dot fluorescent probe is used for preparing human blood Hg²⁺The quantum dot fluorescent probe comprises silicon dioxide microspheres and 10Ca/6Tb: Bi₂O₂S nano-crystal and aminated fluorescent carbon quantum dot, wherein a layer of 10Ca/6Tb Bi is modified after the surface of the silicon dioxide microsphere is aminated₂O₂S nanocrystal of 10Ca/6Tb Bi₂O₂The outer layer of the S nanocrystal is also wrapped and modified with aminated fluorescent carbon quantum dots.

6. Use according to claim 1, characterized in that the silica microspheres, 10Ca/6Tb: Bi₂O₂Amount of S nanocrystalline and aminated fluorescent carbonThe molar ratio of the sub-points is 1:5-10: 10-30; preferably 1:6-8: 12-25.

7. The use of claim 1 or 2, wherein the quantum dot fluorescent probe is prepared by a method comprising the steps of:

1) synthesis of silica microspheres

First, 80.0mL of ethanol, 4.850mL of water, and 3.6mL of 25% aqueous ammonia were sequentially added to a 250.0mL flask, which was heated to 55 ℃ with vigorous stirring.

8. Then a mixed solution of 3.1mL Tetraethoxysilane (TEOS) and 8.0mL ethanol was rapidly added to the flask; keeping the temperature of the solution at 55 ℃, reacting for 5 hours to prepare silicon dioxide microspheres with the particle size of about 40 +/-5 nm, and taking the prepared microspheres as seeds for continuous growth of the microspheres;

2) synthesis and purification of carbon amide quantum dots

Taking 20.0mg of silicon dioxide microspheres to be dissolved in 2.0mL of anhydrous methanol solution which is prepared freshly again,the concentration of 3-aminopropyltriethoxysilane in the freshly prepared anhydrous methanol solution accounts for 10 percent, ultrasonic dispersion is carried out, and then stirring reaction is carried out for 2 hours at room temperature; after the reaction, the aminated silica spheres were washed repeatedly three times with methanol solution and water in this order and dispersed in 10Ca/6Tb: Bi of 2, OmL₂O₂Mixing and stirring the S nanocrystal (5mg/mL) and 2.0mL of EDC (20mg/mL) for 12h at 4 ℃, centrifugally washing for 3 times after the reaction is finished, and washing away the uncoupled nanocrystal to obtain 10Ca/6Tb: Bi₂O₂S nanocrystalline-modified silicon spheres;

9. Human blood Hg²⁺The visual detection kit comprises a quantum dot fluorescent probe, wherein the quantum dot fluorescent probe comprises silicon dioxide microspheres and 10Ca/6Tb: Bi₂O₂S nano-crystal and aminated fluorescent carbon quantum dot, wherein a layer of 10Ca/6Tb Bi is modified after the surface of the silicon dioxide microsphere is aminated₂O₂S nanocrystal of 10Ca/6Tb Bi₂O₂The outer layer of the S nanocrystal is also wrapped and modified with aminated fluorescent carbon quantum dots.

10. The kit of claim 4, wherein the silica microspheres and 10Ca/6Tb: Bi₂O₂The molar ratio of the S nanocrystal to the aminated fluorescent carbon quantum dot is 1:5-10: 10-30; preferably 1:6-8: 12-25.