CN111394090A - Three-layer coated copper cluster ratio fluorescent probe and preparation method and application thereof - Google Patents
Three-layer coated copper cluster ratio fluorescent probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a three-layer-wrapped copper cluster ratio fluorescent probe and a preparation method and application thereof. Compared with the prior art, the probe provided by the invention has the advantages that the stability of the internal standard is improved by virtue of the protection effect of the silicon dioxide of the middle layer, so that the fluorescence intensity of the internal standard is not easily interfered by the outside. When the probe is used for the fluorescence detection of drugs, the selectivity, the sensitivity and the accuracy are greatly improved.
Description
Technical Field
The invention belongs to the technical field of drug detection, and particularly relates to a three-layer coated copper cluster ratiometric fluorescent probe and a preparation method and application thereof.
Background
At present, in the field of drug detection, common instrument methods have the common defects of complex operation, high cost, time and labor waste and the like. The fluorescent spectrometry method for detecting drugs has the advantages of convenient operation, low cost, simplicity, rapidness, intuition and the like. The metal nanocluster has the advantages of small size, good light stability, large Stokes displacement, mild preparation conditions, no toxicity, no harm and the like, and is a fluorescent sensor with a very good application prospect. For example, chinese patent document (CN110609024A) discloses a fluorescence-enhanced two-color visual drug detection probe and a preparation method thereof, wherein two types of copper nanoclusters capable of emitting fluorescence of different colors under single excitation light are disclosed, and by designing a fluorescence-enhanced detection mode, visual drug detection can be realized by using the copper nanoclusters, which has the advantages of high signal-to-noise ratio, high detection sensitivity, and strong reliability.
However, in the field of drug detection, the metal nanoclusters are mostly used as monochromatic fluorescence sensors in which the metal nanoclusters are used as fluorescence probes, and the detection of the target object is realized by measuring the intensity change of a single fluorescence emission wavelength. The construction of ratio-type fluorescence sensors is beneficial to solving the problems and can also improve selectivity and sensitivity, and attention is gradually paid to the field of fluorescence detection at present.
The ratiometric fluorescence sensor generally comprises two or more fluorescence sensors with different emission wavelengths, when a target detection object is added, the fluorescence intensity of one fluorescence sensor is often caused to be strongly changed, but the fluorescence intensity of the other fluorescence sensor has no obvious effect (the intensity of the fluorescence sensor can be used as an internal standard), so that the detection of the target object is realized according to the ratio of the two fluorescence intensities, and the advantages of improving the selectivity and the sensitivity, and being beneficial to eliminating the interference of other factors and improving the detection accuracy are achieved.
At present, the construction of a ratio fluorescence sensor is to simply mix two fluorescent substances with different luminescence characteristics, and the problem of the sensors is that the fluorescence intensity of an internal standard is easily interfered by the outside world, so that the detection accuracy is not enough.
Disclosure of Invention
The invention aims to solve the problems and provide a three-layer coated copper cluster ratiometric fluorescent probe and a preparation method and application thereof.
The invention realizes the purpose through the following technical scheme:
the invention provides a three-layer-wrapped copper cluster ratio fluorescent probe, which comprises an internal standard, silicon dioxide and a copper nanocluster, wherein the silicon dioxide is wrapped outside the internal standard, and the copper nanocluster is wrapped outside the silicon dioxide to form the ratio fluorescent probe with an internal standard @ silicon dioxide @ copper cluster three-layer wrapping structure.
As a further optimization of the invention, the copper nanoclusters are wrapped outside the silicon dioxide by an electrostatic attraction strategy.
As a further optimization scheme of the invention, the internal standard is a carbon point, the internal carbon point has a different light-emitting color from the external copper nanocluster, the internal carbon point does not respond to the drug, and the drug is detected by utilizing the sensitivity of the external copper nanocluster to the drug to be detected.
The invention also provides a preparation method of the three-layer-wrapped copper cluster ratiometric fluorescent probe, which comprises the following steps of:
(1) preparing an internal standard and a copper cluster, and performing surface modification on the copper cluster by using an antibody specifically bound with a drug to be detected to obtain the copper cluster of the modified antibody;
(2) taking ethyl orthosilicate, adding ethanol for prehydrolysis under an alkaline condition, adding the internal standard solution obtained in the step (1), and preparing and obtaining internal standard @ silicon dioxide with a double-layer wrapping structure;
(4) and mixing the internal standard @ silicon dioxide with the copper cluster of the modified antibody to prepare the internal standard @ silicon dioxide @ copper cluster with a three-layer wrapping structure, thereby obtaining the ratiometric fluorescent probe.
As a further optimization scheme of the invention, in the step (1):
the internal standard is a carbon point emitting blue fluorescence, but is not limited to the internal standard, and the preparation method comprises the steps of adding L-proline into ultrapure water, reacting in a polytetrafluoroethylene stainless steel autoclave at 180 ℃, naturally cooling to room temperature after the reaction is finished, filtering by a 0.45 mu M microporous filter membrane to remove large particles and precipitates, and dialyzing by a 5000Da dialysis bag to obtain the internal standard;
the copper cluster is a copper nanocluster emitting orange fluorescence, but is not limited thereto, and the preparation method thereof includes: mixing a copper salt and a surface ligand containing a carboxyl functional group in ultrapure water, adjusting the pH to 3-5 by using an alkaline substance to obtain a mixed solution, adding a poor solvent into the mixed solution, centrifuging to obtain a precipitate, and performing vacuum drying to obtain a copper cluster;
the method for carrying out surface modification on the copper cluster by using the antibody specifically bound with the drug to be detected to obtain the copper cluster of the modified antibody comprises the following steps: mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and an aqueous solution of N-hydroxysuccinimide sodium salt, adding a monoclonal antibody specifically bound with a drug to be detected, and uniformly stirring to obtain a standby antibody solution; dispersing the copper clusters in water to obtain copper cluster dispersion liquid; and mixing the standby antibody solution with the copper cluster dispersion liquid, and incubating at 37 ℃ to finish the modification of the copper cluster surface antibody.
As a further optimized scheme of the invention, in the step (3), the internal standard @ silicon dioxide is mixed with the copper cluster of the modified antibody, and the cationic polymer electrolyte is added into the reaction system at the same time, so that the surface of the copper cluster is modified by the positively charged polymer electrolyte, and the copper cluster and the silicon dioxide can be connected together through an electrostatic attraction strategy after the silicon dioxide surface is generally negatively charged.
As a further optimization scheme of the invention, the cationic polymer electrolyte is selected from one or more of polydiallyl dimethyl ammonium chloride, polyethyleneimine hydrochloride, polyvinyl pyridine and polyvinyl amine, but is not limited thereto.
The invention also provides application of the three-layer-wrapped copper cluster ratiometric fluorescent probe in drug detection.
The invention has the beneficial effects that:
1) the fluorescent material (carbon dots, copper clusters and silicon dioxide) is low in price and rich in source;
2) when the probe is used for fluorescence detection of drugs, compared with a single fluorescence sensing mode, the probe is higher in accuracy and not easy to interfere, the stability of an internal standard is good, and due to the protection effect of the silicon dioxide in the middle layer, the fluorescence intensity of the probe is more stable and not easy to interfere by the outside, so that the selectivity, the sensitivity and the accuracy of the whole composite probe are greatly improved;
3) according to the invention, the silicon dioxide intermediate layer is connected with the clusters by using an electrostatic attraction strategy, so that the change of fluorescence emission of the clusters caused by a covalent bond connection mode is effectively avoided, and the decrease of fluorescence intensity caused by aggregation can be prevented;
4) the invention can also obtain probes with different fluorescence emission performances by changing the ligand of the copper nanocluster.
Drawings
FIG. 1 is a graph of the fluorescence response of a ratiometric fluorescent probe of the invention to ketamine;
FIG. 2 is a graph of the linear relationship of ratiometric fluorescent probes of the present invention to ketamine content;
FIG. 3 is a graph showing the fluorescence response to chloraminoketone when copper nanoclusters are used as monochromatic fluorescent probes;
FIG. 4 is a graph of the linear relationship of copper nanoclusters as monochromatic fluorescent probes to chloraminoketone content;
FIG. 5 is a graph of the fluorescence response of ratiometric fluorescent probes with no packing structure to ketamine;
FIG. 6 is a graph of the linear relationship of ratiometric fluorescent probe without packing structure to ketamine content;
FIG. 7 is a graph of the fluorescence response of a non-electrostatically attracted, three-layer encapsulated ratiometric fluorescent probe to ketamine;
FIG. 8 is a graph of the linear relationship of non-electrostatically attracted three-layer encapsulated ratiometric fluorescent probes to ketamine content.
Detailed Description
Example 1
The embodiment provides a three-layer-wrapped copper cluster ratiometric fluorescent probe, which is characterized in that a central carbon point is used as an internal standard, silicon dioxide is wrapped outside the carbon point to be used as an intermediate layer, and a copper cluster is wrapped outside the intermediate layer of the silicon dioxide by utilizing an electrostatic attraction strategy to obtain the ratiometric fluorescent probe with a three-layer-wrapped structure of the carbon point @ silicon dioxide @ copper cluster. The following detailed description of the method for preparing ratiometric fluorescent probes is provided in connection with specific examples, and it is to be understood that the following detailed description is provided for the purpose of further illustrating the present application and is not to be construed as limiting the scope of the present application, and that certain insubstantial modifications and adaptations of the present application may be made by those skilled in the art based on the teachings of the above application.
1. Material
The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used therein are commercially available products unless otherwise specified.
2. Method of producing a composite material
(1) Preparing carbon dots and copper clusters, and performing antibody surface modification on the copper clusters
Preparation of carbon dots has been reported at present, and the carbon dots emitting blue fluorescence are selected in the embodiment, and the specific method comprises the steps of taking 25ml of ultrapure water solution containing 1.2g of L-proline, reacting in a polytetrafluoroethylene stainless steel autoclave at 180 ℃ for 4h, naturally cooling the solution to room temperature after the reaction is finished, filtering the solution by using a 0.45uM microporous filter membrane to remove larger particles and precipitates, dialyzing for 6h by using a 5000Da dialysis bag, and storing the obtained solution as the carbon dots in a refrigerator at 4 ℃ for later use.
Preparation of copper clusters the copper nanocluster capable of emitting orange fluorescence with a central wavelength of 600nm under 365nm excitation light is selected in the embodiment, and the specific method is that 200 mmol of copper salt and 1-25mol of surface ligand containing carboxyl functional groups are mixed in 6-16m L ultrapure water, the pH is adjusted to 3-5 by using an alkaline substance to obtain a mixed solution, a poor solvent is added into the mixed solution according to the volume ratio of the mixed solution to the poor solvent of 1:25-95, then centrifugation is carried out, and the obtained precipitate is dried in vacuum to obtain the copper nanocluster powder emitting orange fluorescence.
Preparing a spare antibody solution, namely mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with the concentration of 0.9-3.8 mg/L and an aqueous solution of N-hydroxysuccinimide sodium salt with the concentration of 1.1-4.3 mg/L according to the volume ratio of 1:1, then adding a monoclonal antibody specifically bound with the drug to be detected according to the type of the drug to be detected, and continuously stirring for 0.5-2 hours at 37 ℃ to obtain the spare antibody solution, wherein the concentration of the antibody in the spare antibody solution ranges from 50-200 mu M.
And (3) dispersing the copper cluster in water to obtain a dispersion liquid with the concentration of 0.95-3.8mg/m L, adding the dispersion liquid of the copper nanocluster emitting orange fluorescence into the standby antibody solution according to the volume ratio of 1:1-5, and incubating for 3-6 hours at 37 ℃ to finish the modification of the copper nanocluster surface antibody to obtain the modified orange copper nanocluster dispersion liquid.
(2) Preparation of carbon point @ silica
Mixing 50-150ul ammonia water (or other alkaline substances such as NaOH) with mass concentration of 25%, 800ul ultrapure water, 100ul-1000ul TEOS, and 8.7ml ethanol, pre-hydrolyzing for 30min, adding 100ul of above carbon dots, and reacting at room temperature for 12-48 h. And centrifuging to collect the product, washing with ethanol and water respectively to obtain the carbon point @ silicon dioxide, and dispersing the carbon point @ silicon dioxide in 2ml of ultrapure water for later use. The amount range of TEOS is 100ul-1000ul, the reaction time with carbon points is 12-48h, and the thickness of the wrapped silicon dioxide can be adjusted by adjusting the amount range of TEOS and the reaction time with carbon points.
(3) Preparation of carbon dot @ silicon dioxide @ copper cluster
2ml of the carbon point @ silicon dioxide is taken, 1mg of the antibody-modified copper cluster and 0.5-3mg of the cationic polymer electrolyte are added, and the mixture is stirred and reacted for 30min under the condition of 500 revolutions per minute. Wherein the cationic polymer electrolyte is selected from polydiallyl dimethyl ammonium chloride, polyethyleneimine hydrochloride, polyvinyl pyridine, polyvinyl amine, etc.
Example 2
The embodiment provides a preparation method of a three-layer wrapped copper cluster ratiometric fluorescent probe for detecting ketamine, which comprises the following steps:
(1) preparing carbon dots and copper clusters, and performing antibody surface modification on the copper clusters
A process for preparing the carbon dots emitting blue fluorescence includes such steps as putting 25ml of ultra-pure water solution containing L-proline (1.2 g) in stainless-teflon autoclave, reacting at 180 deg.C for 4 hr, cooling to room temp, filtering with 0.45uM millipore filter to remove big particles and deposit, dialyzing in 5000Da dialysis bag for 6 hr to obtain carbon dots, and storing in 4 deg.C refrigerator.
The preparation method of the orange fluorescent copper nanocluster comprises the steps of mixing 360mmol of copper nitrate and 9mol of glutathione in 8m L ultra-pure water, adjusting the pH value to 4 by using 1.5 mol/L of sodium hydroxide to obtain a mixed solution, adding ethanol into the mixed solution according to the volume ratio of the mixed solution to a poor solvent of 1:60, centrifuging, and drying the obtained precipitate in vacuum to obtain the orange fluorescent copper nanocluster powder.
A solution of a candidate antibody was prepared by mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride having a concentration of 1.9 mg/L and an aqueous solution of N-hydroxysuccinimide sodium salt having a concentration of 2.15 mg/L in a volume ratio of 1:1, followed by addition of ketamine monoclonal antibody and continuous stirring at 37 ℃ for 1 hour to obtain a ketamine antibody solution in which the concentration of the antibody was in the range of 100. mu.M.
And (3) copper cluster surface modification antibody, namely dispersing the copper cluster in water to obtain a dispersion liquid with the concentration of 2mg/m L, adding the dispersion liquid of the copper cluster into a ketamine antibody solution according to the volume ratio of 1:1, and incubating for 4 hours at 37 ℃ to finish the modification of the copper nanocluster surface antibody and obtain the modified orange light copper nanocluster dispersion liquid.
(2) Preparation of carbon point @ silica
100ul of ammonia water (with the mass concentration of 25 percent), 800ul of ultrapure water, 300ul of TEOS and 8.7ml of ethanol are taken for prehydrolysis for 30min, 100ul of carbon dots are added, and the reaction is carried out for 24h at room temperature. The product was collected by centrifugation, washed with ethanol and water, respectively, and dispersed in 2ml of ultrapure water for use.
(3) Preparation of carbon dot @ silicon dioxide @ copper cluster
2ml of the carbon dot @ silicon dioxide is taken, 1mg of the copper cluster of the modified antibody and 1mg of PDDA (20% aqueous solution, Mw: 100000-200000g/mol) are added, and the mixture is stirred and reacted for 30min under the condition of 500 revolutions per minute, so that the three-layer-coated copper cluster ratiometric fluorescent probe is obtained.
Ketamine was detected using this ratio fluorescent probe: in the ratiometric fluorescent probes, ketamine solutions with different concentrations (0nM, 10nM, 20nM, 40nM, 60nM, and 80nM) are respectively added, fluorescence emission spectra are recorded under 365nM excitation light, data recording is performed on the intensities of fluorescence emission peaks at 460nM and 600nM, a ratio of the intensity of the fluorescence emission peak at 600nM to the intensity of the fluorescence emission peak at 460nM is taken as a vertical coordinate, the concentration of a target analyte is taken as a horizontal coordinate, a dot diagram is made, linear fitting is performed on the data to obtain a standard curve, and a standard concentration conversion formula is obtained, so that the results are shown in fig. 1 and 2, and a fluorescence response graph of the ratiometric fluorescent probe prepared for the embodiment for ketamine and a linear relation graph of the ketamine content are obtained.
A control group was also set:
control 1: taking the modified orange copper nanocluster as a monochromatic fluorescent probe and used for detecting ketamine solution with different concentration gradients, recording a fluorescence emission spectrum under 365nm exciting light, recording data of the intensity of a fluorescence emission peak at 600nm, taking the ratio of the intensity of the fluorescence emission peak at 600nm when a target object to be detected is added to the fluorescence emission peak at 600nm when the target object to be detected is not added as a vertical coordinate, taking the concentration of the target object to be detected as a horizontal coordinate, making a dot diagram, performing linear fitting on the data to obtain a standard curve, and obtaining a standard concentration conversion formula, wherein the result is shown in fig. 3 and 4 and is a linear relation diagram of a fluorescence response diagram to chloramine and the content to chloramine when the copper nanocluster is taken as the monochromatic fluorescent probe;
control 2: mixing carbon dots emitting blue fluorescence with modified orange copper nanoclusters to obtain a ratio fluorescence probe without a wrapping structure, detecting ketamine solution with different concentration gradients, recording fluorescence emission spectra under 365nm excitation light, recording data of intensities of fluorescence emission peaks at 460nm and 600nm, taking a ratio of the intensity of the fluorescence emission peak at 600nm to the intensity of the fluorescence emission peak at 460nm as a vertical coordinate, taking the concentration of a target object to be detected as a horizontal coordinate, making a point diagram, performing linear fitting on the data to obtain a standard curve, and obtaining a standard concentration conversion formula, wherein the result is shown in fig. 5 and 6, and is a linear relation diagram of the ratio fluorescence of the wrapping structure to the content of ketamine and the ratio fluorescence response diagram of the ketamine.
Control 3: when the carbon dot @ silicon dioxide @ copper cluster three-layer-wrapped ratiometric fluorescent probe is prepared, no cationic polymer electrolyte is added, the non-electrostatic-attraction three-layer-wrapped ratiometric fluorescent probe is obtained and used for detecting ketamine solutions with different concentration gradients, a fluorescence emission spectrum is recorded under 365nm excitation light, data recording is carried out on the intensities of fluorescence emission peaks at 460nm and 600nm, the ratio of the intensity of the fluorescence emission peak at 600nm to the intensity of the fluorescence emission peak at 460nm is taken as a vertical coordinate, the concentration of a target object to be detected is taken as a horizontal coordinate, a dot diagram is made, linear fitting is carried out on the data, a standard curve is obtained, a standard concentration conversion formula is obtained, and the result is shown in fig. 7 and 8 and is a linear relation diagram of the non-electrostatic-attraction three-layer-wrapped ratiometric fluorescent probe for ketamine and the content of p-chloroaminone.
The results show that the probe of the invention has stable fluorescence intensity and higher detection sensitivity to chloramine ketone, and the linear relation equations in figures 2, 4, 6 and 8 show that the slopes of the fitted linear equations are different when the copper clusters in different states detect the chloramine ketone, wherein the slope of the equation in the ratiometric fluorescent probe of the invention is-0.01179, R is-0.011792This indicates that the fluorescence intensity of the three-layer encapsulated ratiometric fluorescent probe of the present invention changes more and the detection sensitivity is higher when the same concentration of ketamine is detected.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (8)
1. The three-layer coated copper cluster ratio fluorescent probe is characterized by comprising an internal standard, silicon dioxide and a copper nanocluster, wherein the silicon dioxide is coated outside the internal standard, and the copper nanocluster is coated outside the silicon dioxide to form the ratio fluorescent probe with an internal standard @ silicon dioxide @ copper cluster three-layer coating structure.
2. The triple-wrapped copper cluster ratiometric fluorescent probe according to claim 1, wherein the copper nanoclusters are wrapped outside silicon dioxide by an electrostatic attraction strategy.
3. The triple-layered encapsulated copper cluster ratiometric fluorescent probe according to claim 1, wherein the internal standard is a carbon dot, and the internal carbon dot has a different color from the external copper nanoclusters, wherein the internal carbon dot does not respond to the drug, and the drug is detected by using the sensitivity of the external copper nanoclusters to the drug to be detected.
4. A method for preparing a triple-wrapped copper cluster ratiometric fluorescent probe according to any one of claims 1 to 3, comprising the steps of:
(1) preparing an internal standard and a copper cluster, and performing surface modification on the copper cluster by using an antibody specifically bound with a drug to be detected to obtain the copper cluster of the modified antibody;
(2) taking ethyl orthosilicate, adding ethanol for prehydrolysis under an alkaline condition, adding the internal standard solution obtained in the step (1), and preparing and obtaining internal standard @ silicon dioxide with a double-layer wrapping structure;
(3) and mixing the internal standard @ silicon dioxide with the copper cluster of the modified antibody to prepare the internal standard @ silicon dioxide @ copper cluster with a three-layer wrapping structure, thereby obtaining the ratiometric fluorescent probe.
5. The method for preparing a triple-wrapped copper cluster ratiometric fluorescent probe according to claim 4, wherein in the step (1):
the internal standard is carbon point emitting blue fluorescence, and the preparation method comprises adding L-proline into ultrapure water, reacting in polytetrafluoroethylene stainless steel autoclave at 180 deg.C, naturally cooling to room temperature after reaction, filtering with 0.45 μ M microporous membrane to remove large particles and precipitate, dialyzing with 5000Da dialysis bag to obtain internal standard;
the copper cluster is a copper nanocluster emitting orange fluorescence, and the preparation method comprises the following steps: mixing a copper salt and a surface ligand containing a carboxyl functional group in ultrapure water, adjusting the pH to 3-5 by using an alkaline substance to obtain a mixed solution, adding a poor solvent into the mixed solution, centrifuging to obtain a precipitate, and performing vacuum drying to obtain a copper cluster;
the method for carrying out surface modification on the copper cluster by using the antibody specifically bound with the drug to be detected to obtain the copper cluster of the modified antibody comprises the following steps: mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and an aqueous solution of N-hydroxysuccinimide sodium salt, adding a monoclonal antibody specifically bound with a drug to be detected, and uniformly stirring to obtain a standby antibody solution; dispersing the copper clusters in water to obtain copper cluster dispersion liquid; and mixing the standby antibody solution with the copper cluster dispersion liquid, and incubating at 37 ℃ to finish the modification of the copper cluster surface antibody.
6. The method for preparing the triple-layer coated copper cluster ratiometric fluorescent probe according to claim 4, wherein in the step (3), the internal standard @ silica is mixed with the modified antibody copper cluster, and the cationic polymer electrolyte is added into the reaction system, so as to link the copper cluster and the silica together through an electrostatic attraction strategy.
7. The method for preparing a three-layer coated copper cluster ratiometric fluorescent probe according to claim 6, wherein the cationic polymer electrolyte is selected from one or more of polydiallyldimethylammonium chloride, polyethyleneimine hydrochloride, polyvinyl pyridine and polyvinyl amine.
8. Use of the triple-wrapped copper cluster ratiometric fluorescent probe of any one of claims 1 to 3 for detecting drugs.
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