CN111721748A - Fluorescent probe for detecting zoledronic acid, preparation method of fluorescent probe, fluorescent sensor, construction method of fluorescent sensor and application of fluorescent sensor - Google Patents
Fluorescent probe for detecting zoledronic acid, preparation method of fluorescent probe, fluorescent sensor, construction method of fluorescent sensor and application of fluorescent sensor Download PDFInfo
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Abstract
The invention discloses a fluorescent probe for detecting zoledronic acid, a preparation method thereof, a fluorescent sensor, a construction method and application thereof, wherein the fluorescent probe is a sulfur quantum dot-Fe3+And (3) compounding fluorescent probes. The invention leads sulfur quantum dots-Fe3+The fluorescent probe is mixed with standard zoledronic acid solutions with different concentrations for reaction, a standard working curve for detecting zoledronic acid is obtained through measurement and drawing by a fluorescence method, and a fluorescence sensor for detecting zoledronic acid is constructed, so that a novel method for rapidly detecting zoledronic acid based on change of a sulfur quantum dot fluorescence signal is established; by Fe3+And the competitive reaction of the zoledronic acid and the sulfur quantum dots is used for regulating and controlling the size of the sulfur quantum dots, so that the change of a fluorescence signal is captured, and the zoledronic acid in a sample is fixedAnd (4) detecting the quantity. The detection method has the advantages of simplicity, rapidness, reliability, low cost and the like, and has been successfully used for determining the content of the zoledronic acid in human serum, urine samples and zoledronic acid injection.
Description
Technical Field
The invention relates to the technical field of fluorescence analysis, in particular to a fluorescent probe for detecting zoledronic acid, a preparation method thereof, a fluorescent sensor, a construction method thereof and application thereof.
Background
Zoledronic Acid (ZA) is a heterocyclic imidazole third-generation bisphosphonate. Bisphosphonates have high calcium affinity and inhibit osteoclasts, and ZA plays an important role in the treatment of metabolic bone diseases. More importantly, it has been extensively expanded to inhibit bone resorption as a priority for the treatment of Paget's disease, postmenopausal osteoporosis and neuropathic pain. In addition, it may have an antitumor effect directly. To date, many traditional methods for detecting zoledronic acid have been developed, including ion exchange high performance liquid chromatography (IEC), ion-pair high performance liquid chromatography, reverse phase liquid chromatography (RP-LC), and liquid chromatography-tandem mass spectrometry (LC/MS), which mostly rely on chromatographic analysis, and have a series of disadvantages, such as complicated sample purification, complicated instruments, and time-consuming process, which limit their application in actual sample determination.
Sulfur is a rich bioactive element, and has the functions of catalysis, oxidation resistance, sterilization, cancer resistance and the like, but the solubility of sulfur in water is poor, so that the application of sulfur in biomedicine is greatly limited, and scientists begin to explore the preparation of sulfur nano materials with small size and stable dispersion in a hydrophilic system. At present, the methods for preparing small-size sulfur nanoparticles mainly include: one-pot hydrothermal method, freeze-drying method, phase interface reaction, oxygen acceleration grading method, ultrasonic promotion method and the like. For example, Zhang et al [ Zhang, c.; zhang, p.; ji, x.; wang, h.; kuang, h.; cao, w.; pan, m.; shi, y.e.; wang, Z., ultrasonic-catalyzed synthesis of luminescent sulfur nanoparticles, chem Commun (Camb)2019,55(86), 13004-. In Song topic group [ Song, y.; tan, j.; wang, g.; gao, p.; lei, j.; zhou, L., Oxygen acellular fluorescent synthesis of high fluorescence quantum dots. chemical Science2020,11(3), 772-. The quantum dot prepared by the method has high fluorescence quantum yield (21.5%), and has good dispersibility in water and common organic solvents. Among them, small-sized sulfur quantum dots generally have photoluminescence characteristics. Therefore, the fluorescence sensor based on the quantum dots, particularly the sulfur quantum dots, has good application prospect in drug analysis as a rapid, sensitive and stable analysis technology. However, due to the lack of a mature method for preparing the sulfur quantum dots, the mechanism of photoluminescence is not very clear, the luminous efficiency is not high enough, and the application of the sulfur quantum dots is limited. Therefore, the practical application of sulfur quantum dots in the field of pharmaceutical analysis is still rare.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a fluorescent probe for detecting zoledronic acid, a preparation method thereof, a fluorescent sensor, a construction method thereof and applications thereof, by establishing a Fe-based fluorescent probe3+A novel fluorescent 'on-off-on' zoledronic acid detection strategy for gathering/dispersing control of Sulfur Quantum Dots (SQDs) is used for improving the sensitivity and speed of detecting the content of zoledronic acid in a sample.
In order to achieve the above objects and other related objects, a first aspect of the present invention provides a fluorescent probe for detecting zoledronic acid, wherein the fluorescent probe is sulfur quantum dot-Fe3+And (3) compounding fluorescent probes.
The second aspect of the invention provides a preparation method of a fluorescent probe for detecting zoledronic acid, which comprises the following steps:
(1) and (3) synthesis of sulfur quantum dots: adding alkali, sulfur powder and a dispersing agent into water, stirring uniformly, carrying out heating reaction to obtain a sulfur nanoparticle solution, mixing the sulfur nanoparticle solution and a hydrogen peroxide solution for reaction, cooling to room temperature after the reaction is finished, and enabling a product to appear blue under the irradiation of an ultraviolet lamp to obtain a sulfur quantum dot solution; the dispersing agent is polyethylene glycol;
(2) preparation of Fe3+A solution;
(3) adding sulfur quantum dot solution and Fe into buffer solution3+Incubating to obtain S quantum dot-Fe3+And (3) compounding fluorescent probes.
Optionally, in the step (1), the sulfur powder is at least one selected from sublimed sulfur powder, elemental sulfur, sulfur blocks, sulfur granules and sulfur powder; preferably, the sulfur powder is sublimed sulfur powder.
Alternatively, in step (1), the base is sodium hydroxide; more preferably, the sodium hydroxide is solid sodium hydroxide.
Optionally, in the step (1), the molecular weight of the polyethylene glycol is 400-1000; preferably, the polyethylene glycol is PEG-400.
Optionally, in the step (1), the ratio of the used amount of the alkali, the sulfur powder and the dispersant is 2-4: 1.0-1.6: 2-4(w/w/v), preferably 4:1.4:3 (w/w/v).
Optionally, in the step (1), the heating reaction is carried out at the temperature of 60-80 ℃ for 60-80 h; preferably, in the preparation method of the sulfur quantum dot solution, the heating reaction temperature is 70 ℃ and the time is 72 hours.
Optionally, in step (1), the concentration of the hydrogen peroxide solution is … … 0.75.75-7.5%, preferably 7.5%.
Optionally, in the step (1), the volume ratio of the sulfur nanoparticle solution to the hydrogen peroxide solution is 2-4:3-5, preferably 3: 4.
Optionally, in the step (1), the mixing reaction of the sulfur nanoparticle solution and the hydrogen peroxide solution is performed at room temperature and normal pressure for 1-3 h.
Optionally, in step (2), for the formulation of Fe3+The raw material of the solution is selected from at least one of ferric sulfate and ferric chloride; for the preparation of Fe3+The solvent of the solution is water, preferably deionized water.
Optionally, in step (2), the Fe3+In solution, Fe3+The concentration of the ions was 0.01 mol/L.
Optionally, in the step (3), the buffer solution is at least one selected from citric acid-sodium citrate; preferably a citric acid-sodium citrate buffer solution.
Optionally, in step (3), the pH of the buffer solution is 3.0 to 6.6, preferably 5.6.
Optionally, in the step (3), the buffer solution and the sulfur amountSub-dot solution and Fe3+The volume ratio of the solution is 1000-2000: 50-100: 50-100, preferably 1000: 75: 90.
optionally, in the step (3), the sulfur quantum dots-Fe3+Fe in composite fluorescent probe system3+The final concentration of the ions is 100-400. mu. mol/L, preferably 300. mu. mol/L.
The invention provides a fluorescence sensor for detecting zoledronic acid, which comprises the fluorescence probe of the first aspect or the fluorescence probe prepared by the preparation method of the second aspect and a standard working curve for detecting zoledronic acid, wherein the standard working curve is obtained by mixing and reacting the fluorescence probe with standard zoledronic acid solutions with different concentrations, and then measuring and drawing the standard working curve by a fluorescence method.
The fourth aspect of the invention provides a method for constructing a fluorescence sensor for detecting zoledronic acid, which comprises the following steps:
(1) mixing the fluorescent probe according to the first aspect or the fluorescent probe prepared by the preparation method according to the second aspect with standard zoledronic acid solutions with different concentrations, and then reacting to obtain sulfur quantum dots-Fe containing zoledronic acid with different concentrations3+-a zoledronic acid system;
(2) measuring by adopting a fluorescence method to obtain sulfur quantum dots-Fe of the zoledronic acid with different concentrations3+-fluorescence spectrum of zoledronic acid system;
(3) sulfur quantum dot-Fe in each fluorescence spectrogram3+The ratio of the fluorescence intensity of the zoledronic acid system to that of the blank control at 440nm is an ordinate, the concentration of the zoledronic acid standard solution is an abscissa, and a standard working curve for detecting zoledronic acid is drawn; wherein the blank control is sulfur quantum dot-Fe with final concentration of 0 mu mol/L of zoledronic acid3+-zoledronic acid system.
Further, the sulfur quantum dots-Fe containing different concentrations of zoledronic acid3+The final concentration of zoledronic acid in the zoledronic acid system is 1, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 500, 1000. mu. mol/L, respectively.
Further, the reaction time is 1-10min, preferably 5 min.
Further, the linear equation of the standard working curve for detecting the zoledronic acid is F/F00.0017c +1.015 (r-0.9971), wherein F is the sulfur quantum dot-Fe3+Fluorescence intensity at 440nm, F, of the zoledronic acid system, measured at an excitation wavelength of 350nm, a slit width of 10nm and a scanning speed of 500nm/min0The fluorescence intensity of the blank under the same conditions, and c is the concentration of zoledronic acid.
Further, the linear range of the standard working curve for detecting the zoledronic acid is 0.1-200 mu mol/L.
The fifth aspect of the present invention provides a method for detecting the content of zoledronic acid in a sample, wherein the method employs the fluorescence sensor according to the third aspect or the fluorescence sensor obtained by the construction method according to the fourth aspect, and the detection process includes the following steps: mixing the buffer solution, sulfur quantum dot solution and Fe3+And (3) uniformly mixing the solution and the sample solution, then reacting, measuring the fluorescence intensity by adopting a fluorescence method, and finally calculating the concentration of the zoledronic acid in the sample according to a standard working curve for detecting the zoledronic acid.
Further, the reaction time is 1-10min, preferably 5 min.
Further, the sample is selected from at least one of serum, urine sample and zoledronic acid injection.
Further, the sample is used after being pretreated, and the pretreatment mode of the sample comprises centrifugation and/or dilution.
Optionally, the centrifugation conditions of the sample are 10000-; the sample is diluted to 100-fold, 1000-fold, preferably 1000-fold of the stock solution.
As described above, the fluorescent probe for detecting zoledronic acid, the preparation method thereof, the fluorescent sensor, the construction method thereof and the application thereof have the following beneficial effects:
the invention relates to Sulfur Quantum Dots (SQDs) and Fe dispersed by polyethylene glycol3+A new method for detecting zoledronic acid is established for a fluorescent probe. The sulfur quantum dots have strong photoluminescence characteristics and can emit blue light under the excitation of ultraviolet light (365 nm); and Fe3+The SQDs can be induced by ion energy to gather, so that the blue luminescence of the SQDs is strongly quenched; however, in the presence of zoledronic acid, zoledronic acid can react with Fe3+Coordinate to thereby suppress Fe3+And (3) the aggregation of the sulfur quantum dots enables the fluorescence of the system to be recovered again. The detection method established by the invention has the advantages of simplicity, convenience, rapidness, reliability, high sensitivity, low cost and the like, is successfully applied to the detection of serum, urine and zoledronic acid injection, and can provide a new idea for the rapid analysis and identification of bone metastasis drugs.
The maximum excitation wavelength of the sulfur quantum dots measured under the conditions that the slit width is 10nm and the scanning speed is 500nm/min is 350nm, the maximum emission wavelength is 440nm, the fluorescence intensity is about 800, and the sulfur quantum dots have good fluorescence performance; fe3+As a fluorescence quencher of the sulfur quantum dots, the fluorescence intensity of the sulfur quantum dots is Fe3+Quenching under aggregation induction; when zoledronic acid is present, due to Fe3+And the fluorescence of the sulfur quantum dots can be recovered again through the interaction with the zoledronic acid functional group.
Drawings
FIG. 1 is a graph showing the results of the selectivity of sulfur quantum dots for different kinds of metal ions in example 1- (3) of the present invention, in which graph A is a fluorescence emission spectrum obtained after adding different kinds of metal ions to the system, and curve a in graph A is sulfur quantum dots-Fe3+And the curve b is the sulfur quantum point-Hg2+The 'control' line in the c-frame amplification part is an independent sulfur quantum dot, and other curves in the c-frame are the fluorescence intensity of the sulfur quantum dot plus other metal ions; and the graph B is a fluorescence intensity change histogram of the system in the presence of different kinds of metal ions.
Fig. 2 shows an atomic force microscopy test picture of samples 1, 2 and 3 of example 1- (4) of the present invention and a corresponding particle size variation graph of quantum dot size measured as sample height, wherein A, D refers to sample 1: sulfur alone quantum dot, B, E refers to sample 2: sulfur Quantum dot-Fe3+C, F denotes sample 3: sulfur Quantum dot-Fe3+-zoledronic acid.
FIG. 3 is a graph showing the results of fluorescence intensity measurements of samples 1, 2 and 3 in example 1- (4) of the present invention, in which curve a indicates that sample 1: sulfur quantum dots alone, curve b refers to sample 2: sulfur Quantum dot-Fe3+Curve c indicates sample 3: sulfur Quantum dot-Fe3+-zoledronic acid.
FIG. 4 shows control group (sulfur quantum dots alone), experimental group 1 (sulfur quantum dots-Fe) in example 2-1 of the present invention3 +) Experimental group 2 (Sulfur Quantum dot-Fe)3+Zoledronic acid) system with Fe3+Fluorescence change results with increasing concentration.
FIG. 5 is a graph showing the results of the reaction time selection test in example 2-2 of the present invention, in which curves a, b, and c respectively indicate the fluorescence intensities of samples 1, 2, and 3 with time.
FIG. 6 is a graph showing the results of pH optimization tests in examples 2 to 3 of the present invention, in which curves a, b, and c respectively indicate the fluorescence intensities of the control group, the experimental group 1, and the experimental group 2 according to the change in pH.
FIG. 7 shows sulfur quantum dots-Fe in examples 2-4 of the present invention3+The composite probe is used for detecting a working curve graph of zoledronic acid, wherein an embedded graph is a standard working curve graph for detecting zoledronic acid.
FIG. 8 shows sulfur quantum dots-Fe in example 3 of the present invention3+The composite probe is used for detecting the selectivity and the anti-interference capability of zoledronic acid, wherein a graph A is a selectivity test result graph, a graph B is an anti-interference capability test result graph, in the graphs, ZA in an abscissa refers to zoledronic acid, and 1-24 refer to the following graphs respectively: ZnSO4,KCl,MgCl2,NaCl,CuCl2,CaCl2,Al(NO3)3Carboxymethyl cellulose, mannitol, sucrose, glucose, α -lactose, α -cyclodextrin, β -cyclodextrin, L-glutamic acid, L-lysine, L-threonine, DL-aspartic acid, L-cysteine, L-valine, L-phenylalanine, L-proline, DL-tartaric acid and soluble starch.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The invention provides a fluorescent probe and a fluorescent sensor for detecting zoledronic acid, and a preparation method and application thereof.
A fluorescent probe for detecting zoledronic acid is a sulfur quantum dot-Fe3+And (3) compounding fluorescent probes.
A preparation method of a fluorescent probe for detecting zoledronic acid comprises the following steps:
(1) and (3) synthesis of sulfur quantum dots: adding alkali, sulfur powder and a dispersing agent into water, stirring uniformly, carrying out heating reaction to obtain a sulfur nanoparticle solution, mixing the sulfur nanoparticle solution and a hydrogen peroxide solution for reaction, cooling to room temperature after the reaction is finished, and enabling a product to appear blue under the irradiation of an ultraviolet lamp to obtain a sulfur quantum dot solution; the dispersing agent is polyethylene glycol;
(2) preparation of Fe3+A solution;
(3) adding sulfur quantum dot solution and Fe into buffer solution3+Incubating to obtain S quantum dot-Fe3+And (3) compounding fluorescent probes.
In the step (1), the sulfur powder may be at least one of sublimed sulfur powder, elemental sulfur, sulfur blocks, sulfur granules and sulfur powder, and the sublimed sulfur powder is specifically used in the following embodiments; the alkali can be at least one of sodium hydroxide, potassium hydroxide, ammonia water or sodium carbonate, and solid sodium hydroxide is specifically adopted in the following examples; polyethylene glycol having a molecular weight of 400-1000 can be used, and PEG-400 is specifically used in the examples below.
In the step (1), the dosage ratio of the alkali, the sulfur powder and the dispersant is 2-4: 1.0-1.6: 2-4(w/w/v), and specifically used in the examples below is 4:1.4:3 (w/w/v).
Wherein, in the step (1), the heating reaction temperature is 60-80 ℃, and the time is 60-80 h; in the following examples, the heating reaction was carried out at 70 ℃ for 72 hours.
Wherein, in the step (1), the concentration of the hydrogen peroxide solution is 0.75-7.5%, and is specifically 7.5% in the following examples.
In the step (1), the volume ratio of the sulfur nanoparticle solution to the hydrogen peroxide solution is 2-4:3-5, and specifically, 3:4 is adopted in the following examples.
In the step (1), the mixing reaction of the sulfur nanoparticle solution and the hydrogen peroxide solution is performed at room temperature and normal pressure, the reaction time is 1-3h, and the specific reaction time in the following examples is 2 h.
Wherein, in the step (2), the Fe is used for preparing Fe3+The raw material of the solution is selected from at least one of ferric sulfate and ferric chloride, and the ferric sulfate is specifically adopted in the following examples; for the preparation of Fe3+The solvent of the solution is water, preferably deionized water.
Wherein, in the step (3), the buffer solution is selected from at least one of citric acid-sodium citrate and acetic acid-sodium acetate; the pH value of the buffer solution is 3.0-6.6; specifically used in the following examples is a citric acid-sodium citrate buffer solution at pH 5.6.
The invention relates to Sulfur Quantum Dots (SQDs) and Fe dispersed by polyethylene glycol3+For fluorescent probe, a sulfur quantum dot and Fe3+The composite fluorescent probe is mixed with standard zoledronic acid solutions with different concentrations for reaction to obtain sulfur quantum dots-Fe containing zoledronic acid with different concentrations3+And a zoledronic acid system, wherein a standard working curve for detecting zoledronic acid is obtained by fluorescence method determination and drawing, so that a fluorescence sensor for detecting zoledronic acid is constructed, and a new method for quickly detecting zoledronic acid based on sulfur quantum dot fluorescence signal change is established. The sulfur quantum dots have strong photoluminescence characteristics and can emit blue light under the excitation of ultraviolet light (365 nm); and Fe3+The SQDs can be induced by ion energy to gather, so that the blue luminescence of the SQDs is strongly quenched; however, in the presence of zoledronic acid, zoledronic acid can react with Fe3+Coordinate to thereby suppress Fe3+And (3) the aggregation of the sulfur quantum dots enables the fluorescence of the system to be recovered again. The detection method established by the invention isHas the advantages of simplicity, rapidness, reliability, high sensitivity, low cost and the like, is successfully applied to the detection of serum, urine and zoledronic acid injection, and can provide a new idea for the rapid analysis and identification of bone metastasis drugs.
The specific implementation process is as follows:
example 1
1. Synthesis of sulfur quantum dots
(1) 4g of flaky NaOH and 1.4g of sublimed sulfur powder are accurately weighed, and 3mL of polyethylene glycol PEG-400 is removed by using a liquid-removing gun.
(2) And mixing the weighed NaOH, sublimed sulfur powder and transferred PEG-400 in 50mL of deionized water, uniformly stirring, transferring into a 100mL round-bottom flask, and reacting in an oil bath kettle at constant temperature of 70 ℃ for 72 hours to obtain the sulfur nanoparticle solution. The product is stored in a refrigerator at 4 ℃ for later use.
(3) The sulfur nanoparticle solution prepared above was mixed with a hydrogen peroxide solution diluted to 7.5% in a ratio of 3:4, carrying out mixed reaction at room temperature and normal pressure for 2 hours; after the reaction is finished, the reaction product is cooled to room temperature, and the product is blue under the irradiation of an ultraviolet lamp (365nm), namely, the sulfur quantum dot solution emitting blue fluorescence is successfully prepared. The product was used in the next step in a refrigerator at 4 ℃.
Since PEG-400 is a very safe biomedical material proven by FDA, the sulfur quantum dot used in the embodiments of the present invention has a unique advantage in biosafety.
2. Sulfur Quantum dot-Fe3+Construction of composite fluorescent probes
(1) Preparing a solution:
in the experimental process, deionized water is used as a solvent for preparing all solutions, and the existing preparation method is adopted. Fe prepared by taking ferric sulfate as raw material3+And (3) solution.
(2) Measurement of excitation and emission wavelengths of sulfur quantum dots:
75 mu L of prepared sulfur quantum dots are put into a 5mL centrifuge tube, deionized water is added to 3mL, the mixture is fully mixed, and the maximum excitation and emission wavelengths of the sulfur quantum dots are tested under an LS 55 type fluorescence spectrophotometer (Perkinelmer company, USA). The slit width was set to 10nm and the scanning speed was 500 nm/min. Finally, the maximum excitation wavelength of the sulfur quantum dots is 350nm, and the maximum emission wavelength of the sulfur quantum dots is 440 nm. The fluorescence intensity is about 800, which shows that the synthesized sulfur quantum dot solution has good fluorescence property.
(3) Selectivity experiment of sulfur quantum dots to metal ions:
control group: 1mL of citric acid-sodium citrate buffer solution with the pH value of 5.6 and 75uL of sulfur quantum dot solution are sequentially added into a 5mL centrifuge tube respectively, and finally deionized water is added to 3mL respectively.
Experimental groups: 1mL of citric acid-sodium citrate buffer solution with pH value of 5.6, 75uL of sulfur quantum dot solution, and 90 uL (0.01mol/L) of some common different kinds of metal ion solutions including Mg are added into a 5mL centrifuge tube in turn2+,Na+,Ag+,Zn2+,K+,Ca2+,Pb2+,Al3+,Cu2+,Cr3+,Ni2+,Co2+,Hg2+,Fe3+. Finally, make up deionized water to 3mL respectively.
The samples were mixed well and reacted for 5min, and the fluorescence spectra of the two groups of samples were measured at an excitation wavelength of 350nm, a slit width of 10nm and a scanning speed of 500nm/min, and the fluorescence intensity values of the components at 440nm were recorded, respectively, with the results shown in FIG. 1. In FIG. 1A, the "control" line inside the enlargement in the c-box is the sulfur quantum dot alone, and the other curves in the c-box are the sulfur quantum dot + other metal ions (Mg)2+,Na+,Ag+,Zn2+,K+,Ca2+,Pb2+,Al3+,Cu2+,Cr3+,Ni2+) It can be seen that the change of the fluorescence intensity of the system is not obvious after other metal ions are added, which indicates that the addition of other metal ions cannot quench the fluorescence of the sulfur quantum dots; curve a is sulfur quantum point-Fe3+And the curve b is the sulfur quantum point-Hg2+，Hg2+Has quenching effect on sulfur quantum dot fluorescence, but has no effect of Fe3+Preferably, and Hg2+The addition of (2) can red-shift the maximum emission wavelength of the system; thus, inTo the sample was added 90. mu.L of 0.01mol/L Fe3+The best quenching effect later, so Fe is selected3+As a fluorescence quencher for sulfur quantum dots.
(4) Sulfur Quantum dot-Fe3+Construction of a composite fluorescent probe:
sample 1: a 5mL centrifuge tube was charged with 1mL of citric acid-sodium citrate buffer solution at ph 5.6, 75uL of sulfur quantum dot solution, and finally 3mL of deionized water was made up.
Sample 2: to a 5mL centrifuge tube were added 1mL of a pH 5.6 citric acid-sodium citrate buffer solution, 75uL of a sulfur quantum dot solution, and 90. mu.L (0.01mol/L) of Fe in that order3+Finally, make up deionized water to 3 mL.
Sample 3: to a 5mL centrifuge tube were added 1mL of a pH 5.6 citric acid-sodium citrate buffer solution, 75uL of a sulfur quantum dot solution, and 90. mu.L (0.01mol/L) of Fe in that order3+240 mu L (0.01mol/L) of zoledronic acid standard solution, and finally, adding deionized water to 3 mL.
The fluorescence spectra of the above three samples were measured under the conditions of an excitation wavelength of 350nm, a slit width of 10nm and a scanning speed of 500nm/min, and the results are shown in FIGS. 2 and 3. As can be seen from FIG. 2, Fe3+The addition of the compound leads the sulfur quantum dots to be aggregated and the particle size to be increased, and the addition of the zoledronic acid leads the particle size of the sulfur quantum dots to be further reduced, thereby verifying the experimental mechanism; also, as can be seen from FIG. 3, Fe3+Has good quenching effect on the fluorescence of the sulfur quantum dots, and the zoledronic acid can recover the fluorescence of the sulfur quantum dots again. The results show that the construction of the sulfur quantum dot-Fe for detecting the zoledronic acid is successful3+And (3) compounding fluorescent probes.
Example 2
Selection and optimization of Experimental conditions
1、Fe3+Selection of concentration:
control group: 1mL of citric acid-sodium citrate buffer solution with pH value of 5.6 and 75uL of sulfur quantum dot solution are sequentially added into a 5mL centrifuge tube respectively, and finally deionized water is added to 3mL respectively (9 groups in total).
Experimental group 1: 1mL of citric acid-citric acid with pH value of 5.6 was added into a 5mL centrifuge tube in sequenceSodium citrate buffer solution, 75uL of sulfur quantum dot solution, 30, 45, 60, 67.5, 75, 82.5, 90, 105, 120. mu.L (0.01mol/L) of Fe3+Finally, make up deionized water to 3mL each (9 total).
Experimental group 2: 1mL of citric acid-sodium citrate buffer solution with pH value of 5.6, 75uL of sulfur quantum dot solution, 30, 45, 60, 67.5, 75, 82.5, 90, 105 and 120 uL (0.01mol/L) of Fe are added into a 5mL centrifuge tube in sequence3+And 240 mu.L of 0.01mol/L zoledronic acid standard solution, and finally, respectively supplementing deionized water to 3mL (9 groups in total).
The samples were mixed well and reacted for 5min, and then the fluorescence spectra of the three groups of samples were measured under the conditions of an excitation wavelength of 350nm, a slit width of 10nm and a scanning speed of 500nm/min, and the results are shown in FIG. 4. From FIG. 4, it can be seen that when Fe is present in the system3+When the concentration is 250-350 mu mol/L, Fe is added into the sample3+The quenching effect is good, and the system fluorescence recovery effect is also good after the zoledronic acid is added. Meanwhile, Fe is labeled in FIG. 43+The fluorescence quenching and recovery of the system at concentrations of 250, 275 and 300. mu.M were 26%, 26% and 28%, respectively. The calculated percentages refer to: sulfur Quantum dot-Fe3+Fluorescence intensity of zoledronic acid and sulphur quantum dots-Fe3+The larger the ratio is, the better the fluorescence quenching and recovery effects are, so that the sulfur quantum dot-Fe is finally selected3+Optimum Fe in the zoledronic acid system3+The concentration was 300. mu. mol/L, i.e., 90. mu.L of 0.01mol/L Fe was added to the sample3+The quenching effect is best after the reaction, and the fluorescence recovery effect of the system is best after the zoledronic acid is added.
2. Selection of reaction time:
sample 1: 1mL of citric acid-sodium citrate buffer solution with pH value of 5.6 and 75uL of sulfur quantum dot solution are added into a 5mL centrifuge tube, and finally deionized water is added to 3 mL.
Sample 2: to a 5mL centrifuge tube were added 1mL of a pH 5.6 citric acid-sodium citrate buffer solution, 75. mu.L of a sulfur quantum dot solution, and 90. mu.L (0.01mol/L) of Fe in that order3+Finally, make up deionized water to 3 mL.
Sample 3: to a 5mL centrifuge tube were added 1mL of a pH 5.6 citric acid-sodium citrate buffer solution, 75. mu.L of a sulfur quantum dot solution, and 90. mu.L (0.01mol/L) of Fe in that order3+240 mu L (0.01mol/L) of zoledronic acid standard solution, and finally, adding deionized water to 3 mL.
The three samples were subjected to kinetic measurements. The measurement time was 10min, and the change in the fluorescence intensity of the sample with time was observed, and the results are shown in FIG. 5 (curves a, b, and c respectively indicate the change in fluorescence intensity of samples 1, 2, and 3 with time), and the optimum reaction time was selected. The experimental result shows that the fluorescence intensity of the three samples does not change greatly along with the prolonging of the reaction time. Therefore, in order to make the reaction sufficient, the optimum time for the reaction in the subsequent experiment was determined to be 5 min.
3. Selecting the pH value:
control group: 1mL of citric acid-sodium citrate buffer solution with pH value of 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6 is added into a 5mL centrifuge tube respectively, 75uL of sulfur quantum dot solution is added, and finally deionized water is added to 3 mL.
Experimental group 1: to a 5mL centrifuge tube were added 1mL of a citric acid-sodium citrate buffer solution having a pH of 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 75 μ L of a sulfur quantum dot solution, and 90 μ L (0.01mol/L) of Fe in this order, respectively3+Finally, make up deionized water to 3 mL.
Experimental group 2: to a 5mL centrifuge tube were added 1mL of a citric acid-sodium citrate buffer solution having a pH of 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 75 μ L of a sulfur quantum dot solution, and 90 μ L (0.01mol/L) of Fe in this order, respectively3+240 mu L (0.01mol/L) of zoledronic acid standard solution, and finally, adding deionized water to 3 mL.
Mixing the above samples, reacting for 5min, and testing fluorescence intensity of the three groups of samples under the conditions of excitation wavelength of 350nm, slit width of 10nm, and scanning speed of 500nm/minAnd the fluorescence intensity at 440nm was recorded. Each fraction was assayed in triplicate. By plotting the relationship between the pH value and the fluorescence intensity, the results are shown in FIG. 6 (curves a, b, c refer to the fluorescence intensity of the control group, the experiment group 1, and the experiment group 2, respectively, varying with the pH), and then finding out the addition of Fe in the system3+The subsequent fluorescence quenching effect is optimal, the system fluorescence recovers the optimal pH value after the zoledronic acid is added, the stability of the system fluorescence around the pH value is considered, and the optimal pH value of the reaction is determined to be 5.6 by comprehensively considering the two factors.
4. Detection of zoledronic acid
To a 5mL centrifuge tube were added 1mL of a pH 5.6 citric acid-sodium citrate buffer solution, 75. mu.L of a sulfur quantum dot solution, and 90. mu.L (0.01mol/L) of Fe in that order3+Finally, supplementing deionized water to 3mL to obtain the sulfur quantum dots-Fe containing the zoledronic acid with different concentrations3+-zoledronic acid system. The final concentration of zoledronic acid in the system is 0,1, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 500 and 1000 [ mu ] mol/L respectively.
Mixing the above samples thoroughly, reacting for 5min, and testing sulfur quantum dot-Fe under the conditions of excitation wavelength of 350nm, slit width of 10nm, and scanning speed of 500nm/min3+Fluorescence intensity of the zoledronic acid system and recording the fluorescence intensity at 440 nm. Each sample was assayed in triplicate. By F/F0On the ordinate, the concentration of zoledronic acid (c)ZA) Plotting a working curve for the detection of zoledronic acid for the abscissa, where F is the fluorescence intensity of a system with a final concentration of zoledronic acid of 1, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 500, 1000. mu. mol/L, F0The results are shown in FIG. 7, which is the fluorescence intensity of the system (i.e., blank) with a final concentration of 0. mu. mol/L zoledronic acid. Finally, the linear equation for detecting the zoledronic acid is F/F00.0017c +1.015 (r-0.9971), which detects a linear range of 0.1-200 μmol/L.
Example 3
Selectivity and interference rejection test
1. And (3) selectivity: are respectively provided withTo a 5mL centrifuge tube were added 1mL of a pH 5.6 citric acid-sodium citrate buffer solution, 75. mu.L of a sulfur quantum dot solution, and 90. mu.L (0.01mol/L) of Fe in that order3+240 mu L (0.01mol/L) of zoledronic acid standard solution, some common inorganic ions, and auxiliary materials possibly contained in the medicine, such as some amino acids, starch, saccharides and the like, and finally, deionized water is added to 3 mL. Mixing uniformly, reacting for 5min, and measuring the fluorescence intensity respectively.
2. Anti-interference: to a 5mL centrifuge tube were added 1mL of a pH 5.6 citric acid-sodium citrate buffer solution, 75. mu.L of a sulfur quantum dot solution, and 90. mu.L (0.01mol/L) of Fe in that order3+240 mu L (0.01mol/L) of zoledronic acid standard solution, meanwhile, adding some common inorganic ions and auxiliary materials possibly contained in the medicine, such as some amino acids, starch, saccharides and the like, into each sample, and finally, supplementing deionized water to 3 mL. Mixing uniformly, reacting for 5min, and measuring the fluorescence intensity respectively.
Whether the selectivity and the anti-interference capability of the method for detecting the zoledronic acid are good or not is judged through the influence of each interferent in the system on the fluorescence intensity. As can be seen from the test result chart of selectivity and anti-interference ability shown in FIG. 8, each interferent has good selectivity for detecting zoledronic acid by the method, and has strong anti-interference ability. The result shows that the method can be applied to the detection of the content of the zoledronic acid in an actual sample.
Example 4
Determination of zoledronic acid content in real sample
Serum, urine sample and zoledronic acid injection are respectively used as actual samples, and the standard recovery rates of zoledronic acid in the three actual samples are respectively determined in a linear range.
The experimental method comprises the following steps:
serum and urine samples are pretreated by centrifugation (15000rpm for 15min), dilution (gradually diluted to 1000 times of the stock solution) and the like, and the treatment mode of the zoledronic acid injection is carried out according to the instruction.
1. Serum: to a 5mL centrifuge tube, 1mL of a citric acid-sodium citrate buffer solution having a pH of 5.6 was added in this order, 500 μ L of a serum diluted 1000 times, 75 μ L of a sulfur quantum dot solution, and 90 μ L of a sulfur quantum dot solutionL (0.01mol/L) Fe3+And standard solutions of zoledronic acid at various concentrations. The final concentrations of the zoledronic acid standard solutions were: 120, 150 and 180 mu mol/L.
After the sample has reacted sufficiently (5min), the fluorescence intensity of the sample is measured at an excitation wavelength of 350nm, a slit width of 10nm and a scanning speed of 500nm/min, and the fluorescence intensity at 440nm is recorded. Each sample was assayed in triplicate. And then respectively calculating the standard recovery rate.
2. A urine sample: to a 5mL centrifuge tube were added 1mL of a citric acid-sodium citrate buffer solution having a pH of 5.6, 500 μ L of 1000-fold diluted urine, 75 μ L of a sulfur quantum dot solution, and 90 μ L (0.01mol/L) of Fe in this order3+And standard solutions of zoledronic acid at various concentrations. The final concentrations of the zoledronic acid standard solutions were: 120, 150 and 180 mu mol/L.
After the sample has reacted sufficiently (5min), the fluorescence intensity of the sample is measured at an excitation wavelength of 350nm, a slit width of 10nm and a scanning speed of 500nm/min, and the fluorescence intensity at 440nm is recorded. Each sample was assayed in triplicate. And then respectively calculating the standard recovery rate.
3. Zoledronic acid injection: to a 5mL centrifuge tube were added 1mL of a pH 5.6 citric acid-sodium citrate buffer solution, 75. mu.L of a sulfur quantum dot solution, and 90. mu.L (0.01mol/L) of Fe in that order 3+20 mu mol/L (final concentration) of zoledronic acid injection and standard solutions of zoledronic acid with different concentrations. The final concentrations of the zoledronic acid standard solutions were: 100, 130 and 160 mu mol/L.
After the sample has reacted sufficiently (5min), the fluorescence intensity of the sample is measured at an excitation wavelength of 350nm, a slit width of 10nm and a scanning speed of 500nm/min, and the fluorescence intensity at 440nm is recorded. Each sample was assayed in triplicate. The normalized recovery was then calculated separately and the results are shown in tables 1 and 2.
Table 1 test results of the system for detecting human serum and urine samples by adding standard recovery
TABLE 2 test results of standard recovery for zoledronic acid injection
As can be seen from tables 1 and 2, the recovery rate of zoledronic acid in serum is 97.4-99.3%, the recovery rate of zoledronic acid in urine is 92.1-103.3%, and the recovery rate of zoledronic acid in injection is 99.1-108.5%.
In conclusion, the invention provides a sulfur quantum dot-Fe-based material3+A novel method for fluorescence analysis of the zoledronic acid content of a sensor. By Fe3+And the competitive reaction of the zoledronic acid and the sulfur quantum dots is used for regulating and controlling the size of the sulfur quantum dots, so that the change of a fluorescence signal is captured, and the quantitative detection of the zoledronic acid is realized. The method is simple, rapid and sensitive. The method is successfully applied to the quantitative detection of the zoledronic acid in practical samples, and has good application prospect in drug analysis.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. The fluorescent probe for detecting zoledronic acid is characterized by being a sulfur quantum dot-Fe3+And (3) compounding fluorescent probes.
2. A preparation method of a fluorescent probe for detecting zoledronic acid is characterized by comprising the following steps:
(1) and (3) synthesis of sulfur quantum dots: adding alkali, sulfur powder and a dispersing agent into water, stirring uniformly, carrying out heating reaction to obtain a sulfur nanoparticle solution, mixing the sulfur nanoparticle solution and a hydrogen peroxide solution for reaction, cooling to room temperature after the reaction is finished, and enabling a product to appear blue under the irradiation of an ultraviolet lamp to obtain a sulfur quantum dot solution; the dispersing agent is polyethylene glycol;
(2) preparation of Fe3+A solution;
(3) adding sulfur quantum dot solution and Fe into buffer solution3+Incubating to obtain S quantum dot-Fe3+And (3) compounding fluorescent probes.
3. The method of claim 2, wherein: in the step (1), the sulfur powder is at least one selected from sublimed sulfur powder, elemental sulfur, sulfur blocks, sulfur granules and sulfur powder;
and/or, in the step (1), the alkali is sodium hydroxide;
and/or, in the step (1), the molecular weight of the polyethylene glycol is 400-1000;
and/or in the step (1), the dosage ratio of the alkali, the sulfur powder and the dispersing agent is 2-4: 1.0-1.6: 2-4 (w/w/v);
and/or in the step (1), the heating reaction temperature is 60-80 ℃, and the time is 60-80 h;
and/or, in the step (1), the concentration of the hydrogen peroxide solution is 0.75-7.5%;
and/or in the step (1), the volume ratio of the sulfur nanoparticle solution to the hydrogen peroxide solution is 2-4: 3-5;
and/or in the step (1), the mixing reaction of the sulfur nanoparticle solution and the hydrogen peroxide solution is carried out at room temperature and normal pressure for 1-3 h;
and/or, in step (2), for the preparation of Fe3+The raw material of the solution is selected from at least one of ferric sulfate and ferric chloride; for the preparation of Fe3+The solvent of the solution is water;
and/or, in step (2), theFe3+In solution, Fe3+The concentration of the ions is 0.01 mol/L;
and/or, in the step (3), the buffer solution is selected from at least one of citric acid-sodium citrate and acetic acid-sodium acetate;
and/or, in the step (3), the pH value of the buffer solution is 3.0-6.6;
and/or, in the step (3), the buffer solution, the sulfur quantum dot solution and Fe3+The volume ratio of the solution is 1000-2000: 50-100: 50-100 parts of;
and/or, in the step (3), the sulfur quantum dots-Fe3+Fe in composite fluorescent probe system3+The final concentration of the ions was 100-400. mu. mol/L.
4. A fluorescence sensor for detecting zoledronic acid, which is characterized by comprising the fluorescence probe of claim 1 or the fluorescence probe prepared by the preparation method of any one of claims 2 to 3 and a standard working curve for detecting zoledronic acid, wherein the standard working curve is obtained by measuring and drawing by a fluorescence method after the fluorescence probe is subjected to mixed reaction with standard solutions of zoledronic acid with different concentrations.
5. A construction method of a fluorescence sensor for detecting zoledronic acid is characterized by comprising the following steps:
(1) mixing the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 3 with standard solutions of zoledronic acid with different concentrations, and then reacting to obtain sulfur quantum dots-Fe containing different concentrations of zoledronic acid3+-a zoledronic acid system;
(2) measuring by adopting a fluorescence method to obtain sulfur quantum dots-Fe of the zoledronic acid with different concentrations3+-fluorescence spectrum of zoledronic acid system;
(3) sulfur quantum dot-Fe in each fluorescence spectrogram3+-the ratio of the fluorescence intensity of the zoledronic acid system to that of the blank at 440nm is plotted on the ordinate and the concentration of the zoledronic acid standard solution is plotted on the abscissa, detecting zoledronic acidStandard working curve of acid.
6. The construction method according to claim 5, wherein: the concentration of the standard zoledronic acid solution is 0,1, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 500 and 1000 mu mol/L respectively, wherein the standard zoledronic acid solution with the concentration of 0 mu mol/L is a blank control solution;
and/or the reaction time is 1-10 min.
7. The construction method according to claim 5, wherein: the linear equation of the standard working curve for detecting the zoledronic acid is F/F00.0017c +1.015 (r-0.9971), wherein F is the sulfur quantum dot-Fe3+Fluorescence intensity at 440nm, F, of the zoledronic acid system, measured at an excitation wavelength of 350nm, a slit width of 10nm and a scanning speed of 500nm/min0The fluorescence intensity of the blank under the same conditions, and c is the concentration of zoledronic acid;
and/or the linear range of the standard working curve for detecting the zoledronic acid is 0.1-200 mu mol/L.
8. A method for detecting the content of zoledronic acid in a sample is characterized in that: the fluorescence sensor according to claim 4 or the fluorescence sensor obtained by the construction method according to any one of claims 5 to 7 is used for detecting the content of zoledronic acid in a sample, and the detection process comprises the following steps: mixing the buffer solution, sulfur quantum dot solution and Fe3+And (3) uniformly mixing the solution and the sample solution, then reacting, then measuring by adopting a fluorescence method to obtain corresponding fluorescence intensity, and finally calculating according to a standard working curve for detecting the zoledronic acid to obtain the concentration of the zoledronic acid in the sample.
9. The detection method according to claim 8, characterized in that: the reaction time is 1-10 min;
and/or, the sample is selected from at least one of serum, urine sample and zoledronic acid injection;
and/or the sample is used after being pretreated, and the pretreatment mode of the sample comprises centrifugation and/or dilution.
10. The detection method according to claim 9, characterized in that: the centrifugation conditions of the sample are 10000-; the sample was diluted 1000 times to 100 times of the stock solution.
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