CN111504961A - Fluorescent sensor based on glutathione gold nanoclusters and application thereof - Google Patents
Fluorescent sensor based on glutathione gold nanoclusters and application thereof Download PDFInfo
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Abstract
The invention provides a glutathione gold nanocluster-based fluorescence sensor and application thereof, wherein glutathione is simultaneously used as a reducing agent and a stabilizing agent, a chemical reduction method is adopted to prepare glutathione gold nanoclusters (GSH-AuNCs), the synthesized gold nanoclusters are small in particle size and good in dispersity, the synthesized gold nanoclusters have a maximum fluorescence emission peak at 581nm under the excitation wavelength of 360nm, and when Pb exists in a GSH-AuNCs solution2+Of time, Pb2+The fluorescence of the system can be enhanced by forming aggregates through interaction with glutathione on the gold nanoclusters. To GSH-AuNCs-Pb2+After phytic acid is added into the composite system, the phytic acid and Pb are added2+Coordination of (b), Pb2+The fluorescence intensity begins to decrease as gold nanoclusters are detached. Based on the method, a fluorescence sensing method for rapidly detecting the phytic acid is constructed, and the method has the advantages of rapid response, simplicity in operation, higher selectivity and better development potential.
Description
Technical Field
The invention relates to a method for rapidly detecting phytic acid, in particular to a fluorescent sensor based on a glutathione gold nanocluster and application thereof.
Background
Phytic Acid (PA), also known as phytic acid, is a simple cyclic carbohydrate with six phosphate groups per carbon atom. The phytic acid can be used as a preservative in food, and has very beneficial effects on reducing the content of serum cholesterol and triglyceride, preventing the risk of colon cancer and the like. However, when it is ingested in excess, phytic acid can combine with some essential mineral elements to form phytate, resulting in decreased bioavailability of minerals and thus mineral deficiency in humans and animals. Therefore, the development of a plurality of effective methods for detecting the phytic acid is of great significance. At present, many detection methods for phytic acid include high performance liquid chromatography, high performance ion chromatography, nuclear magnetic resonance, inductively coupled plasma atomic emission spectrometry, spectrophotometry, flame atomic absorption, and the like. These analytical methods have advantages and disadvantages, and although the detection sensitivity is high and the stability is good, the application is limited to a certain extent due to the complicated operation, long time consumption, high cost and professional technical personnel. With the introduction of fluorescent nano materials, the fluorescence spectrum analysis method is developed more greatly. The gold nanocluster, as a typical representative of fluorescent nanomaterials, has the excellent characteristics of simplicity and convenience in synthesis, ultramicro size, good fluorescence, strong stability and the like, and thus has gained wide attention. The gold nanoclusters are synthesized by a template method, and quantitative detection of substances can be realized by researching the relation between the fluorescence intensity emitted by the gold nanoclusters before and after the target is added and the concentration of the target. Compared with the traditional detection means, the method has the advantages of simpler operation, strong timeliness, good selectivity and potential application value.
Disclosure of Invention
The invention aims to provide a fluorescence sensor based on a glutathione gold nanocluster and application thereof aiming at overcoming the defects of inconvenient operation, low sensitivity and the like of a conventional detection method for phytic acid in the prior art.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a fluorescence sensor based on glutathione gold nanoclusters and application thereof comprise the following steps:
s1, preparation of glutathione gold nanocluster GSH-AuNCs
Freshly prepared 2.50m L mM HAuCl at a concentration of 20mM4Mixing the aqueous solution, a 100mM GSH aqueous solution with the concentration of 0.75m L and 21.75m L ultrapure water at 25 ℃ for 5min, transferring the reaction mixed solution into a constant-temperature heating magnetic stirrer with the temperature of 70 ℃ for reaction, and carrying out mild stirring for 24h to obtain a GSH-AuNCs aqueous solution which is light yellow under a fluorescent lamp and emits strong orange fluorescence under an ultraviolet lamp, wherein all glass instruments used in the preparation process are soaked overnight by chromic acid washing solution, then are fully washed by the ultrapure water, and are placed in an oven for drying;
s2, detecting phytic acid by fluorescence
GSH-AuNCs solution obtained by diluting 2m L by 50 times with Tris-HCl buffer solution and Pb2+Mixing, reacting for 1min, adding phytic acid solution with different concentrations into the mixed solution, incubating for 3min, after the reaction is complete, taking 360nm as excitation wavelength, with excitation and emission slits of 5nm and 10nm respectively, and measuring fluorescence emission spectrum of the incubation solution within 475 nm-700 nm; then, drawing a working curve by taking the concentration of the phytic acid as an abscissa and the relative fluorescence value F/FA of the system as an ordinate, and performing linear fitting to obtain a one-dimensional linear equation; the phytic acid content of milk powder and glutinous rice samples after pretreatment of extraction, purification and the like is determined by the same method, phytic acid standard solutions with different levels are added into the samples by a standard addition method, and the standard addition recovery rate of the samples is determined.
Pb in step S22+The concentration was 35. mu.M.
The pH of the Tris-HCl buffer described in step S2 was 7.0.
GSH-AuNCs and Pb in step S22+The full reaction time is 1 min; phytic acid and GSH-AuNCs-Pb2+The incubation time of the mixed solution was 3 min.
The invention has the beneficial effects that:
the method is characterized in that a template method is adopted to synthesize the gold nanoclusters, and quantitative detection of the phytic acid substances is realized through the relation between the fluorescence intensity emitted by the gold nanoclusters and the concentration of a target object before and after the target object is added into the system. Compared with the traditional detection means, the detection method provided by the invention is simpler to operate, and has the advantages of good selectivity and strong timeliness.
Drawings
FIG. 1 is a photograph of the UV-VIS absorption spectrum (a) and the fluorescence emission spectrum (b) of GSH-AuNCs and their irradiation with a fluorescent lamp (I) and an ultraviolet lamp (II) according to an embodiment of the present invention;
FIG. 2 is a transmission electron micrograph and a particle size distribution of synthesized GSH-AuNCs according to an embodiment of the present invention;
FIG. 3 is a fluorescence emission spectrum of GSH-AuNCs under different conditions in the example of the present invention (in the figure, a-g are GSH-AuNCs-Pb in sequence)2+、GSH-AuNCs-Pb2+-PA、GSH-AuNCs、GSH-AuNCs-PA、Pb2+PA and Pb2+-fluorescence spectrum of PA);
FIG. 4 shows the addition of 3. mu.M phytic acid to GSH-AuNCs-Pb in accordance with the present invention2+Transmission electron microscope images before and after the composite system (a is before adding phytic acid, and b is after adding phytic acid);
FIG. 5 shows different concentrations of Pb in examples of the present invention2+Fluorescence emission spectra (a) and relative fluorescence intensity change maps (b) of GSH-AuNCs in the presence of F0And FARespectively indicates the absence and presence of Pb in the system2+The fluorescence intensity of GSH-AuNCs at 581 nm);
FIG. 6 is a graph showing the change in fluorescence intensity of a system under different pH conditions in an example of the present invention;
FIG. 7 shows GSH-AuNCs-Pb in the example of the present invention2+(a) And GSH-AuNCs-Pb2+-pa (b) relative fluorescence intensity plots at different times;
FIG. 8 shows GSH-AuNCs-Pb in the presence of various concentrations of phytic acid (0. mu.M, 0.5. mu.M, 1.0. mu.M, 1.5. mu.M, 2.0. mu.M, 2.5. mu.M, 3.0. mu.M, 3.5. mu.M, 4.0. mu.M) in the examples of the present invention2+Fluorescence emission spectrogram (a) of a composite system and relative fluorescence intensity (F/F) of phytic acid and the system at different concentrationsA) Relationship diagram (b) (in the diagram, F and FAIndicates that GSH-AuNCs-Pb in the presence and absence of phytic acid, respectively2+Fluorescence intensity of complex system at 581 nm);
FIG. 9 shows GSH-AuNCs-Pb in the presence of different interferents in an example of the invention2+Relative fluorescence of complex systemsIntensity profile (concentration of each substance in the profile was 3.0. mu.M).
Detailed Description
The invention is further described below with reference to the figures and examples.
Example 1: a fluorescence sensor based on glutathione gold nanoclusters and application thereof comprise the following steps:
s1, preparation of glutathione gold nanocluster GSH-AuNCs
Freshly prepared 2.50m L mM HAuCl at a concentration of 20mM4Mixing the aqueous solution, a 100mM GSH aqueous solution with the concentration of 0.75m L and 21.75m L ultrapure water at 25 ℃ for 5min, transferring the reaction mixed solution into a constant-temperature heating magnetic stirrer with the temperature of 70 ℃ for reaction, and carrying out mild stirring for 24h to obtain a GSH-AuNCs aqueous solution which is light yellow under a fluorescent lamp and emits strong orange fluorescence under an ultraviolet lamp, wherein all glass instruments used in the preparation process are soaked overnight by chromic acid washing solution, then are fully washed by the ultrapure water, and are placed in an oven for drying;
s2, detecting phytic acid by fluorescence
2ml of a 50-fold dilution of GSH-AuNCs solution in Tris-HCl buffer and a final concentration of 35. mu. MPb2+Mixing, reacting for 1min, adding phytic acid solution with different concentrations into the mixed solution, incubating for 3min, after the reaction is complete, taking 360nm as excitation wavelength, with excitation and emission slits of 5nm and 10nm respectively, and measuring fluorescence emission spectrum of the incubation solution within 475 nm-700 nm; then, drawing a working curve by taking the concentration of the phytic acid as an abscissa and the relative fluorescence value (F/FA) of the system as an ordinate, and performing linear fitting to obtain a linear equation of unity; the phytic acid content of milk powder and glutinous rice samples after pretreatment of extraction, purification and the like is determined by the same method, phytic acid standard solutions with different levels are added into the samples by a standard addition method, and the standard addition recovery rate of the samples is determined.
Pb in step S22+The concentration is 35 μ M;
the Tris-HClpH in step S2 is 7.0;
GSH-AuNCs and Pb in step S22+Mixing for 1 min; phytic acid and GSH-AuNCs-Pb2+Mixed solutionThe incubation time of (3) was 3 min.
Example 2: the following detailed description of the invention with reference to the drawings and specific examples shows the following specific processes:
s11. instrument and reagent
Type F-7000 fluorescence photometers (Hitachi, Japan); UV-2450 ultraviolet-visible spectrophotometer (Shimadzu corporation, Japan); pHS-3E type acidimeters (Shanghai Lei magnetic Instrument works); JEM-2100 high-resolution transmission electron microscope (Japan Electron Co., Ltd.); DF-101S heat collection type constant temperature heating magnetic stirrer (Steud City Wai Limited liability company); millipore silicon water purification system (millipore corporation, france); finnpipette adjustable pipettor.
HAuCl4(purity is more than or equal to 99.9 percent, Shanghai-sourced leaf Biotechnology Co., Ltd.) is prepared into 1.0 percent stock solution by using ultrapure water, reduced glutathione (Aladdin reagent), lead nitrate (Shanghai Sanpu chemical Co., Ltd.), phytic acid solution (Aladdin reagent), L-histidine (national drug group chemical Co., Ltd.), L-lysine (Aladdin reagent) and anhydrous sodium carbonate (national drug group chemical Co., Ltd.).
S12. test method
1) Preparation of glutathione gold nanoclusters (GSH-AuNCs)
Freshly prepared 2.50m L mM HAuCl at a concentration of 20mM4The aqueous solution and 0.75m L aqueous solution of GSH with the concentration of 100mM are mixed with 21.75m L ultrapure water at 25 ℃ for 5min, then the reaction mixture is moved to a constant temperature heating magnetic stirrer with 70 ℃ for reaction, and the mixture is stirred gently for 24h to obtain the GSH-AuNCs aqueous solution which is light yellow under a fluorescent lamp and emits strong orange fluorescence under an ultraviolet lamp.
2) Optimization of detection conditions
During the course of an analytical measurement of a target, a change in the detection conditions can affect the response of the constructed sensor to the target, and thus the sensitivity of the detection. In order to obtain the optimal condition for detecting the phytic acid and improve the detection efficiency, a single-factor control variable is adoptedQuantitative method, sequentially investigating Pb2+The concentration, the pH value and the reaction time of the compound (A) to the fluorescence intensity of the system.
3) Fluorescence detection of phytic acid
GSH-AuNCs solution diluted 50-fold with 2m L in Tris-HCl (pH 7.0) buffer and brought to a final concentration of 35. mu.MPb2+Mixing, reacting for 1min, adding phytic acid solution with different concentrations, and incubating for 3 min. After the reaction is completed, the fluorescence emission spectrum of the hatching solution within the range of 475 nm-700 nm is measured by taking 360nm as the excitation wavelength and 5nm and 10nm as the excitation and emission slits respectively. And then drawing a working curve by taking the phytic acid concentration as an abscissa and taking the relative fluorescence value (F/FA) of the system as an ordinate.
4) Detection of actual samples
Accurately weighing 1g of ground milk powder and Oryza Glutinosa sample, adding 1.2% HCl-10% Na2SO4The mixed solution (40m L) is stirred uniformly and then is placed statically for 2h, then the clear solution is centrifuged to take the volume to 50m L, 2m L is taken and the extract liquid treated by the method is mixed with 2m L15 percent trichloroacetic acid, then the mixture is placed in a refrigerator at 4 ℃ for standing for 2h, then the mixture is centrifuged to collect the supernatant liquid, and the volume is increased to 25m L by using water for subsequent detection.
S13. result and discussion
1) Characterization of glutathione gold nanoclusters (GSH-AuNCs)
Characterization of GSH-AuNCs
FIG. 1 shows GSH-AuNCs ultraviolet-visible absorption spectrum and fluorescence emission spectrum, from 1a, GSH-AuNCs has a shoulder peak at about 400nm, and no obvious absorption peak at about 520nm, which indicates that no SPR absorption peak similar to gold nanoparticles appears in the sample, and the size of the synthesized nano-cluster is small. 1b is the fluorescence spectrum of GSH-AuNCs, which has a maximum emission peak at 581nm at an excitation wavelength of 360 nm. The inset shows that GSH-AuNCs is bright yellow under fluorescent light and emits strong orange fluorescence visible to the naked eye under 365nm ultraviolet lamp illumination. Fig. 2 is a Transmission Electron Microscope (TEM) image, and it can be seen from the figure that the gold nanoclusters have uniform particle size distribution, good dispersibility, and an average particle size of about 1.6nm (inset).
Detection research of phytic acid by fluorescent sensor constructed by GSH-AuNCs
FIG. 3 is a fluorescence spectrum of gold nanoclusters under different conditions. When Pb is added into the gold nanocluster solution2+Then, Pb2 +Can interact with GSH in GSH-AuNCs to form GSH-AuNCs-Pb2+The combination leads the gold nano-cluster to generate obvious aggregation, and the fluorescence intensity is enhanced; after 3 μ M phytic acid is added, the phytic acid and Pb are mixed2+Coordination of (2) so that Pb is present2+The fluorescence intensity of the gold nanoclusters is reduced after the gold nanoclusters are separated from the surface of the gold nanoclusters. The results of comparative experiments carried out at the same time show that no Pb exists2+Under the condition (2), the phytic acid has little influence on the fluorescence intensity of the gold nanoclusters. In addition, phytic acid and Pb2+And Pb2+And the mixed solution of the phytic acid and the phytic acid has no obvious fluorescent signal. Meanwhile, it can be seen from the transmission electron micrograph (FIG. 4) that GSH-AuNCs-Pb is present in the presence of phytic acid2+The combination changes from an aggregation state to a dispersion state, corresponding to a fluorescence spectroscopy phenomenon. Thus, at Pb2+With the assistance of the method, the phytic acid can be detected by using the fluorescent sensor constructed by the GSH-AuNCs.
2) Optimization of the Experimental conditions
a.Pb2+Is optimized
Determination of optimum Pb2+The concentration is crucial to improve the sensitivity of the system. Pb2+When the concentration is too low, the fluorescence enhancement phenomenon cannot be obviously generated, and the detection is not facilitated. And Pb2+When the concentration is too high, the original fluorescence can be recovered only by using phytic acid with higher concentration, which is not beneficial to improving the sensitivity of the system. As can be seen from FIG. 5, when Pb is reached2+The fluorescence intensity at a concentration in the range of 0-35. mu.M follows Pb2+The concentration is increased gradually, and when the concentration is higher than 35 mu M, the fluorescence intensity is basically kept unchanged, which indicates that Pb is combined on the surface of the gold nanocluster2+Has been substantially saturated, and Pb is selected2+The concentration was 35. mu.M as the optimum concentration.
Optimization of pH
The influence of pH in the range of 5 to 12 on the fluorescence intensity of the system was examined in three cases. Respectively GSH-AuNCs and GSH-AuNCs-Pb2+And GSH-AuNCs-Pb2+Influence of pH on system fluorescence in the presence of 2. mu.M phytic acid. As can be seen from FIG. 6, GSH-AuNCs and Pb were observed at pH 72+The fluorescence intensity is strongest during mixing, and the fluorescence intensity is reduced most obviously after the phytic acid is added. Thus, a Tris-HCl buffer solution with a pH of 7 was selected for subsequent assay conditions.
c. Selection of reaction time
FIG. 7 shows the reaction time vs. GSH-AuNCs-Pb, respectively2+And GSH-AuNCs-Pb2+-the effect of PA. As can be seen from FIG. 7a, when Pb was added to the system2+Then, the relative fluorescence intensity of the gold nanoclusters is rapidly increased, and the fluorescence intensity is basically unchanged after 1min, so that 1min is selected as Pb2+Reaction time with GSH-AuNCs. FIG. 7b shows that GSH-AuNCs and Pb are reacted with each other2+After 2 mu M phytic acid is added into the mixed solution, the fluorescence intensity of the system begins to decrease and basically reaches stability after 3min, so that 3min is selected as the reaction time for detecting the target phytic acid.
3) Fluorescence detection of phytic acid
Under the optimal experimental conditions, 2m L GSH-AuNCs-Pb is added2+The mixed solution is added with phytic acid solution with different concentrations, the fluorescence spectrum of the mixed solution is shown in figure 8a, and the fluorescence intensity is gradually reduced along with the increase of the phytic acid concentration. FIG. 8b shows the relationship between the concentration of phytic acid and the relative fluorescence intensity (F/FA) of the system, wherein the concentration of phytic acid is in a linear relationship with the relative fluorescence intensity of the system within the range of 0.5-4.0 μ M, and the linear relationship is that Y is-0.1597C +1.0490(R is-0.1597C + 1.0490)20.9934); the lowest detection limit was 0.3. mu.M.
4) Selectivity test
To examine the selectivity of the method, several interfering substances including anions (CH) were separately determined3COO-、CO3 2-、HPO4 2-、SO3 2-、H2PO4 -、HCO3 -、PO4 3-And P2O7 4-) Amino acids (Met, Ala, His, Cys, Ser and L ys) and cations (Al)3+、Zn2+、Ca2+、Ba2+、K+、Mg2+And NH4 +) Influence on the detection system. As shown in FIG. 9, only phytic acid was effective in reducing GSH-AuNCs-Pb2+The fluorescence of the combined body and other interfering substances have no obvious influence on the overall fluorescence of the system, and the method is proved to have better selectivity.
5) Detection of actual samples
In order to study the detection capability of the designed fluorescent sensor on phytic acid in actual samples, the fluorescent sensor is used for detecting milk powder and glutinous rice samples. According to the experimental method, the recovery rates of phytic acid in the milk powder and the glutinous rice samples are measured by a standard addition method, spectral data are recorded and analyzed, and the results are shown in the following table 1, wherein the average recovery rates of the glutinous rice and the milk powder samples are respectively 83.2% -110.9%, 98.1% -112.8%, and the relative standard deviations are respectively 2.05% -3.53% and 2.25% -4.75%. Meanwhile, the method is relatively consistent with the result measured by ultraviolet spectroscopy, and shows that the fluorescence sensing detection method established in the research has great potential in quantitatively detecting the phytic acid content in an actual sample.
TABLE 1 analysis results and recovery of samples
S14. conclusion
The invention successfully prepares the gold nanocluster emitting strong orange fluorescence by taking glutathione as a template, and the synthesized GSH-AuNCs have small size and good dispersibility. Based on Pb2+The fluorescent sensor can form an aggregate with the gold nanocluster to effectively enhance the fluorescence of GSH-AuNCs, and the addition of the phytic acid causes the phenomenon that the fluorescence of the system is weakened, so that the fluorescent sensor is constructed to realize the rapid detection of the phytic acid. The method is efficient, easy to operate and good in selectivity, obtains a satisfactory recovery rate in actual sample analysis, and has a good application prospect.
Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention.
Claims (4)
1. A fluorescence sensor based on glutathione gold nanoclusters and application thereof comprise the following steps:
s1, preparation of glutathione gold nanocluster GSH-AuNCs
Mixing freshly prepared HAuCl4 aqueous solution with the concentration of 2.50m L being 20mM, GSH aqueous solution with the concentration of 0.75m L being 100mM and ultrapure water with the concentration of 21.75m L at 25 ℃ for 5min, transferring the reaction mixed solution into a constant-temperature heating magnetic stirrer with the temperature of 70 ℃ for reaction, and carrying out mild stirring for 24h to obtain GSH-AuNCs aqueous solution which is light yellow under a fluorescent lamp and emits strong orange fluorescence under an ultraviolet lamp, wherein glass instruments used in the preparation process are all soaked overnight by chromic acid washing liquid, then are fully washed by the ultrapure water, and are placed in an oven for drying and then used;
s2, detecting phytic acid by fluorescence
GSH-AuNCs solution obtained by diluting 2m L by 50 times with Tris-HCl buffer solution and Pb2+Mixing, reacting for 1min, adding phytic acid solution with different concentrations into the mixed solution, incubating for 3min, after the reaction is complete, taking 360nm as excitation wavelength, with excitation and emission slits of 5nm and 10nm respectively, and measuring fluorescence emission spectrum of the incubation solution within 475 nm-700 nm; then taking the concentration of phytic acid as an abscissa, and taking the relative fluorescence value F/F of the systemADrawing a working curve for a vertical coordinate, and performing linear fitting to obtain a linear equation of unity; the phytic acid content of milk powder and glutinous rice samples after pretreatment of extraction, purification and the like is determined by the same method, phytic acid standard solutions with different levels are added into the samples by a standard addition method, and the standard addition recovery rate of the samples is determined.
2. Root of herbaceous plantThe fluorescence sensor based on glutathione gold nanoclusters and the application thereof as claimed in claim 1, wherein Pb is in step S22+The concentration was 35. mu.M.
3. The glutathione gold nanocluster-based fluorescence sensor and the use thereof as claimed in claim 1, wherein the Tris-HCl buffer solution in step S2 has a pH of 7.0.
4. The fluorescence sensor based on glutathione gold nanoclusters of claim 1, wherein the GSH-AuNCs and Pb are mixed in step S22+The full reaction time is 1 min; phytic acid and GSH-AuNCs-Pb2+The incubation time of the mixed solution was 3 min.
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