CN108896521B - Fluorescence analysis method for antioxidant activity of black tea - Google Patents
Fluorescence analysis method for antioxidant activity of black tea Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention discloses a fluorescence analysis method for the antioxidant activity of black tea, which comprises the preparation of quinolyl cyanine derivatives (CyQ), the preparation of fluorescent probe (CyQ-Cys) solution, metal selectivity experiment, Fe3+The detection test, the preparation of the black tea standby liquid, the test of the black tea standby liquid and the like. The invention uses 4-methylquinoline and dialdehyde condensing agent to react to obtain quinolyl cyanine derivatives (CyQ), and then the quinolyl cyanine derivatives (CyQ) and cysteine (Cys) react through halogen-sulfhydryl nucleophilic substitution and rearrangement to obtain fluorescent probes (CyQ-Cys), while ferric ions (Fe)3+) The fluorescence signal of the inducible fluorescent probe (CyQ-Cys) is obviously enhanced, and the change of the fluorescence signal and Fe3+The concentration is in good linear relation, and the fluorescent probe (CyQ-Cys) can rapidly (less than 30s) and recognize Fe with high specificity3+Without interference from common ions.
Description
Technical Field
The invention relates to the field of black tea antioxidation, in particular to a fluorescence analysis method for black tea antioxidation activity.
Background
The antioxidant substances such as flavone and catechin in the black tea can effectively inhibit the formation of active oxygen, and have good antioxidant activity, and the determination method mainly comprises a free radical scavenging capacity method, an oxidation free radical absorption capacity method and an iron ion reduction method. Wherein, Fe3+Is reduced to Fe by antioxidant2+Then, the antioxidant activity was detected by the change in absorbance due to complexation with tris-pyridyltriazine (TPTZ), but the sensitivity of the spectrophotometry was low. Therefore, the improvement of the iron ion reduction method and the development of a high-sensitivity and high-specificity fluorescence analysis method for detecting the antioxidant activity of the dark tea have very important significance.
On the other hand, the metal ions can be coordinated with carboxyl, amino and sulfydryl in molecules of biological thiol such as Glutathione (GSH) and cysteine (Cys), the detection of the metal ions is realized by changing the optical signal of a detection system, and the Fe3+ can be rapidly identified with high selectivity by effectively utilizing the principle without being interfered by Fe2 +.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a fluorescence analysis method for the antioxidant activity of black tea.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fluorescence analysis method for the antioxidant activity of black tea specifically comprises the following steps:
preparation of S1, 4-methyl-N-ethylquinolyl cyanine derivative (CyQ): dissolving 4-methyl-N-ethyl quinoline, N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride and anhydrous sodium acetate in ethanol, reacting at 80 ℃ for 1h under the protection of nitrogen, cooling, performing rotary evaporation to remove the solvent, stirring in chloroform and sodium acetate solution for 1h, filtering, performing vacuum drying, and performing column chromatography separation to obtain a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ), which is convenient for subsequent use;
s2, preparation of fluorescent probe (CyQ-Cys) solution: preparing a 10mol/L solution of a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ) by using N, N-dimethylformamide, preparing a 200mmol/L solution of cysteine (Cys) by using deionized water, sequentially adding a 10mol/L solution of a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ) into a centrifuge tube, dropwise adding a 200mmol/L solution of cysteine (Cys) and a phosphate buffer solution with a 10mmol/L, pH value of 7, uniformly mixing, diluting with N, N-dimethylformamide, reacting for 3min, then recording the change of a fluorescence signal by using a fluorescence spectrophotometer, and obtaining a fluorescent probe (CyQ-Cys) mother solution through halogen-sulfhydryl nucleophilic substitution and rearrangement reaction;
s3, metal selectivity experiment: the metal salt is prepared into 1.0mmol/L solution by deionized water for standby. Adding the fluorescent probe (CyQ-Cys) solution prepared in the step S2 into the fluorescence pool, and measuring the fluorescenceEmitting spectrum, adding different metal ion solutions with different amounts, reacting for 3min, detecting fluorescence emission spectrum, recording fluorescence signal change with fluorescence spectrophotometer, and detecting fluorescent probe (CyQ-Cys) for Fe3+In a specific response of, Fe3+The anti-interference capability of the fluorescent probe (CyQ-Cys) when coexisting with other metal ions;
S4、Fe3+the detection test of (2): adding the solution of the fluorescent probe (CyQ-Cys) prepared in the step S2 into a fluorescence pool, measuring the fluorescence emission spectrum of the solution, and then adding Fe with different concentrations3+Detecting the fluorescence emission spectrum of the solution, recording the change of the fluorescence signal by using a fluorescence spectrophotometer, and detecting Fe with different concentrations3+The corresponding change condition of the solution fluorescence intensity;
s5, preparing a black tea standby liquid: weighing untreated Anhua dark tea and fermented Anhua dark tea, soaking in boiling water for 10min, soaking the residue in boiling water for 2 times, mixing the filtrates, concentrating, adding into a volumetric flask, and diluting to constant volume with distilled water to obtain dark tea solution;
s6, testing the black tea standby liquid: the fluorescent probe (CyQ-Cys) solution prepared in the step S2 and the black tea stock solution prepared in the step S5 are added into a fluorescence pool, the fluorescence emission spectrum of the solution is detected, and the change of a fluorescence signal is recorded by a fluorescence spectrophotometer.
Preferably: in S1, the molar mass ratio of the 4-methyl-N-ethyl quinoline, the N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride and the anhydrous sodium acetate is 4: 1: 4, the developing solvent for column chromatography separation is methanol and dichloromethane, and the volume ratio of the methanol to the dichloromethane is 1: 9.
Preferably: in S2, the volume ratio of the quinolyl cyanine derivative (CyQ) solution to the phosphate buffered saline solution is 1: 25.
Preferably: in S3, the metal salt is Al3+、Fe2+、Cu2+，Zn2+，Hg2+，Pb2+，Mn2+，Ag+Or Co2+A metal salt.
Preferably: in S5, the mass ratio of the Anhua dark tea, the fermented Anhua dark tea and the water is 1: 10.
Preferably: in S1-S6, the water is double distilled water.
Preferably: in S2, S3, S4, S6, test conditions of the emission spectrum: the slit width of the excitation light and the slit width of the emission light are both 10nm, the voltage is 500V, the excitation wavelength is 460nm, and the parallel test is carried out for 3 times.
The method greatly improves the iron ion reduction method, and develops a high-sensitivity and high-specificity fluorescence analysis method for detecting the antioxidant activity of the dark tea.
Compared with the prior art, the fluorescence analysis method for the antioxidant activity of the dark tea provided by the invention is characterized in that 4-methyl-N-ethyl quinoline and N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene are subjected to fluorescence analysis]Aniline hydrochloride reaction to obtain 4-methyl-N-ethyl quinolyl cyanine derivatives (CyQ), and subsequent reaction of 4-methyl-N-ethyl quinolyl cyanine derivatives (CyQ) with cysteine (Cys) via halogen-sulfhydryl nucleophilic substitution and rearrangement reaction to obtain fluorescent probes (CyQ-Cys), while iron ions (Fe)3+) The fluorescence signal of the inducible fluorescent probe (CyQ-Cys) is obviously enhanced, and the change of the fluorescence signal and Fe3+The concentration is in good linear relation, and the fluorescent probe (CyQ-Cys) can rapidly (less than 30s) and recognize Fe with high specificity3+Without interference from common ions. The fluorescent analysis method for the antioxidant activity of the black tea is constructed by utilizing the fact that the antioxidant active substances in the black tea can reduce iron ions to weaken the fluorescent recovery degree of a detection system, and is applied to the analysis of actual samples, and the result is reliable, quick and sensitive.
Drawings
FIG. 1 is a schematic diagram of the synthetic route of quinoline cyanine dye of the present invention;
FIG. 2 is a diagram showing fluorescence emission spectra of cysteine (Cys) before and after addition of quinolinyl cyanine derivative (CyQ) according to the present invention;
FIG. 3 is a graph showing the effect of different concentrations of cysteine (Cys) on the change in fluorescence intensity of quinolinyl cyanine derivatives (CyQ) according to the present invention;
FIG. 4 shows the addition of Fe according to the present invention3+Front and backUV-VIS absorption spectrum of fluorescent probe (CyQ-Cys);
FIG. 5 shows the addition of Fe according to the present invention3+A schematic diagram of fluorescence emission spectra of a front fluorescent probe (CyQ-Cys) and a back fluorescent probe (CyQ-Cys);
FIG. 6 shows the pH value versus Fe in the present invention3+FIG. 8 is a graph showing the effect of fluorescence intensity of the fluorescent probe (CyQ-Cys) before and after treatment;
FIG. 7 shows Fe of the present invention3+Stability profiles of fluorescent probes (CyQ-Cys) before and after treatment;
FIG. 8 shows the fluorescent probe (CyQ-Cys) and Fe of the present invention3+A schematic of the kinetic curve of the incubation;
FIG. 9 is a graph showing fluorescence emission spectra of different ion incubated fluorescent probes (CyQ-Cys) of the present invention;
FIG. 10 shows Fe of the present invention3+A fluorescence intensity diagram showing the change of fluorescence intensity of the fluorescent probe (CyQ-Cys) before and after adding different metal ion solutions;
FIG. 11 shows different concentrations of Fe according to the present invention3+Schematic representation of the effect on the fluorescence emission spectra of the fluorescent probe (CyQ-Cys);
FIG. 12 shows excess Fe according to the present invention3+A linear relationship with the change of fluorescence intensity of the fluorescent probe (CyQ-Cys);
FIG. 13 is a diagram showing fluorescence emission spectra of a quinolinyl cyanine derivative (CyQ) of the present invention after incubation with Glutathione (GSH), cysteine (Cys) and N-acetyl cysteine (NAC);
FIG. 14 shows the fluorescent probe (CyQ-Cys) and Fe of the present invention3+The working curve of (1);
FIG. 15 is a schematic diagram of the recognition mechanism of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
A fluorescence analysis method for the antioxidant activity of black tea specifically comprises the following steps:
preparation of S1, 4-methyl-N-ethylquinolyl cyanine derivative (CyQ): dissolving 4-methyl-N-ethyl quinoline, N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride and anhydrous sodium acetate in ethanol, reacting at 80 ℃ for 1h under the protection of nitrogen, cooling, performing rotary evaporation to remove the solvent, stirring in chloroform and sodium acetate solution for 1h, filtering, performing vacuum drying, and performing column chromatography separation to obtain a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ), reacting 4-methyl-N-ethyl quinoline with N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride to obtain a quinolyl cyanine derivative (CyQ), the subsequent use is convenient;
s2, preparation of fluorescent probe (CyQ-Cys) solution: preparing a 10mol/L solution of a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ) by using N, N-dimethylformamide, preparing a 200mmol/L solution of cysteine (Cys) by using deionized water, sequentially adding a 10mol/L solution of a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ) into a centrifuge tube, dropwise adding a 200mmol/L solution of cysteine (Cys) and a phosphate buffer solution with a 10mmol/L, pH value of 7, uniformly mixing, diluting with N, N-dimethylformamide, reacting for 3min, then recording the change of a fluorescence signal by using a fluorescence spectrophotometer, and obtaining a fluorescent probe (CyQ-Cys) mother solution through halogen-sulfhydryl nucleophilic substitution and rearrangement reaction;
s3, metal selectivity experiment: the metal salt is prepared into 1.0mmol/L solution by deionized water for standby. Adding the prepared fluorescent probe (CyQ-Cys) solution in the step S2 into a fluorescence cell, measuring the fluorescence emission spectrum, adding different metal ion solutions with different amounts, reacting for 3min, detecting the fluorescence emission spectrum, recording the change of a fluorescence signal by using a fluorescence spectrophotometer, and detecting the application of the fluorescent probe (CyQ-Cys) to Fe3+In particular, Fe3+The anti-interference capability of the fluorescent probe (CyQ-Cys) when coexisting with other metal ions;
S4、Fe3+the detection test of (2): adding the solution of the fluorescent probe (CyQ-Cys) prepared in the step S2 into a fluorescence pool, measuring the fluorescence emission spectrum of the solution, and then adding Fe with different concentrations3+The solution is prepared by mixing a solvent and a solvent,detecting its fluorescence emission spectrum, recording the change of fluorescence signal with fluorescence spectrophotometer, and detecting Fe with different concentrations3+The corresponding change condition of the solution fluorescence intensity;
s5, preparing a black tea standby liquid: weighing untreated Anhua dark tea and fermented Anhua dark tea, and soaking in boiling water for 10 min. Soaking the residue in boiling water for 2 times, mixing filtrates, concentrating, adding into volumetric flask, and diluting with distilled water to desired volume to obtain black tea solution;
s6, testing the black tea standby liquid: the fluorescent probe (CyQ-Cys) solution prepared in the step S2 and the black tea stock solution prepared in the step S5 are added into a fluorescence pool, the fluorescence emission spectrum of the solution is detected, and the change of a fluorescence signal is recorded by a fluorescence spectrophotometer.
Construction and spectral characterization of fluorescent probe (CyQ-Cys):
according to the characteristic that biological thiol can be complexed with metal ions, a nucleophilic substitution reaction and a rearrangement mechanism between thiol and halogen are utilized, the quinolyl cyanine derivatives (CyQ) are combined with cysteine (Cys) to construct fluorescent probes (CyQ-Cys), as shown in FIGS. 2 and 3, the fluorescence intensity of the compounds CyQ is gradually reduced with the increase of the concentration of cysteine (Cys), when the concentration of Cys is 110mol/L, the fluorescence quenching is most obvious, and the optimal value is obtained in subsequent experiments; in addition, fluorescent probes (CyQ-Cys) were studied with Fe3+The change conditions of the ultraviolet-visible absorption spectrum and the fluorescence emission spectrum before and after the action; as shown in FIG. 4, the fluorescent probe (CyQ-Cys) produced a strong absorption peak at 460nm with Fe3+After the action, the intensity of an absorption peak is obviously weakened; as shown in FIG. 5, the fluorescent probe (CyQ-Cys) was used in combination with Fe3+After the action, the fluorescence signal at 662nm of a near infrared region is obviously enhanced; changes in the spectroscopic signals indicated that the fluorescent probe (CyQ-Cys) was associated with Fe3+Exhibits a fluorescent 'off-on' process before and after the action, and is beneficial to Fe3+The fluorescence detection of (3).
Analysis of the influence of pH:
considering that the isoelectric point of the quinolinylcyanine derivative (CyQ) is 5.05, the fluorescent probe (CyQ-Cys) is used as Fe3+Based mainly on Fe3+With carboxyl, amino and mercapto groupsThe coordination among the groups, and the pH of the solution changes the existence mode of the amino group, so that the combination mode of the quinolyl cyanine derivative (CyQ) and the cysteine (Cys) is changed, and the fluorescence response of the fluorescent probe (CyQ-Cys) is influenced; FIGS. 6 and 7 show the interaction of fluorescent probe (CyQ-Cys) with Fe at different pH conditions3+The relationship curve of the fluorescence intensity and the pH value at the maximum emission wavelength before and after the action; as shown in FIG. 6, the fluorescent probe (CyQ-Cys) exhibited a weaker fluorescence signal at pH 7, but Fe3+The addition of the fluorescent probe obviously improves the fluorescence intensity of a detection system; however, cysteine (Cys) does not effectively quench the fluorescence intensity of the quinolyl cyanine derivative (CyQ) in the pH range of 6.0 to 6.5, despite the addition of Fe3+The fluorescence signal is enhanced, but the change of the fluorescence intensity can be ignored; whereas cysteine (Cys) caused a significant decrease in the fluorescence intensity of quinolinylcyanine derivatives (CyQ) at pH 8.0, the addition of Fe3+The fluorescence signal of the system cannot be recovered; the reason is that under acidic conditions, amino groups in cysteine (Cys) molecules have positive charges and are difficult to compete for replacing sulfydryl groups, so that fluorescent signals are difficult to quench, and Fe3+Will form Fe (OH) under alkaline conditions3Thereby reducing the coordination with the fluorescent probe (CyQ-Cys) and preventing the recovery of the system fluorescent signal; as can be seen from FIG. 7, the fluorescence signal of the fluorescent probe (CyQ-Cys) remained stable for 30min, and it was found that the fluorescence signal was stable with Fe3+The fluorescence signal after the action is also kept constant, which indicates that the fluorescent probe (CyQ-Cys) and Fe3+The stability is strong before and after the action; therefore, the method can detect Fe under the condition of pH 73+And (4) content.
And (3) kinetic process analysis:
the pH was fixed at 7 at room temperature, and Fe was added after the fluorescence signal of the fluorescent probe (CyQ-Cys) had leveled off3+Further examination of fluorescent probe (CyQ-Cys) and Fe3+The change of fluorescence emission spectrum in the reaction process; as shown in FIG. 8, the fluorescence intensity of the fluorescent probe (CyQ-Cys) at 662nm rapidly increased within 30-60 s, and the fluorescence intensity peaked and remained substantially constant after about 20 s; the results showed that the fluorescent probe (CyQ-Cys) was associated with Fe3+There is good complexation and reactionShould be rapid to enable rapid detection of Fe3 +.
Ion selective analysis:
to verify the fluorescent probe (CyQ-Cys) versus Fe3+High selectivity of (1), we select Fe3+And other common ions are used as identification objects, and the change of fluorescence intensity before and after the common ions and the fluorescent probes (CyQ-Cys) are considered; FIG. 9 shows that some common metal ions (e.g., Al)3+，Fe2+) The fluorescence intensity of the fluorescent probe (CyQ-Cys) is increased negligibly, while Cu2+，Zn2+，Hg2+，Pb2+，Mn2+，Ag+，Co2+The plasma further weakens the fluorescence intensity of the system; in sharp contrast, Fe3+The addition of (2) obviously improves the fluorescence intensity of the detection system, and shows that the fluorescent probe (CyQ-Cys) is used for Fe3+The detection has better specific response; in particular, Fe3+In the presence of other metal ions, the fluorescent probe (CyQ-Cys) is used for the reaction of Fe3+Still shows strong anti-interference capability, and the result is shown in fig. 10; thus, the fluorescent probe (CyQ-Cys) is a highly selective fluorescence-enhanced Fe3+A near infrared fluorescent probe.
Fe3+Detection and analysis of (2):
from FIGS. 11 and 12, to examine Fe3+Effect on the fluorescence intensity of the fluorescent Probe (CyQ-Cys), different concentrations of Fe were added to the detection System3+And a corresponding fluorescence emission spectrum was obtained, the result being shown in fig. 11. The fluorescent probe (CyQ-Cys) exhibited a weaker fluorescent signal at 662nm, but with Fe3+The increase of the concentration and the corresponding gradual increase of the fluorescence intensity of the Fe3+Still enhances the fluorescence intensity of the system, and Fe3+The concentration is respectively in the range of 40-280mol/L and 280-900mol/L and shows good linear relation with the fluorescence intensity change of the fluorescent probe (CyQ-Cys), and the linear regression equation is fitted to be respectively that Y is 0.0034x +0.9682(R is R)20.9958) and Y0.010 x +0.9227 (R)20.9968) with a minimum detection limit of 18.2 mol/L.
Research and analysis of recognition mechanism:
to further demonstrate that the fluorescent probe (CyQ-Cys) was associated with Fe3+The action mechanism of (1) is to examine the combination mode of the quinolyl cyanine derivatives (CyQ) and cysteine (Cys), as shown in FIG. 13; after the quinolyl cyanine derivatives (CyQ) react with cysteine (Cys), the fluorescence intensity is remarkably quenched, and the quinolyl cyanine derivatives still show stronger fluorescence intensity after reacting with N-acetyl cysteine, which indicates that the quinolyl cyanine derivatives (CyQ) are combined with the cysteine (Cys) through halogen-sulfhydryl nucleophilic substitution reaction, and then amino competes for substituting sulfhydryl to obtain the fluorescent probe (CyQ-Cys); subsequently, we immobilized the fluorescent probe (CyQ-Cys) with Fe3+Further demonstrated that the fluorescent probe (CyQ-Cys) was associated with Fe by plotting a complex curve between the two3+The complexation ratio of (A) is shown in FIG. 14. With Fe3+The fluorescence intensity of the system is gradually enhanced by increasing the mole fraction; when Fe3+When the molar fraction of (b) is 0.5, the fluorescent probe (CyQ-Cys) is in contact with Fe3+The complex of (A) exhibited maximum fluorescence emission, indicating that the fluorescent probe (CyQ-Cys) was associated with Fe3+The complexing ratio is 1: 1; CyQ-Cys and Fe3+The action process is shown in FIG. 15, which is-SH, -NH, -COOH and Fe in CyQ-Cys molecule3+And (4) coordination.
Fe3+The actual sample detection of (2):
the method is further evaluated to detect Fe by using river water test objects3+The reliability of (2). Calculating Fe according to linear regression equation3+The results are shown in table 1 below.
TABLE 1 the following table shows Fe in a river water sample3+Measurement of (2)
The result shows that the method detects Fe in the environmental water sample3+The added standard recovery rate and the relative standard deviation meet the requirements and can detect Fe3+The application prospect of (1); in particular, Fe2+Is very easy to be oxidized into Fe3+Therefore, the method can be applied to the detection of the iron content in the actual water sample.
And (3) detecting the antioxidant activity of the dark tea:
referring to an iron ion reduction method, taking a tea aqueous extract as a test object, further investigating the feasibility of detecting the antioxidant activity of the dark tea by the method, wherein the result is shown in the following table 2; the result shows that the fluorescence intensity of the detection system is reduced along with the increase of the volume fraction of the water extract, and the weaker the fluorescence intensity is, the stronger the reducibility of the system is; the black tea has stronger antioxidant activity, and the antioxidant activity of the Anhua black tea is stronger than that of the fermented Anhua black tea.
TABLE 2 reducing power of Anhua dark tea and Anhua dark tea after fermentation
And (4) conclusion: the invention synthesizes quinolyl cyanine derivatives (CyQ), and adopts a strategy of quenching fluorescent signals of quinolyl cyanine derivatives (CyQ) caused by halogen-sulfhydryl nucleophilic substitution reaction to construct a near-infrared fluorescent probe (CyQ-Cys) which can rapidly identify Fe3+(< 30s), has the advantages of high selectivity and high sensitivity; cysteine (Cys) -mediated 'off-on' type near-infrared fluorescent probe-based Fe detection method3+The method of using black tea antioxidant substances to reduce Fe3+Is Fe2+The method is applied to the analysis of actual samples of the golden flower dark tea, and the result is reliable.
In summary, the following steps: compared with the traditional detection technology, the fluorescence analysis method for the antioxidant activity of the dark tea has the following advantages: high sensitivity, high specificity, high detection speed and the like.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A fluorescence analysis method for the antioxidant activity of black tea is characterized by comprising the following steps: the method specifically comprises the following steps:
preparation of S1, 4-methyl-N-ethylquinolyl cyanine derivative (CyQ): dissolving 4-methyl-N-ethyl quinoline, N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride and anhydrous sodium acetate in ethanol, reacting at 80 ℃ for 1h under the protection of nitrogen, cooling, performing rotary evaporation to remove the solvent, stirring in chloroform and sodium acetate solution for 1h, filtering, performing vacuum drying, and performing column chromatography separation to obtain a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ), which is convenient for subsequent use;
s2, preparation of fluorescent probe (CyQ-Cys) solution: preparing a 10mol/L solution of a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ) by using N, N-dimethylformamide, preparing a 200mmol/L solution of cysteine (Cys) by using deionized water, sequentially adding a 10mol/L solution of a 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ) into a centrifuge tube, dropwise adding a 200mmol/L solution of cysteine (Cys) and a phosphate buffer solution with a 10mmol/L, pH value of 7, uniformly mixing, diluting with N, N-dimethylformamide, reacting for 3min, then recording the change of a fluorescence signal by using a fluorescence spectrophotometer, and obtaining a fluorescent probe (CyQ-Cys) mother solution through halogen-sulfhydryl nucleophilic substitution and rearrangement reaction;
s3, metal selectivity experiment: preparing 1.0mmol/L solution of metal salt with deionized water, adding the prepared fluorescent probe (CyQ-Cys) solution in the step S2 into a fluorescence cell, measuring the fluorescence emission spectrum, adding different metal ion solutions with different amounts, reacting for 3min, detecting the fluorescence emission spectrum, recording the change of fluorescence signals with a fluorescence spectrophotometer, and detecting the application of the fluorescent probe (CyQ-Cys) to Fe3+In a specific response of, Fe3+The anti-interference capability of the fluorescent probe (CyQ-Cys) when coexisting with other metal ions;
S4、Fe3+the detection test of (2): adding the solution of the fluorescent probe (CyQ-Cys) prepared in the step S2 into a fluorescence pool, measuring the fluorescence emission spectrum of the solution, and then adding Fe with different concentrations3+Detecting the fluorescence emission spectrum of the solution, recording the change of the fluorescence signal by using a fluorescence spectrophotometer, and detecting Fe with different concentrations3+Solutions ofThe corresponding change of the fluorescence intensity;
s5, preparing a black tea standby liquid: weighing untreated Anhua dark tea and fermented Anhua dark tea, soaking in boiling water for 10min, soaking the residue in boiling water for 2 times, mixing the filtrates, concentrating, adding into a volumetric flask, and diluting to constant volume with distilled water to obtain dark tea solution;
s6, testing the black tea standby liquid: the fluorescent probe (CyQ-Cys) solution prepared in the step S2 and the black tea stock solution prepared in the step S5 are added into a fluorescence pool, the fluorescence emission spectrum of the solution is detected, and the change of a fluorescence signal is recorded by a fluorescence spectrophotometer.
2. The fluorescence analysis method for the antioxidant activity of the dark tea as claimed in claim 1, wherein: in S1, the molar mass ratio of the 4-methyl-N-ethyl quinoline, the N- [ (3- (anilinomethylene) -2-chloro-1-cyclohexene-1-yl) methylene ] aniline hydrochloride and the anhydrous sodium acetate is 4: 1: 4, the developing solvent for column chromatography separation is methanol and dichloromethane, and the volume ratio of the methanol to the dichloromethane is 1: 9.
3. The fluorescence analysis method for the antioxidant activity of the dark tea as claimed in claim 2, wherein: in S2, the volume ratio of the 4-methyl-N-ethyl quinolyl cyanine derivative (CyQ) solution to the phosphate buffer solution is 1: 25.
4. The fluorescence analysis method for the antioxidant activity of the dark tea as claimed in claim 1, wherein: in S3, the metal salt is Al3+、Fe2+、Cu2+，Zn2+，Hg2+，Pb2+，Mn2+，Ag+Or Co2+A metal salt.
5. The fluorescence analysis method for the antioxidant activity of the dark tea as claimed in claim 1, wherein: in S5, the mass ratio of the Anhua dark tea, the fermented Anhua dark tea and the water is 1: 10.
6. The fluorescence analysis method for the antioxidant activity of the dark tea as claimed in claim 1, wherein: in S1-S6, the water is double distilled water.
7. The fluorescence analysis method for the antioxidant activity of the dark tea as claimed in claim 1, wherein: in S2, S3, S4, S6, test conditions of the emission spectrum: the slit width of the excitation light and the slit width of the emission light are both 10nm, the voltage is 500V, the excitation wavelength is 460nm, and the parallel test is carried out for 3 times.
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