CN103868899A - Fluorescence detection method for kojic acid in food and environmental sample - Google Patents
Fluorescence detection method for kojic acid in food and environmental sample Download PDFInfo
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- CN103868899A CN103868899A CN201410068969.2A CN201410068969A CN103868899A CN 103868899 A CN103868899 A CN 103868899A CN 201410068969 A CN201410068969 A CN 201410068969A CN 103868899 A CN103868899 A CN 103868899A
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Abstract
The invention relates to a fluorescence detection method for the content of kojic acid in a food and environmental sample, and belongs to the technical field of fluorescence detection. The fluorescence detection method provided by the invention takes a copper nanocluster covered by protein as a fluorescence probe and the kojic acid which is also a detected object as a fluorescence quencher. The fluorescence detection method specifically comprises the following steps of preparing the copper nanocluster fluorescence probe, preparing kojic acid standard solutions with a series of concentrations, measuring the fluorescence intensity of the kojic acid standard solutions and the copper nanocluster before and after reaction, and establishing a standard curve; and measuring the relative changing value of the fluorescence intensity of a to-be-measured sample and the copper nanocluster before and after reaction, and comparing the standard curve so as to obtain the content of the kojic acid in the to-be-measured sample. Compared with an existing kojic acid detection method, the kojic acid fluorescence analysis method provided by the invention has the advantages that the response is rapid, the operation is simple and convenient, the cost is low, and the sensitivity is high and the like; meanwhile, because of the high selectivity of the method, when the kojic acid in the food and environmental sample is detected, a preprocessing process of a complicated sample is not needed.
Description
Technical field
The present invention relates to a kind of fluorescence detection method for Food and environment sample kojic acid content, belong to fluorescence analysis detection technique field.
Background technology
Kojic acid, 5-hydroxyl-2-methylol-pyrans-4-ketone, is a kind of common faintly acid organism that most of aspergillus and Penicillium fungi produce.In food industry, owing to having the chemistry of inhibition and the effect of microbial degradation, kojic acid is generally used as food additives and antistaling agent and is generally used.But research shows that kojic acid has the potential hazards such as carcinogenicity, teratogenesis and genetoxic.In view of the foregoing, take kojic acid as detecting target, set up accurate, rapid, sensitive analyzing detecting method significant.
The detection method of kojic acid mainly contains the precipitation method, colourimetry, liquid phase chromatography and electrochemical method at present.These detection methods respectively have relative merits, for example precipitation method and colourimetry, although more directly perceived, selectivity and sensitivity are poor.Liquid phase chromatography has high separating efficiency and high sensitivity, but operates consuming time loaded down with trivial details.Electrochemical methods is easy and simple to handle, highly sensitive, but poor stability.Spectrographic method is commonly use sensitive, one of modern instrumental analysis method fast and accurately.Wherein, fluorometry due to highly sensitive, selectivity good, the feature such as simple and efficient to handle has obvious detection advantage, obtains extensive concern in the field such as environmental monitoring and food security.Meanwhile, novel fluorescent nano material infiltration is fused in analysis science, has greatly promoted the development of fluorescence analysis detection method.Metal nanometer cluster is as a kind of novel fluorescence nano material, have that good biocompatibility, light stability are strong, stoke shift is large and the feature such as flicker free on all correlation time yardstick, greatly improve its performance as fluorescence probe, significantly promoted the sensitivity of existing analytical approach.But, at present also without any the relevant report that fluorescence analysis is detected for kojic acid.
Therefore, based on the fluorescent characteristic of metal nanometer cluster excellence, fluorescence analysis and nanometer technology are combined, set up for kojic acid highly sensitive, fast, fluorescence detection method cheaply, detect and the work tool such as environmental water sample monitoring is of great significance for current food quality.
Summary of the invention
The object of the present invention is to provide the fluorescent method of kojic acid in a kind of detection Food and environment sample of simple, quick, economic, sensitive and high selectivity.
Principle of the present invention is: the present invention uses the copper nanocluster of protein coated as fluorescence radiation material, and kojic acid, as detected material, is also fluorescence quencher simultaneously.The present invention realizes the detection to kojic acid by kojic acid to the specificity fluorescent quenching effect of copper nanocluster.
As mentioned above copper nanocluster, is characterized in that, in described copper nanocluster, the existence form of copper is zeroth order and divalence and deposits, and can be under the exciting of the light of different wave length the fluorescence of transmitting the same band.
The light of described different wave length comprises ultraviolet light and visible ray.
The method comprises following concrete steps:
(1) prepare copper nanocluster fluorescence probe;
(2) configure the kojic acid standard solution of a series of concentration;
(3) Criterion working curve: the fluorescence intensity in determination step (2) before and after kojic acid standard solution and the reaction of copper nanocluster, according to being related to Criterion curve between kojic acid concentration and fluorescence intensity relative changing value;
(4) quantitatively detect: measure the fluorescence intensity change of testing sample and copper nanocluster reaction front and back, contrast the typical curve obtaining in step (3), obtain the content of kojic acid in testing sample.
In step (1), the preparation process of described copper nanocluster fluorescence probe comprises the steps: that by bivalent cupric ion and protein mol ratio be 0.25～0.35:1, and by 10～50 mM proteinometers in every premium on currency, bivalent cupric ion and protein are added to the water, obtain mixed solution.Mixed solution is that 10～30 ℃ and rotating speed are under 80～200 revs/min in temperature, after stirring 2～3 minutes, adds NaOH solution that system pH is adjusted to 12～13.Mixed solution stirs 8～10 hours to complete reaction at 50～65 ℃.Solution after reaction is placed in to 4 ℃ of aqueous solution and dialyses more than 36h, remove impurity.
Described bivalent cupric ion solution preferentially adopts copper sulphate, copper nitrate or other similar water dissolubility mantoquita; Described protein preferentially adopts bovine serum albumin(BSA) and lysozyme; The preferred 0.267:1 of the mol ratio of bivalent cupric ion and protein in described preparation process.
In step (2), the collocation method of described kojic acid standard items is: the standard model storing solution of preparing kojic acid with the acetate buffer of 10～100mmol/L (pH is 6.0-8.0), storing solution concentration is 0.1mmol/L to 10mmol/L, with acetate buffer, storing solution is configured to the standard solution of variable concentrations, after the kojic acid standard solution of getting variable concentrations fully mixes with copper nanocluster, carries out fluorometric investigation.
In step (3), the reaction time of described reaction is preferably 5～10 minutes, and the temperature of reaction of described reaction is preferably 10～40 ℃;
The measurement of described fluorescence intensity can realize by any a fluorescence spectrophotometer;
Described fluorescence intensity relative changing value is according to formula (F
0-F)/F
0× 100% calculates, wherein F
0represent the fluorescence intensity of copper nanocluster while not adding kojic acid, F represents to add the fluorescence intensity of copper nanocluster after kojic acid; The test condition of described fluorescence intensity is as follows: fluorescent emission wavelength is the crest of copper nanocluster fluorescence emission spectrum, the crest that fluorescence exciting wavelength is corresponding excitation spectrum.
Compared with existing detection method, the invention has the advantages that:
(1) fluorescence radiation material of the present invention is copper nanocluster, has the advantages such as with low cost, good biocompatibility and fluorescent stability be good, and kojic acid is had to good selectivity and higher sensitivity;
(2) fluorometry that the present invention sets up, can fast and effeciently detect kojic acid, has improved sample detection efficiency, has shortened the sample detection time, and simple and efficient to handle;
(3) the method can be used in the detection of kojic acid in Food and environment sample, can exempt loaded down with trivial details sample pretreatment process, make detect more economically, easy.
Accompanying drawing explanation
Fig. 1 is copper nanocluster of the present invention and the reacted fluorescence emission spectrogram of variable concentrations kojic acid.
Fig. 2 is detectability and the range of linearity that fluorescence detection method of the present invention is measured kojic acid.
Embodiment
Embodiment 1
(1) prepare copper nanocluster fluorescence probe: 1mL20mM copper-bath is joined in 5mL15mM Bovine Serum Albumin in Aqueous Solution.Mixed solution is under 100 revs/min at 25 ℃ and rotating speed, and after stirring 2～3 minutes, adding NaOH solution to make system pH is 12.Mixed solution stirs 8 hours to complete reaction at 55 ℃.Solution after reaction is placed in to 4 ℃ of aqueous solution 36h that dialyses, removes impurity, with 10 times of the gained copper nanocluster dilutions of dialyse of the acetate buffer of 20mmol/L (pH=7.0);
(2) configuration kojic acid standard solution: the standard model storing solution of preparing kojic acid with the acetate buffer of 20mmol/L (pH=7.0), storing solution concentration is 0.1mmol/L to 10mmol/L, with acetate buffer, storing solution is configured to the standard solution of variable concentrations;
(3) Criterion working curve: step (2) gained kojic acid standard items and copper nanocluster are fully mixed, react the fluorescence intensity F of mensuration system before reaction after 5 minutes at 25 ℃
0with reacted fluorescence intensity F, by formula (F
0-F)/F
0× 100% calculates fluorescence intensity relative changing value, according to being related to Criterion curve between kojic acid concentration and fluorescence intensity relative changing value, the results are shown in Figure 1 and Fig. 2.
(4) quantitatively detect: get soy sauce 1mL, it is diluted respectively to 10 times with the acetate buffer of 20mmol/L (pH=7.0), centrifugal rear filtration, gets supernatant as testing sample.The fluorescence intensity relative changing value who measures testing sample and copper nanocluster reaction front and back, contrasts the typical curve obtaining in step (3), obtains the content of kojic acid in testing sample.
Embodiment 2
(1) prepare copper nanocluster fluorescence probe: 1mL20mM copper acetate solution is joined in 5mL15mM Lysozyme in Aqueous Solution.Mixed solution is under 150 revs/min at 25 ℃ and rotating speed, and after stirring 2～3 minutes, adding NaOH solution to make system pH is 13.Mixed solution stirs 8 hours to complete reaction at 55 ℃.Solution after reaction is placed in to 4 ℃ of aqueous solution 36h that dialyses, removes impurity, with 10 times of the gained copper nanocluster dilutions of dialyse of the acetate buffer of 50mmol/L (pH=7.0);
(2) configuration kojic acid standard solution: the standard model storing solution of preparing kojic acid with the acetate buffer of 50mmol/L (pH=7.0), storing solution concentration is 0.1mmol/L to 10mmol/L, with acetate buffer, storing solution is configured to the standard solution of variable concentrations;
(3) Criterion working curve: step (2) gained kojic acid standard items and copper nanocluster are fully mixed, react the fluorescence intensity F of mensuration system before reaction after 5 minutes at 25 ℃
0with reacted fluorescence intensity F, by formula (F
0-F)/F
0× 100% calculates fluorescence intensity relative changing value, according to being related to Criterion curve between kojic acid concentration and fluorescence intensity relative changing value, the results are shown in attached Fig. 1 and 2.
(4) quantitatively detect: get vinegar 1mL, it is diluted respectively to 10 times with the acetate buffer of 50mmol/L (pH=7.0), centrifugal rear filtration, gets supernatant as testing sample.The fluorescence intensity relative changing value who measures testing sample and copper nanocluster reaction front and back, contrasts the typical curve obtaining in step (3), obtains the content of kojic acid in testing sample.
The present invention is equally applicable to the qualitative analysis of kojic acid in other water-soluble food and environmental water sample and quantitatively detects.Foregoing is all within protection domain of the present invention.
Claims (10)
1. the fluorescence detection method for Food and environment sample kojic acid; It is characterized in that using the copper nanocluster of protein coated as fluorescence radiation material, kojic acid, as detected material, is realized the detection to kojic acid by kojic acid to the specificity fluorescent quenching effect of copper nanocluster.
2. the method for claim 1, is characterized in that comprising the steps:
(1) prepare copper nanocluster fluorescence probe;
(2) configure the kojic acid standard solution of a series of concentration;
(3) Criterion working curve: the fluorescence intensity in determination step (2) before and after kojic acid standard solution and the reaction of copper nanocluster, according to fluorescence intensity relative changing value Criterion curve;
(4) quantitatively detect: measure the fluorescence intensity change of testing sample and copper nanocluster reaction front and back, contrast the typical curve obtaining in step (3), obtain the content of kojic acid in testing sample.
3. the method for claim 1, it is characterized in that in described step (1), the preparation process of described copper nanocluster fluorescence probe comprises the steps: that by bivalent cupric ion and protein mol ratio be 0.25～0.35:1, and by 10～50 mM proteinometers in every premium on currency, bivalent cupric ion and protein are added to the water, obtain mixed solution; Mixed solution is that 10～30 ℃ and rotating speed are under 80～200 revs/min in temperature, after stirring 2～3 minutes, adds NaOH solution that system pH is adjusted to 12～13; Mixed solution stirs 8～10 hours to complete reaction at 50～65 ℃; Solution after reaction is placed in to 4 ℃ of aqueous solution and dialyses more than 36h, remove impurity.
4. method as claimed in claim 2, is characterized in that described bivalent cupric ion solution adopts copper sulphate, copper nitrate or other similar water dissolubility mantoquita.
5. method as claimed in claim 2, is characterized in that described protein adopts bovine serum albumin(BSA) and lysozyme.
6. method as claimed in claim 4, is characterized in that in described preparation process, the mol ratio of bivalent cupric ion and protein is 0.267:1.
7. the method for claim 1, it is characterized in that in described step (2), the collocation method of described kojic acid standard items is: the standard model storing solution of the acetate buffer that is 6.0-8.0 with 10～100mmol/L, pH preparation kojic acid, storing solution concentration is 0.1mmol/L to 10mmol/L, with acetate buffer, storing solution is configured to the standard solution of variable concentrations, after the kojic acid standard solution of getting variable concentrations fully mixes with copper nanocluster, carries out fluorometric investigation.
8. the method for claim 1, is characterized in that in described step (3), the reaction time is 5～10min, and temperature of reaction is 10～40 ℃.
9. the method for claim 1, is characterized in that the measurement of described fluorescence intensity realizes by any a fluorescence spectrophotometer; Fluorescence intensity relative changing value is according to formula (F
0-F)/F
0× 100% calculates, wherein F
0represent the fluorescence intensity of copper nanocluster while not adding kojic acid, F represents to add the fluorescence intensity of copper nanocluster after kojic acid.
10. method as claimed in claim 9, is characterized in that the test condition of described fluorescence intensity is as follows: fluorescent emission wavelength is the crest of copper nanocluster fluorescence emission spectrum, the crest that fluorescence exciting wavelength is corresponding excitation spectrum.
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| CN108115127A (en) * | 2017-11-29 | 2018-06-05 | 上海师范大学 | A kind of Nanometer Copper biomimetic material and preparation method and application |
| CN108827921A (en) * | 2018-06-08 | 2018-11-16 | 山西大学 | A kind of the room temperature phosphorimetry detection method and application of lysozyme |
| CN113960007A (en) * | 2021-10-29 | 2022-01-21 | 杭州食疗晶元生物科技有限公司 | Method for identifying organic food based on food structure energy and processing energy storage |
| CN114965410A (en) * | 2022-05-26 | 2022-08-30 | 北京工商大学 | Method for rapidly detecting CH3SH content in meat product by using BSA-CuNCs fluorescence sensing strategy |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105562705A (en) * | 2015-12-21 | 2016-05-11 | 江苏科技大学 | Method for synthesizing copper quantum dots based on protein and application of copper quantum dots |
| CN105750561A (en) * | 2016-03-17 | 2016-07-13 | 湖北大学 | Method for purifying copper nano-cluster |
| CN108115127A (en) * | 2017-11-29 | 2018-06-05 | 上海师范大学 | A kind of Nanometer Copper biomimetic material and preparation method and application |
| CN108827921A (en) * | 2018-06-08 | 2018-11-16 | 山西大学 | A kind of the room temperature phosphorimetry detection method and application of lysozyme |
| CN108827921B (en) * | 2018-06-08 | 2021-03-30 | 山西大学 | Room-temperature phosphorescence detection method for lysozyme and application |
| CN113960007A (en) * | 2021-10-29 | 2022-01-21 | 杭州食疗晶元生物科技有限公司 | Method for identifying organic food based on food structure energy and processing energy storage |
| CN113960007B (en) * | 2021-10-29 | 2024-03-05 | 杭州食疗晶元生物科技有限公司 | Method for identifying organic food based on food structure energy and processing energy storage |
| CN114965410A (en) * | 2022-05-26 | 2022-08-30 | 北京工商大学 | Method for rapidly detecting CH3SH content in meat product by using BSA-CuNCs fluorescence sensing strategy |
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Application publication date: 20140618 |