CN103743711A - Method for detecting gibberellins in food by using cyclodextrin and fluorescence resonance energy transfer technology - Google Patents
Method for detecting gibberellins in food by using cyclodextrin and fluorescence resonance energy transfer technology Download PDFInfo
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- CN103743711A CN103743711A CN201410000021.3A CN201410000021A CN103743711A CN 103743711 A CN103743711 A CN 103743711A CN 201410000021 A CN201410000021 A CN 201410000021A CN 103743711 A CN103743711 A CN 103743711A
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Abstract
The invention discloses a method for detecting gibberellins in a food by using cyclodextrin and a fluorescence resonance energy transfer technology. The fluorescence intensity of a receptor butyl rhodamine B of an energy transfer system is enhanced by using including action of cyclodextrin, so as to build a method for determining the content of the gibberellins in the food. The concentration of the gibberellins is in the range of 40-760 nanograms per milliliter, and forms a good linear relationship with fluorescence quenching amount. The detection limit of the method is 8.9 nanograms per milliliter. By adopting the method, the defects of low sensitivity, narrow application range and the like in detection in the prior art are overcome, the sensitivity and the selectivity are improved, the detection of low-concentration gibberellins in the food is convenient and fast.
Description
Technical field
The present invention relates to a kind of method that comprises trace gibberellin in effect and FRET (fluorescence resonance energy transfer) technology fast detecting food of utilizing cyclodextrin.
Background technology
Gibberellin is the class plant hormone extensively existing, and is mainly used in promoting the growth of agricultural product.The kind of gibberellin has more than more than 100 kinds, wherein the most general with gibberellin GA3.The countries such as studies have found that, gibberellin residual rear metabolism in human body is incomplete, thereby diseases induced, American-European have formulated relevant criterion to the residual quantity of gibberellin GA3.The method that detects at present gibberellin both at home and abroad mainly contains high performance liquid chromatography, immunoassay, electrochemical process etc.Though above method sensitivity is higher, severe reaction conditions, instrument costliness, and the running time is long.Therefore, seek to have fast, accurately, in the higher detection food of selectivity, the method for gibberellin is significant.And FRET (fluorescence resonance energy transfer) method is as a kind of novel fluorescence detection technique, than other fluorescent methods, there is higher sensitivity and selectivity, utilize the special hydrophobic tubular structure of cyclodextrin simultaneously, give body and the acceptor of energy transfer system are closely contained in molecule cavity, effectively strengthen the efficiency that energy shifts.This method is utilized the detection characteristic of its uniqueness, thereby sets up quick, sensitive, to detect accurately gibberellin new method.Domestic and international application FRET (fluorescence resonance energy transfer) method there is not yet report in the detection of gibberellin at present.
Summary of the invention
The object of this invention is to provide a kind of method simple, highly sensitive, selectivity is good, easily and fast to method that in food, trace gibberellin detects.
Thinking of the present invention: give body using lactochrome as fluorescence energy transfer, butyl rhodamine b is as acceptor, forms the energy transfer system of stable performance, and lactochrome passes to butyl rhodamine b by energy, becomes the hypersensitive fluorescence probe that fluorescence intensity is higher.And the cyclodextrin fluorescence intensity of enhanced sensitivity acceptor butyl rhodamine b effectively, and can improve the efficiency of FRET (fluorescence resonance energy transfer), add after gibberellin, make the fluorescence intensity quencher of butyl rhodamine b, and the concentration of its quenching value and gibberellin is good linear relationship within the scope of 40 ~ 760 micrograms per litre.Thereby the cyclodextrin of setting up detection gibberellin comprises energy transfer method.
The concrete mechanism of the present invention: because cyclodextrin has special hydrophobicity cavity, and be tubular structure, thereby can effectively comprise energy transfer system to body and acceptor, the two the intermolecular distance of furthering, improves the efficiency that energy shifts.In system, add after gibberellin gradually, acceptor butyl rhodamine b, with positive charge, combines by electrostatic interaction with the gibberellin with negative electricity, makes the regular quencher of fluorescence.
Concrete steps are:
1, detection method:
In 10 10 milliliters of color comparison tubes, all add respectively 100 microlitres 1.0 × 10
-5the riboflavin solution of mol/L, 120 microlitres 1.0 × 10
-4the butyl rhodamine b solution of mol/L, 1 milliliter 1.0 × 10
-3the poly-vinyl alcohol solution of the beta-schardinger dextrin-solution of mol/L and 20 microlitre 0.1 grams per liters, in these 10 10 milliliters of color comparison tubes, add respectively again the Gibberellins solution of 40 ~ 760 nanograms/milliliter, with the citric acid solution that sodium hydrogen phosphate-volumetric molar concentration that pH=6.6 volumetric molar concentration is 0.2 mol/L is 0.1 mol/L, be settled to scale respectively, react and with RF-5301PC fluorophotometer, carry out fluorescence intensity detection after 3 minutes, excitation wavelength is 360 nanometers, excites and launch slit width to be 5 nanometers.
2, the drafting of working curve:
In 10 10 milliliters of color comparison tubes, all add respectively 100 microlitres 1.0 × 10
-5the riboflavin solution of mol/L, 120 microlitres 1.0 × 10
-4the butyl rhodamine b solution of mol/L, 1 milliliter 1.0 × 10
-3the beta-schardinger dextrin-solution of mol/L, the poly-vinyl alcohol solution of 20 microlitre 0.1 grams per liters, in these 10 10 milliliters of color comparison tubes, add respectively again the Gibberellins solution of 40 ~ 760 micrograms per litre, with the citric acid solution that sodium hydrogen phosphate-volumetric molar concentration that pH=6.6 volumetric molar concentration is 0.2 mol/L is 0.1 mol/L, be settled to scale respectively, fully shake up rear placing response 3 minutes, the light intensity that resonates detects; The concentration c of gibberellin within the scope of 40 ~ 760 nanograms/milliliter with fluorescent quenching amount (Δ I
f) being good linear relationship, its equation of linear regression is: Δ I
f=0.2503c+0.7416, linearly dependent coefficient r=0.9997.
The present invention has overcome prior art and has existed when detecting the shortcoming of reaction time length, complex operation, poor selectivity, instrument costliness, has improved better sensitivity and selectivity, for the more accurate fast and easy of detection of low concentration gibberellin in food.
Accompanying drawing explanation
Fig. 1 is that the embodiment of the present invention is 1.0 × 10
-7the butyl rhodamine b of mol/L, 1.2 × 10
-4the lactochrome of mol/L, 1.0 × 10
-4the beta-schardinger dextrin-of mol/L, 2.0 × 10
-4in the polyvinyl alcohol (PVA) of grams per liter and the sodium hydrogen phosphate of pH=6.6 (0.2 mol/L)-citric acid (0.1 mol/L) buffer solution, the fluorescent quenching spectrogram of the gibberellin of variable concentrations to butyl rhodamine b.Wherein a is respectively the gibberellin of 40,120,200,280,360,440,520,600,680,760 nanograms/milliliter to the fluorescent quenching spectrogram of butyl rhodamine b to j.
Fig. 2 is the graph of a relation of the fluorescent quenching amount of butyl rhodamine b in the energy transfer system that comprises of embodiment of the present invention GA content and beta-schardinger dextrin-.
Embodiment
Embodiment:
1, detection method:
In 10 10 milliliters of color comparison tubes, all add respectively 100 microlitres 1.0 × 10
-5the riboflavin solution of mol/L, 120 microlitres 1.0 × 10
-4the butyl rhodamine b solution of mol/L, 1 milliliter 1.0 × 10
-3the poly-vinyl alcohol solution of the beta-schardinger dextrin-solution of mol/L and 20 microlitre 0.1 grams per liters, in these 10 10 milliliters of color comparison tubes, add respectively again the Gibberellins solution of 40,120,200,280,360,440,520,600,680,760 nanograms/milliliter, use respectively sodium hydrogen phosphate (0.2 mol/L)-citric acid (0.1 mol/L) buffer solution of pH=6.6 to be settled to scale, react and with RF-5301PC fluorophotometer, carry out fluorescence intensity detection after 3 minutes, excitation wavelength is 360 nanometers, excites and launch slit width to be 5nm.
2, the drafting of working curve:
In 10 10 milliliters of color comparison tubes, all add respectively 100 microlitres 1.0 × 10
-5the riboflavin solution of mol/L, 120 microlitres 1.0 × 10
-4the butyl rhodamine b solution of mol/L, 1 milliliter 1.0 × 10
-3the poly-vinyl alcohol solution of the beta-schardinger dextrin-solution of mol/L and 20 microlitre 0.1 grams per liters, in these 10 10 milliliters of color comparison tubes, add respectively again the Gibberellins solution of 40,120,200,280,360,440,520,600,680,760 nanograms/milliliter, use respectively sodium hydrogen phosphate (0.2 mol/L)-citric acid (0.1 mol/L) buffer solution of pH=6.6 to be settled to scale, fully shake up rear placing response 3 minutes, the light intensity that resonates detects; The concentration c of gibberellin within the scope of 40 ~ 760 nanograms/milliliter with fluorescent quenching amount (Δ I
f) being good linear relationship, its equation of linear regression is: Δ I
f=0.2503c+0.7416, linearly dependent coefficient r=0.9997.
3, the detection of GA content in food:
Get commercially available milk sample appropriate, centrifuging 15 minutes, using centrifugal subnatant with 50 millis rub/liter 1000 times of phosphate buffer dilutions as solution to be measured.
Getting appropriate liquid to be measured measures sample by experimental technique operation, carry out standard simultaneously and add recovery test, result is as shown in table 1, its standard deviation RSD≤2.4% (n=6), recovery of standard addition is 98.3% ~ 103.2%, and illustration method has higher accuracy and good precision.
Table 1: sample determination and mark-on recovery test data
| Sample | Measured value (n=6) ng/mL | RSD(n=6)% | Add scalar ng/mL | Record total amount ng/mL | Recovery % |
| |
9.92 | 2.40 | 20.00 | 29.58 | 98.30 |
| Milk 2 | 12.61 | 1.82 | 40.00 | 52.31 | 99.26 |
| Milk 3 | 16.92 | 1.74 | 60.00 | 78.84 | 103.20 |
Claims (1)
1. a method of utilizing gibberellin in cyclodextrin and FRET (fluorescence resonance energy transfer) technology for detection food, is characterized in that concrete steps are as follows:
1, detection method:
In 10 10 milliliters of color comparison tubes, all add respectively 100 microlitres 1.0 × 10
-5the riboflavin solution of mol/L, 120 microlitres 1.0 × 10
-4the butyl rhodamine b solution of mol/L, 1 milliliter 1.0 × 10
-3the poly-vinyl alcohol solution of the beta-schardinger dextrin-solution of mol/L and 20 microlitre 0.1 grams per liters, in these 10 10 milliliters of color comparison tubes, add respectively again the Gibberellins solution of 40 ~ 760 nanograms/milliliter, with the citric acid solution that sodium hydrogen phosphate-volumetric molar concentration that pH=6.6 volumetric molar concentration is 0.2 mol/L is 0.1 mol/L, be settled to scale respectively, react and with RF-5301PC fluorophotometer, carry out fluorescence intensity detection after 3 minutes, excitation wavelength is 360 nanometers, excites and launch slit width to be 5 nanometers;
2, the drafting of working curve:
In 10 10 milliliters of color comparison tubes, all add respectively 100 microlitres 1.0 × 10
-5the riboflavin solution of mol/L, 120 microlitres 1.0 × 10
-4the butyl rhodamine b solution of mol/L, 1 milliliter 1.0 × 10
-3the beta-schardinger dextrin-solution of mol/L, the poly-vinyl alcohol solution of 20 microlitre 0.1 grams per liters, in these 10 10 milliliters of color comparison tubes, add respectively again the Gibberellins solution of 40 ~ 760 micrograms per litre, with the citric acid solution that sodium hydrogen phosphate-volumetric molar concentration that pH=6.6 volumetric molar concentration is 0.2 mol/L is 0.1 mol/L, be settled to scale respectively, fully shake up rear placing response 3 minutes, the light intensity that resonates detects; The concentration c of gibberellin within the scope of 40 ~ 760 nanograms/milliliter with fluorescent quenching amount (Δ I
f) being good linear relationship, its equation of linear regression is: Δ I
f=0.2503c+0.7416, linearly dependent coefficient r=0.9997.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316501A (en) * | 2014-10-20 | 2015-01-28 | 南昌大学 | Method for detecting thiocyanate ion based on fluorescence resonance energy transfer |
| CN105842210A (en) * | 2016-03-23 | 2016-08-10 | 南昌大学 | Thrombin detection method based on bio-dots and Au NPs fluorescence resonance energy transfer |
| WO2023246200A1 (en) * | 2022-06-24 | 2023-12-28 | 公安部物证鉴定中心 | Octogen photonic crystal sensor, preparation method therefor and application thereof |
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| WO2003008927A2 (en) * | 2001-07-17 | 2003-01-30 | Glycominds Ltd. | Methods and substrates for monitoring binding to a solid phase using proximity based signal generation assays |
| EP1651959B1 (en) * | 2003-08-08 | 2008-03-12 | Pablo Wessig | Molecular probe and material for the detection of an analyte, and use thereof |
| CN103237892A (en) * | 2010-08-30 | 2013-08-07 | 三星泰科威株式会社 | Oligonucleotides for detecting listeria spp. and use thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316501A (en) * | 2014-10-20 | 2015-01-28 | 南昌大学 | Method for detecting thiocyanate ion based on fluorescence resonance energy transfer |
| CN105842210A (en) * | 2016-03-23 | 2016-08-10 | 南昌大学 | Thrombin detection method based on bio-dots and Au NPs fluorescence resonance energy transfer |
| CN105842210B (en) * | 2016-03-23 | 2018-08-24 | 南昌大学 | Blood coagulation enzyme assay method based on biological quantum dot and Au NPs fluorescence resonance energy transfer |
| WO2023246200A1 (en) * | 2022-06-24 | 2023-12-28 | 公安部物证鉴定中心 | Octogen photonic crystal sensor, preparation method therefor and application thereof |
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