CN103743711B - Utilize the method for gibberellin in cyclodextrin and FRET (fluorescence resonance energy transfer) technology for detection food - Google Patents
Utilize the method for gibberellin in cyclodextrin and FRET (fluorescence resonance energy transfer) technology for detection food Download PDFInfo
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- CN103743711B CN103743711B CN201410000021.3A CN201410000021A CN103743711B CN 103743711 B CN103743711 B CN 103743711B CN 201410000021 A CN201410000021 A CN 201410000021A CN 103743711 B CN103743711 B CN 103743711B
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Abstract
The invention discloses a kind of method utilizing gibberellin in cyclodextrin and FRET (fluorescence resonance energy transfer) technology for detection food.What utilize cyclodextrin comprises effect, the fluorescence intensity of the acceptor butyl rhodamine b of energy transfer system is strengthened, thus establishes a kind of method measuring GA content in food.Is the concentration of gibberellin 40 ~ 760? within the scope of nanograms/milliliter with fluorescent quenching amount be good linear relationship, method detects and is limited to 8.9 nanograms/milliliter.Instant invention overcomes prior art and there is the shortcomings such as sensitivity is low, range of application is narrow when detecting, improve sensitivity and selectivity, fast convenient for the detection of low concentration gibberellin in food.
Description
Technical field
The present invention relates to a kind of method utilizing the effect that comprises of cyclodextrin and FRET (fluorescence resonance energy transfer) technology to detect trace gibberellin in food fast.
Background technology
Gibberellin is the class plant hormone extensively existed, and is mainly used in the growth promoting agricultural product.The kind of gibberellin has more than more than 100 kinds, wherein the most general with gibberellin GA3.Studies have found that, the countries such as after gibberellin is residual in human body, metabolism is incomplete, thus diseases induced, American-European have formulated relevant criterion to the residual quantity of gibberellin GA3.The method detecting gibberellin at present both at home and abroad mainly contains high performance liquid chromatography, immunoassay, electrochemical process etc.Though above method sensitivity is higher, severe reaction conditions, expensive equipment, and the running time is long.Therefore, seek to have fast, accurately, in the detection food that selectivity is higher, the method for gibberellin is significant.And FRET (fluorescence resonance energy transfer) method is as a kind of novel fluorescence detection technique, compared to other fluorescent methods, there is higher sensitivity and selectivity, utilize the hydrophobic tubular structure that cyclodextrin is special simultaneously, give body and the acceptor of energy transfer system are closely contained in molecule cavity, effectively strengthen the efficiency of energy trasfer.This method utilizes the examine repair of its uniqueness, thus sets up quick, sensitive, to detect gibberellin accurately new method.There is not been reported in the detection of gibberellin for current domestic and international application FRET (fluorescence resonance energy transfer) method.
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 the method that trace gibberellin in food detects.
Thinking of the present invention: give body using lactochrome as fluorescence energy transfer, butyl rhodamine b is as acceptor, and form the energy transfer system of stable performance, energy transferring to butyl rhodamine b, is become the hypersensitive fluorescence probe that fluorescence intensity is higher by lactochrome.And cyclodextrin can the fluorescence intensity of enhanced sensitivity acceptor butyl rhodamine b effectively, and the efficiency of FRET (fluorescence resonance energy transfer) can be improved, after adding 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.Thus the cyclodextrin setting up detection gibberellin comprises Energy Transfer.
The concrete mechanism of the present invention: because cyclodextrin has special hydrophobic cavity, and in tubular structure, thus can effectively comprise energy transfer system to body and acceptor, further the two intermolecular distance, improves the efficiency of energy trasfer.After adding gibberellin gradually in system, acceptor butyl rhodamine b, with positive charge, is combined by electrostatic interaction with the gibberellin with negative electricity, makes the regular quencher of fluorescence.
Concrete steps are:
1, detection method:
100 microlitres 1.0 × 10 are added all respectively in 10 10 milliliters of color comparison tubes
-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 and the poly-vinyl alcohol solution of 20 microlitre 0.1 grams per liters, the Gibberellins solution of 40 ~ 760 nanograms/milliliter is added again respectively in these 10 10 milliliters of color comparison tubes, scale is settled to respectively 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, react and carry out fluorescence intensity detection with RF-5301PC fluorophotometer after 3 minutes, excitation wavelength is 360 nanometers, excites and launch slit width to be 5 nanometers.
2, the drafting of working curve:
100 microlitres 1.0 × 10 are added all respectively in 10 10 milliliters of color comparison tubes
-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, the Gibberellins solution of 40 ~ 760 micrograms per litre is added again respectively in these 10 10 milliliters of color comparison tubes, scale is settled to respectively 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, fully shake up rear placing response 3 minutes, carry out resonance light intensity detection; The concentration c of gibberellin within the scope of 40 ~ 760 nanograms/milliliter with fluorescent quenching amount (Δ I
f) in good linear relationship, its equation of linear regression is: Δ I
f=0.2503c+0.7416, linearly dependent coefficient r=0.9997.
Instant invention overcomes prior art exists reaction time length, complex operation, poor selectivity, expensive equipment shortcoming when detecting, improve sensitivity and selectivity better, for the detection more accurately fast and easy 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 sodium hydrogen phosphate (0.2 mol/L)-citric acid (0.1 mol/L) buffer solution of pH=6.6, the gibberellin of variable concentrations is to the fluorescence quenching spectrum figure of butyl rhodamine b.Wherein a to j is respectively the fluorescence quenching spectrum figure of gibberellin to butyl rhodamine b of 40,120,200,280,360,440,520,600,680,760 nanograms/milliliter.
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:
100 microlitres 1.0 × 10 are added all respectively in 10 10 milliliters of color comparison tubes
-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 and the poly-vinyl alcohol solution of 20 microlitre 0.1 grams per liters, the Gibberellins solution of 40,120,200,280,360,440,520,600,680,760 nanograms/milliliter is added again respectively in these 10 10 milliliters of color comparison tubes, the sodium hydrogen phosphate of pH=6.6 (0.2 mol/L)-citric acid (0.1 mol/L) buffer solution is used to be settled to scale respectively, react and carry out fluorescence intensity detection with RF-5301PC fluorophotometer after 3 minutes, excitation wavelength is 360 nanometers, excites and launch slit width to be 5nm.
2, the drafting of working curve:
100 microlitres 1.0 × 10 are added all respectively in 10 10 milliliters of color comparison tubes
-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 and the poly-vinyl alcohol solution of 20 microlitre 0.1 grams per liters, the Gibberellins solution of 40,120,200,280,360,440,520,600,680,760 nanograms/milliliter is added again respectively in these 10 10 milliliters of color comparison tubes, the sodium hydrogen phosphate of pH=6.6 (0.2 mol/L)-citric acid (0.1 mol/L) buffer solution is used to be settled to scale respectively, fully shake up rear placing response 3 minutes, carry out resonance light intensity detection; The concentration c of gibberellin within the scope of 40 ~ 760 nanograms/milliliter with fluorescent quenching amount (Δ I
f) in 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, centrifugal subnatant is rubbed in the least with 50/liter phosphate buffer dilute 1000 times as solution to be measured.
Get appropriate liquid to be measured empirically method operation sample is measured, carry out Standard entertion recovery test simultaneously, 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 % |
| Milk 1 | 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. utilize a method for gibberellin in cyclodextrin and FRET (fluorescence resonance energy transfer) technology for detection food, it is characterized in that concrete steps are as follows:
1, detection method:
100 microlitres 1.0 × 10 are added all respectively in 10 10 milliliters of color comparison tubes
-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 and the poly-vinyl alcohol solution of 20 microlitre 0.1 grams per liters, the Gibberellins solution of 40 ~ 760 nanograms/milliliter is added again respectively in these 10 10 milliliters of color comparison tubes, scale is settled to respectively 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, react and carry out fluorescence intensity detection with RF-5301PC fluorophotometer after 3 minutes, excitation wavelength is 360 nanometers, excites and launch slit width to be 5 nanometers;
2, the drafting of working curve:
100 microlitres 1.0 × 10 are added all respectively in 10 10 milliliters of color comparison tubes
-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, the Gibberellins solution of 40 ~ 760 nanograms/milliliter is added again respectively in these 10 10 milliliters of color comparison tubes, scale is settled to respectively 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, fully shake up rear placing response 3 minutes, carry out fluorescence intensity detection; The concentration c of gibberellin within the scope of 40 ~ 760 nanograms/milliliter with fluorescent quenching amount (Δ I
f) in 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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| CN104316501A (en) * | 2014-10-20 | 2015-01-28 | 南昌大学 | Method for detecting thiocyanate ion based on 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 |
| CN115078323A (en) * | 2022-06-24 | 2022-09-20 | 公安部物证鉴定中心 | HMX photonic crystal sensor and preparation method and application thereof |
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| EP1651959B1 (en) * | 2003-08-08 | 2008-03-12 | Pablo Wessig | Molecular probe and material for the detection of an analyte, and use thereof |
| CN102590170A (en) * | 2012-02-28 | 2012-07-18 | 江南大学 | Method for simultaneously detecting mercury ion and/or silver ion in water solution based on fluorescence resonance energy transfer |
| 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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Patent Citations (3)
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| 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 |
| CN102590170A (en) * | 2012-02-28 | 2012-07-18 | 江南大学 | Method for simultaneously detecting mercury ion and/or silver ion in water solution based on fluorescence resonance energy transfer |
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| Title |
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| Chemical Sensors Based on Cyclodextrin Derivatives;T. Ogoshi et al.;《Sensors》;20080825;第8卷;第4961-4982页 * |
| Cyclodextrin Supramolecular Complex as a Water-Soluble Ratiometric Sensor for Ferric Ion Sensing;M. Xu et al.;《Langmuir》;20091023;第26卷(第6期);第4529-4534页 * |
| 固相萃取-高效液相色谱测定番茄中的赤霉素GA_3残留;黄红林等;《分析科学学报》;20050228;第21卷(第01期);第75-77页 * |
| 植物源性食品中赤霉素(GA_3)分析方法的研究进展;吴凤琪等;《食品工业科技》;20110331;第32卷(第03期);第435-437页 * |
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