CN105486788A - Kit for determining zeatin nucleoside content and method thereof - Google Patents
Kit for determining zeatin nucleoside content and method thereof Download PDFInfo
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- CN105486788A CN105486788A CN201511016071.1A CN201511016071A CN105486788A CN 105486788 A CN105486788 A CN 105486788A CN 201511016071 A CN201511016071 A CN 201511016071A CN 105486788 A CN105486788 A CN 105486788A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8637—Peak shape
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention discloses a kit for determining zeatin nucleoside content and a method thereof. The reagent contained in the kit contains a first reagent, a second reagent, a third reagent, a fourth reagent and a fifth reagent, wherein the first reagent is prepared by mixing methyl alcohol, acetic acid and distilled water; the second reagent is formed by petroleum ether; the third reagent is formed by methyl alcohol; the fourth reagent is formed by glacial acetic acid; the fifth reagent is formed by zeatin nucleoside standard substance. The method of adopting the kit provided by the invention for determining the zeatin nucleoside content is simple and easy in sample pretreatment process; the petroleum ether can be coated on the sample for detection just after being extracted, so that the detection time is greatly saved; the ingredients of the extracting solution are simple; only a mixture solution (pH=3) of methyl alcohol and water needs to be extracted, so that the detection cost is greatly lowered; the determining step is automatically performed by the instrument; hundreds of samples can be continuously detected; accurate data can be read; each sample only spends ten minutes; and compared with other methods, the time is greatly shortened.
Description
Technical field
The invention belongs to life science field, relate to a kind of kit, be specifically related to a kind of ribosylzeatin assay kit and method thereof.
Background technology
Plant hormone (planthormone) refer to plant privileged site synthesis, and transport the micro-content organism of other positions of plant to remarkable regulating action the whole vital movement of plant to from synthesising part, be also referred to as natural plant hormone or plant endogenous hormones.The plant endogenous hormones of generally acknowledging at present has five large class, i.e. auxins, cytokinin, gibberellin class, abscisic acid, ethene.
Ribosylzeatin is the most active and general basic element of cell division existed with native form.The basic element of cell division can promote cell division, the propagation of induced bud, suppresses the formation of root, the expression of delaying senility course and activated gene and promote general metabolism.To the plant of maturation, n cell mitogen has high hormonal readiness at most vigorous period.
The method of existing detection hormone can be divided into three major types, biological detection method on the market at present, immunoassay technology and efficient liquid phase detection technique.Although biological detection method is simple, higher to the purity requirement of sample, need complicated sample pre-treatments, simultaneously selectivity and repeatability poor.The method one of immune detection is easily subject to the interference of cross reaction, two carry out the not versatility of antibody and preparation time longer, the whole time cycle is very long.Chromatographic technique is develop rapidly in recent years, more and more extensive in the detection of plant hormone.But now on the market based on the detection technique of high performance liquid chromatography, the pre-treatment for sample is generally loaded down with trivial details, and loss percentage is higher.
Summary of the invention
In order to overcome the defect of prior art, the present invention aims to provide a kind of ribosylzeatin assay kit and method thereof, can calculate the content of ribosylzeatin fast and accurately.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A kind of ribosylzeatin assay kit, comprises following reagent:
Reagent one, liquid × 1 bottle, 4 DEG C of preservations, are mixed by methyl alcohol, acetic acid and distilled water, are placed in 60mL reagent bottle;
Reagent two, liquid × 1 bottle, 4 DEG C of preservations, are made up of sherwood oil, are placed in 100mL reagent bottle;
Reagent three, liquid × 1 bottle, 4 DEG C of preservations, are made up of methyl alcohol, are placed in 50mL reagent bottle;
Reagent four, liquid × 1 bottle, 4 DEG C of preservations, are made up of glacial acetic acid, are placed in 50mL reagent bottle;
Reagent five, ribosylzeatin (ZR) standard items × 1 ,-20 DEG C of preservations, are made up of ribosylzeatin, are placed in 1.5mLEP pipe.
Further, in described reagent one, the volume of methyl alcohol is 45mL, and the volume of acetic acid is 3mL, and the volume of distilled water is 12mL;
Further, in described reagent two, the temperature of sherwood oil is 30-60 DEG C, and volume is 100mL;
Further, in described reagent three, the volume of methyl alcohol is 50mL;
Further, in described reagent four, the volume of glacial acetic acid is 50mL;
Further, in described reagent five, the quality of ribosylzeatin is 0.5mg.
Based on a ribosylzeatin content assaying method for high performance liquid chromatography, comprise the following steps:
The preparation of step 1 instrument and articles for use;
High performance liquid chromatograph, low speed centrifuge, Nitrogen evaporator, solvent Suction filtration device, organic system syringe-driven filter (50,0.22 μm), water system filter membrane (0.45 μm), organic system filter membrane (0.45 μm), C18 post (4.6 × 250mm), adjustable pipette, sample bottle (50,2mL), hplc grade methanol (500mL) and ultrapure water;
The removal of impurities process of step 2 solvent;
By the water system filter membrane suction filtration of 500mL ultrapure water with 0.45 μm, by the organic system filter membrane suction filtration of 500mL methyl alcohol with 0.45 μm, remove the impurity in ultrapure water and methyl alcohol respectively, prevent from blocking chromatographic column;
The preparation of step 3 mobile phase;
Get the mixing of the ultrapure water after the methyl alcohol after 200mL removal of impurities and 300mL removal of impurities, and add reagent four described in 3mL, mixing, is made into mobile phase;
The deaeration of step 4 solvent;
By ultrasonic 20 minutes of the mobile phase for preparing, to slough the bubble in solvent, prevent from blocking chromatographic column;
The extraction of step 5 ribosylzeatin;
Take 0.1g sample, put into mortar and grind, after described reagent one, the 4 DEG C of lixiviates adding 1mL precooling are spent the night, adopt centrifugal 10 minutes of eccentricity 8000g, take out supernatant; Reagent one lixiviate described in residue 0.5mL 2 hours, centrifugal rear taking-up supernatant; Merge twice supernatant, 40 DEG C of reduction vaporizations, to not containing organic phase (about surplus 0.3mL aqueous solution), add reagent two described in 0.5mL, extraction decolouring 3 times, discards upper strata ether phase, lower floor's 40 DEG C of evaporated under reduced pressure, add reagent three described in 0.1mL, dissolve mixing, to be measured after adopting syringe-driven filter to filter;
The preparation of step 6 standard items;
1mL methyl alcohol is added in described reagent five, be made into 500 μ g/mL mother liquors, described mother liquor methyl alcohol is diluted to respectively the ribosylzeatin standard solution of 40 μ g/mL, 30 μ g/mL, 20 μ g/mL, 10 μ g/mL and 5 μ g/mL, to be measured after adopting syringe-driven filter to filter the ribosylzeatin standard solution of five groups of variable concentrations respectively;
Step 7 ribosylzeatin assay operation steps;
1) open computer, detecting device and pump, install chromatographic column, open software, sample size 10 μ L is set in method group, flow velocity 0.8mL/min, column temperature 35 DEG C, retention time 15min, determined wavelength 254nm, store method group that setting completed;
2) start infusion pump, make mobile phase by chromatographic column, after baseline stability, start application of sample;
3) the ribosylzeatin standard solution 10 μ L of five groups of variable concentrations is added respectively, separable ribosylzeatin in 15min, the retention time of ribosylzeatin is about 6min, (pillar is different, retention time is variant), calculate the peak area of the ribosylzeatin standard items of five groups of variable concentrations;
4) add sample 10 μ L, detect the peak area of solution at corresponding retention time place;
The calculating of step 8 ribosylzeatin content;
With standard concentration (μ g/mL) for horizontal ordinate, peak area is that ordinate calculates ribosylzeatin typical curve, sample peak area is substituted into typical curve, calculation sample ribosylzeatin content.
The invention has the beneficial effects as follows:
1, the Sample pretreatment process of ribosylzeatin content method adopting kit of the present invention to carry out is simple to operation, only needs petroleum ether extraction just can sample detection, greatly saves the test duration.
The extract composition of the ribosylzeatin content method 2, adopting kit of the present invention to carry out is simple, only needs methyl alcohol to follow the mixed solution (pH=3) of water to extract, greatly reduces testing cost.
The determination step of the ribosylzeatin content method 3, adopting kit of the present invention to carry out is all automation equipment operation, can continuous detecting sample up to a hundred, read accurate data, and each sample only needs the ten minutes, compares the additive method time greatly to shorten.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technological means of the present invention, and can be implemented according to the content of instructions, describe in detail below with preferred embodiment of the present invention.The specific embodiment of the present invention is provided in detail by following examples.
Embodiment
Below in conjunction with embodiment, describe the present invention in detail.
A kind of ribosylzeatin assay kit, comprises following reagent:
Reagent one, liquid × 1 bottle, 4 DEG C of preservations, are mixed by 45mL methyl alcohol, 3mL acetic acid and 12mL distilled water, are placed in 60mL reagent bottle;
Reagent two, liquid × 1 bottle, 4 DEG C of preservations, are made up of the sherwood oil of 100mL temperature at 30-60 DEG C, are placed in 100mL reagent bottle;
Reagent three, liquid × 1 bottle, 4 DEG C of preservations, are made up of 50mL methyl alcohol, are placed in 50mL reagent bottle;
Reagent four, liquid × 1 bottle, 4 DEG C of preservations, are made up of 50mL glacial acetic acid, are placed in 50mL reagent bottle;
Reagent five, ribosylzeatin (ZR) standard items × 1 ,-20 DEG C of preservations, are made up of 0.5mg ribosylzeatin, are placed in 1.5mLEP pipe.
Ribosylzeatin has absorption peak under 254nm, can utilize its content of high effective liquid chromatography for measuring.
Utilize the ribosylzeatin content assaying method of mentioned reagent box based on high performance liquid chromatography, comprise the following steps:
The preparation of step 1 instrument and articles for use;
High performance liquid chromatograph, low speed centrifuge, Nitrogen evaporator, solvent Suction filtration device, organic system syringe-driven filter (50,0.22 μm), water system filter membrane (0.45 μm), organic system filter membrane (0.45 μm), C18 post (4.6 × 250mm), adjustable pipette, sample bottle (50,2mL), hplc grade methanol (500mL) and ultrapure water;
The removal of impurities process of step 2 solvent;
By the water system filter membrane suction filtration of 500mL ultrapure water with 0.45 μm, by the organic system filter membrane suction filtration of 500mL methyl alcohol with 0.45 μm, remove the impurity in ultrapure water and methyl alcohol respectively, prevent from blocking chromatographic column;
The preparation of step 3 mobile phase;
Get the mixing of the ultrapure water after the methyl alcohol after 200mL removal of impurities and 300mL removal of impurities, and add reagent four described in 3mL, mixing, is made into mobile phase;
The deaeration of step 4 solvent;
By ultrasonic 20 minutes of the mobile phase for preparing, to slough the bubble in solvent, prevent from blocking chromatographic column;
The extraction of step 5 ribosylzeatin;
Take 0.1g sample, put into mortar and grind, after described reagent one, the 4 DEG C of lixiviates adding 1mL precooling are spent the night, adopt centrifugal 10 minutes of eccentricity 8000g, take out supernatant; Reagent one lixiviate described in residue 0.5mL 2 hours, centrifugal rear taking-up supernatant; Merge twice supernatant, 40 DEG C of reduction vaporizations, to not containing organic phase (about surplus 0.3mL aqueous solution), add reagent two described in 0.5mL, extraction decolouring 3 times, discards upper strata ether phase, lower floor's 40 DEG C of evaporated under reduced pressure, add reagent three described in 0.1mL, dissolve mixing, to be measured after adopting syringe-driven filter to filter;
The preparation of step 6 standard items;
1mL methyl alcohol is added in described reagent five, be made into 500 μ g/mL mother liquors, described mother liquor methyl alcohol is diluted to respectively the ribosylzeatin standard solution of 40 μ g/mL, 30 μ g/mL, 20 μ g/mL, 10 μ g/mL and 5 μ g/mL, to be measured after adopting syringe-driven filter to filter the ribosylzeatin standard solution of five groups of variable concentrations respectively;
Step 7 ribosylzeatin assay operation steps;
1) open computer, detecting device and pump, install chromatographic column, open software, sample size 10 μ L is set in method group, flow velocity 0.8mL/min, column temperature 35 DEG C, retention time 15min, determined wavelength 254nm, store method group that setting completed;
2) start infusion pump, make mobile phase by chromatographic column, after baseline stability, start application of sample;
3) the ribosylzeatin standard solution 10 μ L of five groups of variable concentrations is added respectively, separable ribosylzeatin in 15min, the retention time of ribosylzeatin is about 6min, (pillar is different, retention time is variant), calculate the peak area of the ribosylzeatin standard items of five groups of variable concentrations;
4) add sample 10 μ L, detect the peak area of solution at corresponding retention time place;
The calculating of step 8 ribosylzeatin content;
With standard concentration (μ g/mL) for horizontal ordinate, peak area is that ordinate calculates ribosylzeatin typical curve, sample peak area is substituted into typical curve, calculation sample ribosylzeatin content.
Points for attention:
1, in order to avoid use procedure center pillar presses through greatly, the ascending adjustment of flow velocity.
When 2, finishing using, first wash chromatographic column 30min with the methanol aqueous solution of 50%, then with pure methanol wash column chromatographic column 30min.
3, the extension rate of standard items will middle ribosylzeatin concentration be determined per sample, and in sample, the peak area of ZR must drop within the peak area of ribosylzeatin standard items of variable concentrations, and this standard items extension rate is a reference.
Above-described embodiment, just in order to technical conceive of the present invention and feature are described, its objective is and is one of ordinary skilled in the art can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.The change of every equivalence done by the essence of content of the present invention or modification, all should be encompassed in protection scope of the present invention.
Claims (3)
1. a ribosylzeatin assay kit, is characterized in that, comprises following reagent:
Reagent one, liquid × 1 bottle, 4 DEG C of preservations, are mixed by methyl alcohol, acetic acid and distilled water, are placed in 60mL reagent bottle;
Reagent two, liquid × 1 bottle, 4 DEG C of preservations, are made up of sherwood oil, are placed in 100mL reagent bottle;
Reagent three, liquid × 1 bottle, 4 DEG C of preservations, are made up of methyl alcohol, are placed in 50mL reagent bottle;
Reagent four, liquid × 1 bottle, 4 DEG C of preservations, are made up of glacial acetic acid, are placed in 50mL reagent bottle;
Reagent five, ribosylzeatin standard items × 1 ,-20 DEG C of preservations, are made up of ribosylzeatin, are placed in 1.5mLEP pipe.
2. ribosylzeatin assay kit according to claim 1, is characterized in that:
In described reagent one, the volume of methyl alcohol is 45mL, and the volume of acetic acid is 3mL, and the volume of distilled water is 12mL;
In described reagent two, the temperature of sherwood oil is 30-60 DEG C, and volume is 100mL;
In described reagent three, the volume of methyl alcohol is 50mL;
In described reagent four, the volume of glacial acetic acid is 50mL;
In described reagent five, the quality of ribosylzeatin is 0.5mg.
3. adopt a ribosylzeatin content assaying method for kit as claimed in claim 2, it is characterized in that, based on high performance liquid chromatography, comprise the following steps:
The preparation of step 1 instrument and articles for use;
High performance liquid chromatograph, low speed centrifuge, Nitrogen evaporator, solvent Suction filtration device, organic system syringe-driven filter, water system filter membrane, organic system filter membrane, C18 post, adjustable pipette, sample bottle, hplc grade methanol and ultrapure water;
The removal of impurities process of step 2 solvent;
By the water system filter membrane suction filtration of 500mL ultrapure water with 0.45 μm, by the organic system filter membrane suction filtration of 500mL methyl alcohol with 0.45 μm, remove the impurity in ultrapure water and methyl alcohol respectively, prevent from blocking chromatographic column;
The preparation of step 3 mobile phase;
Get the mixing of the ultrapure water after the methyl alcohol after 200mL removal of impurities and 300mL removal of impurities, and add reagent four described in 3mL, mixing, is made into mobile phase;
The deaeration of step 4 solvent;
By ultrasonic 20 minutes of the mobile phase for preparing, to slough the bubble in solvent, prevent from blocking chromatographic column;
The extraction of step 5 ribosylzeatin;
Take 0.1g sample, put into mortar and grind, after described reagent one, the 4 DEG C of lixiviates adding 1mL precooling are spent the night, adopt centrifugal 10 minutes of eccentricity 8000g, take out supernatant; Reagent one lixiviate described in residue 0.5mL 2 hours, centrifugal rear taking-up supernatant; Merge twice supernatant, 40 DEG C of reduction vaporizations, to not containing organic phase, add reagent two described in 0.5mL, extraction decolouring 3 times, and discard upper strata ether phase, lower floor's 40 DEG C of evaporated under reduced pressure, add reagent three described in 0.1mL, dissolve mixing, to be measured after adopting syringe-driven filter to filter;
The preparation of step 6 standard items;
1mL methyl alcohol is added in described reagent five, be made into 500 μ g/mL mother liquors, described mother liquor methyl alcohol is diluted to respectively the ribosylzeatin standard solution of 40 μ g/mL, 30 μ g/mL, 20 μ g/mL, 10 μ g/mL and 5 μ g/mL, to be measured after adopting syringe-driven filter to filter the ribosylzeatin standard solution of five groups of variable concentrations respectively;
Step 7 ribosylzeatin assay operation steps;
1) open computer, detecting device and pump, install chromatographic column, open software, sample size 10 μ L is set in method group, flow velocity 0.8mL/min, column temperature 35 DEG C, retention time 15min, determined wavelength 254nm, store method group that setting completed;
2) start infusion pump, make mobile phase by chromatographic column, after baseline stability, start application of sample;
3) add the ribosylzeatin standard solution 10 μ L of five groups of variable concentrations respectively, separable ribosylzeatin in 15min, the retention time of ribosylzeatin is 6min, calculates the peak area of the ribosylzeatin standard items of five groups of variable concentrations;
4) add sample 10 μ L, detect the peak area of solution at corresponding retention time place;
The calculating of step 8 ribosylzeatin content;
Take standard concentration as horizontal ordinate, peak area is that ordinate calculates ribosylzeatin typical curve, sample peak area is substituted into typical curve, calculation sample ribosylzeatin content.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109324139A (en) * | 2018-12-14 | 2019-02-12 | 广西中烟工业有限责任公司 | Ribosylzeatin liquid-liquid extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf |
| CN113588850A (en) * | 2021-07-27 | 2021-11-02 | 苏州梦犀生物医药科技有限公司 | Cytokinin oxidase activity detection kit and detection method |
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| WO2008097197A1 (en) * | 2007-02-05 | 2008-08-14 | National University Of Singapore | Putative cytokinin receptor and methods for use thereof |
| CN103048404A (en) * | 2012-12-19 | 2013-04-17 | 武汉大学 | Quantitative detection method for endogenous cytokinins in plant sample |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109324139A (en) * | 2018-12-14 | 2019-02-12 | 广西中烟工业有限责任公司 | Ribosylzeatin liquid-liquid extraction-liquid chromatography-tandem mass spectrometry measuring method in a kind of tobacco leaf |
| CN113588850A (en) * | 2021-07-27 | 2021-11-02 | 苏州梦犀生物医药科技有限公司 | Cytokinin oxidase activity detection kit and detection method |
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Application publication date: 20160413 |