CN105699507A - ATP content determination kit and method thereof - Google Patents
ATP content determination kit and method thereof Download PDFInfo
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- CN105699507A CN105699507A CN201610039407.4A CN201610039407A CN105699507A CN 105699507 A CN105699507 A CN 105699507A CN 201610039407 A CN201610039407 A CN 201610039407A CN 105699507 A CN105699507 A CN 105699507A
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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
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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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- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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Abstract
The invention discloses an ATP content determination kit and a method thereof. The kit comprises a reagent I prepared by dissolving concentrated perchloric acid in distilled water, a reagent II prepared by dissolving sodium hydroxide in distilled water, a reagent III prepared from sodium phosphate dibasic dodecahydrate, a reagent IV prepared from sodium dihydrogen phosphate dihydrate and a reagent V prepared from ATP. The method improves the extraction process of ATP and is capable of removing influence of impurities in a sample by firstly adding an acidic extraction solution into the sample for fully extracting and then adding an alkaline extraction solution for neutralizing. The minimum detection limit of the method reaches up to 0.1Mu mol/mL; compared with a conventional spectrophotometric method, the minimum detection limit is significantly improved. The reagent components of the kit only comprise absolute methanol, disodium hydrogen phosphate and sodium dihydrogen phosphate; the detection cost is greatly reduced. The method is qualitative and quantitative in through-peak area and retention time; the detection accuracy is significantly improved.
Description
Technical field
The invention belongs to life science, relate to a kind of test kit, be specifically related to a kind of ATP assay test kit and method thereof。
Background technology
ATP is widely present in animal, plant, microorganism and cultivation cell, is bio-energy currency, and energy charge is to describe the major parameter of cellular energy metabolism state。Measure ATP content and calculate energy charge, it is possible to reflection energy metabolism state。
The method of the existing detection in current market ATP content is the spectrophotography based on phosphomolybdic acid colorimetric。The method utilizes creatine kinase catalysis creatine and ATP reaction to generate phosphagen, and detection phosphagen content, reacts ATP content with this at 700 nm。But the method there is also following shortcoming:
1, detection sensitivity is low, and lowest detection is limited to 1 μm of ol/mL。
2, the interference being subject in sample impurity, affects detection accuracy。
3, reaction system is subject to temperature, and the impact of pH value is relatively big, causes measurement result poor stability, and repeatability is bad。
4, need the creatine kinase reagent used expensive, relatively costly。
Summary of the invention
In order to make up the deficiencies in the prior art, it is desirable to provide a kind of ATP assay test kit and method thereof, can fast and accurately ATP content be measured。
For realizing above-mentioned technical purpose, reaching above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A kind of ATP assay test kit, including following reagent:
Reagent one, liquid × 1 bottle, 4 DEG C of preservations, a certain amount of dense perchloric acid make after being dissolved in distilled water, be placed in 60mL reagent bottle；
Reagent two, liquid × 1 bottle, 4 DEG C of preservations, a certain amount of sodium hydroxide make after being dissolved in distilled water, be placed in 60mL reagent bottle；
Reagent three, powder × 1 bottle, it is made up of a certain amount of disodium hydrogen phosphate dodecahydrate, is placed in 15mL reagent bottle；
Reagent four, powder × 1 bottle, it is made up of a certain amount of two hypophosphite monohydrate sodium dihydrogens, is placed in 10mL reagent bottle；
Reagent five, powder × 2, every is made up of a certain amount of ATP, is placed in 1.5mLEP pipe。
Further, in described reagent one, the volume of dense perchloric acid is the dense perchloric acid of 2mL, and the volume of distilled water is 58mL；
Further, the quality of the sodium hydroxide in described reagent two is 0.96g, and the volume of distilled water is 60mL；
Further, the quality of the disodium hydrogen phosphate dodecahydrate in described reagent three is 11.2833g；
Further, the quality of two hypophosphite monohydrate disodium hydrogens in described reagent four is 10.689g；
Further, the quality of the ATP in every EP pipe in described reagent five is 5mg。
ATP has absworption peak under 254nm, it is possible to use its content of high effective liquid chromatography for measuring。
A kind of adopt mentioned reagent box and the ATP content assaying method 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, solvent Suction filtration device, 50 0.22 μm of water system syringe-driven filters, 0.45 μm of water system filter membrane, 0.45 μm of organic system filter membrane, the C18 post of 4.6 × 150mm, adjustable pipette, the sample bottle of 50 2mL, the hplc grade methanol of 300mL and distilled water；
The preparation of step 2 mobile phase buffer substrate；
Pour in the volumetric flask of 1000mL after described reagent three and described reagent four being dissolved with a certain amount of distilled water, be settled to 1000mL with distilled water, form mobile phase buffer substrate；
The remove impurity of step 3 solvent；
By distilled water 1000mL, mobile phase buffer substrate 1000mL respectively with the water system filter membrane sucking filtration of 0.45 μm, by the methanol 300mL organic system filter membrane sucking filtration with 0.45 μm, to remove the impurity in solvent, it is prevented that blocking chromatographic column；
The preparation of step 4 mobile phase；
By mobile phase buffer substrate complete for sucking filtration with methanol with proportioning for 99.9:0.1(v/v) mix, namely take 999mL buffer substrate and 1mL methanol mixed；
Step 5 methanol complete for sucking filtration is become with distilled water proportioning respectively 100% methanol, 10% methanol, distilled water, each 250mL；
The deaeration of step 6 solvent；
By the ultrasonic 20min respectively of the solvent in step 4 and step 5, to slough the bubble in every kind of solvent, it is prevented that blocking chromatographic column；
The extraction of step 7ATP；
1) process of tissue: according to liver mass (g): reagent one volume (mL) is 1:(5 ~ 10) ratio, carry out ice bath homogenate, adopting centrifuge 4000g, under room temperature, centrifugal 15min, takes supernatant, add isopyknic described reagent two, mixing, adopts centrifuge 4000g, centrifugal 15min under room temperature, taking supernatant, syringe-driven filter is to be measured after filtering；
2) process of cell or antibacterial: first collect antibacterial or cell in centrifuge tube, abandon supernatant after centrifugal；According to antibacterial or cell quantity (104Individual): reagent one volume (mL) is the ratio of 500 ~ (1000:1), ultrasonic disruption antibacterial or cell；Then adopting centrifuge 4000g, under room temperature, centrifugal 15min, takes supernatant, adds isopyknic described reagent two, mixing, then adopts centrifuge 4000g, and under room temperature, centrifugal 15min, takes supernatant, and syringe-driven filter is to be measured after filtering；
The preparation of step 8 standard substance；
Described reagent five adds 1mL distilled water, it is made into the mother solution of 5mg/mL, described mother solution distilled water is diluted to respectively the ATP standard solution of 100 μ g/mL, 80 μ g/mL, 60 μ g/mL, 40 μ g/mL and 20 μ g/mL, and to be measured after syringe-driven filter filters respectively；
Step 9ATP assay operating procedure；
1) open computer, detector and pump, install chromatographic column, open software, method group arranges sample size 20 μ L, flow velocity 1mL/min, retention time 10min, detect wavelength 254nm, store method group that setting completed；
2) with the methanol of 100% after step 6 processes, 10% methanol, distilled water cross chromatographic column 30min by methanol concentration order from big to small；
3) cross pillar with the mobile phase after step 6 processes, after baseline stability, start application of sample；
4) being separately added into each 20 μ L of ATP standard solution of 100 μ g/mL, 80 μ g/mL, 60 μ g/mL, 40 μ g/mL and 20 μ g/mL, in 10min, the retention time of separable ATP, ATP is 3min, calculates the peak area of the ATP standard substance of 5 kinds of variable concentrations；
5) add sample 20 μ L, detect the peak area of ATP at corresponding retention time place；
The calculating of step 10ATP content；
With standard concentration (μ g/mL) for abscissa, peak area is that vertical coordinate calculates ATP standard curve；Sample peak area is substituted into standard curve, calculates sample ATP content。
The invention has the beneficial effects as follows:
1, the method for the present invention improves the extraction process of ATP, after being initially charged acidic extraction liquid and fully extracting, adds in alkalescence extracting solution and can remove the impact of impurity in sample。
2, the lowest detectable limit of the method for the present invention reaches 0.1 μm of ol/mL, compares tradition spectrophotography and significantly improves。
3, the reagent cost of the test kit of the present invention is only absolute methanol, disodium hydrogen phosphate and sodium dihydrogen phosphate, and testing cost is substantially reduced。
4, the method for the present invention is distinguished qualitatively and quantitatively by peak area and retention time, significantly improves detection accuracy。
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, and can be practiced according to the content of description, describes in detail with presently preferred embodiments of the present invention below。The specific embodiment of the present invention is shown in detail in by following example。
Detailed description of the invention
Below in conjunction with embodiment, describe the present invention in detail。
A kind of ATP assay test kit, including following reagent:
Reagent one, liquid × 1 bottle, 4 DEG C of preservations, the dense perchloric acid of 2mL make after being dissolved in 58mL distilled water, be placed in 60mL reagent bottle；
Reagent two, liquid × 1 bottle, 4 DEG C of preservations, 0.96g sodium hydroxide make after being dissolved in 60mL distilled water, be placed in 60mL reagent bottle；
Reagent three, powder × 1 bottle, it is made up of 11.2833g disodium hydrogen phosphate dodecahydrate, is placed in 15mL reagent bottle；
Reagent four, powder × 1 bottle, it is made up of 10.689g bis-hypophosphite monohydrate sodium dihydrogen, is placed in 10mL reagent bottle；
Reagent five, powder × 2, every is made up of the ATP of 5mg, is placed in 1.5mLEP pipe。
ATP has absworption peak under 254nm, it is possible to use its content of high effective liquid chromatography for measuring。
A kind of adopt mentioned reagent box and the ATP content assaying method 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, solvent Suction filtration device, 50 0.22 μm of water system syringe-driven filters, 0.45 μm of water system filter membrane, 0.45 μm of organic system filter membrane, the C18 post of 4.6 × 150mm, adjustable pipette, the sample bottle of 50 2mL, the hplc grade methanol of 300mL and distilled water；
The preparation of step 2 mobile phase buffer substrate；
Pour in the volumetric flask of 1000mL after described reagent three and described reagent four being dissolved with a certain amount of distilled water, be settled to 1000mL with distilled water, form mobile phase buffer substrate (note: the powder in reagent bottle to be rinsed well)；
The remove impurity of step 3 solvent；
By distilled water 1000mL, mobile phase buffer substrate 1000mL respectively with the water system filter membrane sucking filtration of 0.45 μm, by the methanol 300mL organic system filter membrane sucking filtration with 0.45 μm, to remove the impurity in solvent, it is prevented that blocking chromatographic column；
The preparation of step 4 mobile phase；
By mobile phase buffer substrate complete for sucking filtration with methanol with proportioning for 99.9:0.1(v/v) mix, namely take 999mL buffer substrate and 1mL methanol mixed；
Step 5 methanol complete for sucking filtration is become with distilled water proportioning respectively 100% methanol, 10% methanol, distilled water, each 250mL；
The deaeration of step 6 solvent；
By the ultrasonic 20min respectively of the solvent in step 4 and step 5, to slough the bubble in every kind of solvent, it is prevented that blocking chromatographic column；
The extraction of step 7ATP；
1) process of tissue: according to liver mass (g): reagent one volume (mL) is 1:(5 ~ 10) ratio (suggestion weigh about 0.1g tissue, add 1mL reagent one), carry out ice bath homogenate, adopt centrifuge 4000g, centrifugal 15min under room temperature, take supernatant, add isopyknic described reagent two, mixing, adopt centrifuge 4000g, under room temperature, centrifugal 15min, takes supernatant, and syringe-driven filter is to be measured after filtering；
2) process of cell or antibacterial: first collect antibacterial or cell in centrifuge tube, abandon supernatant after centrifugal；According to antibacterial or cell quantity (104Individual): reagent one volume (mL) is the ratio (suggestion 5,000,000 antibacterials or cell add 1mL reagent one) of 500 ~ (1000:1), ultrasonic disruption antibacterial or cell (ice bath, power 20% or 200W, ultrasonic 3s, interval 10s, repeat 30 times)；Then adopting centrifuge 4000g, under room temperature, centrifugal 15min, takes supernatant, adds isopyknic described reagent two, mixing, then adopts centrifuge 4000g, and under room temperature, centrifugal 15min, takes supernatant, and syringe-driven filter is to be measured after filtering；
The preparation of step 8 standard substance；
Described reagent five adds 1mL distilled water, it is made into the mother solution of 5mg/mL, described mother solution distilled water is diluted to respectively the ATP standard solution of 100 μ g/mL, 80 μ g/mL, 60 μ g/mL, 40 μ g/mL and 20 μ g/mL, and to be measured after syringe-driven filter filters respectively；
Step 9ATP assay operating procedure；
1) open computer, detector and pump, install chromatographic column, open software, method group arranges sample size 20 μ L, flow velocity 1mL/min, retention time 10min, detect wavelength 254nm, store method group that setting completed；
2) with the methanol of 100% after step 6 processes, 10% methanol, distilled water cross chromatographic column 30min by methanol concentration order from big to small；
3) cross pillar with the mobile phase after step 6 processes, after baseline stability, start application of sample；
4) each 20 μ L of ATP standard solution of 100 μ g/mL, 80 μ g/mL, 60 μ g/mL, 40 μ g/mL and 20 μ g/mL it are separately added into, separable ATP in 10min, the retention time of ATP is 3min, (pillar is different, retention time is variant), calculate the peak area of the ATP standard substance of 5 kinds of variable concentrations；
5) add sample 20 μ L, detect the peak area of ATP at corresponding retention time place；
The calculating of step 10ATP content；
With standard concentration (μ g/mL) for abscissa, peak area is that vertical coordinate calculates ATP standard curve；Sample peak area is substituted into standard curve, calculates sample ATP content。
Points for attention:
1, in order to avoid use procedure center pillar presses through greatly, the ascending adjustment of flow velocity。
When 2, using complete, owing to mobile phase contains buffer salt, rinse pillar 1 hour with water, to prevent buffer salt Crystallization Plugging chromatographic column, then wash chromatographic column 30min with the methanol of 10%, the methanol of 100% by methanol concentration order from small to large。
3, the extension rate of standard substance middle ATP concentration to be determined per sample, and in sample, the peak area of ATP must drop within the peak area of ATP standard substance of variable concentrations, and this standard substance extension rate is a reference。
Above-described embodiment, simply to illustrate that the technology of the present invention is conceived and feature, its objective is to be in that to allow one of ordinary skilled in the art will appreciate that present disclosure and to implement according to this, can not limit the scope of the invention with this。The change of every equivalence done by the essence of present invention or modification, all should be encompassed in protection scope of the present invention。
Claims (3)
1. an ATP assay test kit, it is characterised in that include following reagent:
Reagent one, liquid × 1 bottle, 4 DEG C of preservations, a certain amount of dense perchloric acid make after being dissolved in distilled water, be placed in 60mL reagent bottle；
Reagent two, liquid × 1 bottle, 4 DEG C of preservations, a certain amount of sodium hydroxide make after being dissolved in distilled water, be placed in 60mL reagent bottle；
Reagent three, powder × 1 bottle, it is made up of a certain amount of disodium hydrogen phosphate dodecahydrate, is placed in 15mL reagent bottle；
Reagent four, powder × 1 bottle, it is made up of a certain amount of two hypophosphite monohydrate sodium dihydrogens, is placed in 10mL reagent bottle；
Reagent five, powder × 2, every is made up of a certain amount of ATP, is placed in 1.5mLEP pipe。
2. ATP assay test kit according to claim 1, it is characterised in that:
In described reagent one, the volume of dense perchloric acid is the dense perchloric acid of 2mL, and the volume of distilled water is 58mL；
The quality of the sodium hydroxide in described reagent two is 0.96g, and the volume of distilled water is 60mL；
The quality of the disodium hydrogen phosphate dodecahydrate in described reagent three is 11.2833g；
The quality of two hypophosphite monohydrate disodium hydrogens in described reagent four is 10.689g；
The quality of the ATP in every EP pipe in described reagent five is 5mg。
3. the ATP content assaying method adopting test kit as claimed in claim 2, it is characterised 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, solvent Suction filtration device, 50 0.22 μm of water system syringe-driven filters, 0.45 μm of water system filter membrane, 0.45 μm of organic system filter membrane, the C18 post of 4.6 × 150mm, adjustable pipette, the sample bottle of 50 2mL, the hplc grade methanol of 300mL and distilled water；
The preparation of step 2 mobile phase buffer substrate；
Pour in the volumetric flask of 1000mL after described reagent three and described reagent four being dissolved with a certain amount of distilled water, be settled to 1000mL with distilled water, form mobile phase buffer substrate；
The remove impurity of step 3 solvent；
By distilled water 1000mL, mobile phase buffer substrate 1000mL respectively with the water system filter membrane sucking filtration of 0.45 μm, by the methanol 300mL organic system filter membrane sucking filtration with 0.45 μm, to remove the impurity in solvent, it is prevented that blocking chromatographic column；
The preparation of step 4 mobile phase；
By mobile phase buffer substrate complete for sucking filtration with methanol with proportioning for 99.9:0.1(v/v) mix, namely take 999mL buffer substrate and 1mL methanol mixed；
Step 5 methanol complete for sucking filtration is become with distilled water proportioning respectively 100% methanol, 10% methanol, distilled water, each 250mL；
The deaeration of step 6 solvent；
By the ultrasonic 20min respectively of the solvent in step 4 and step 5, to slough the bubble in every kind of solvent, it is prevented that blocking chromatographic column；
The extraction of step 7ATP；
1) process of tissue: according to liver mass (g): reagent one volume (mL) is 1:(5 ~ 10) ratio, carry out ice bath homogenate, adopting centrifuge 4000g, under room temperature, centrifugal 15min, takes supernatant, add isopyknic described reagent two, mixing, adopts centrifuge 4000g, centrifugal 15min under room temperature, taking supernatant, syringe-driven filter is to be measured after filtering；
2) process of cell or antibacterial: first collect antibacterial or cell in centrifuge tube, abandon supernatant after centrifugal；According to antibacterial or cell quantity (104Individual): reagent one volume (mL) is the ratio of 500 ~ (1000:1), ultrasonic disruption antibacterial or cell；Then adopting centrifuge 4000g, under room temperature, centrifugal 15min, takes supernatant, adds isopyknic described reagent two, mixing, then adopts centrifuge 4000g, and under room temperature, centrifugal 15min, takes supernatant, and syringe-driven filter is to be measured after filtering；
The preparation of step 8 standard substance；
Described reagent five adds 1mL distilled water, it is made into the mother solution of 5mg/mL, described mother solution distilled water is diluted to respectively the ATP standard solution of 100 μ g/mL, 80 μ g/mL, 60 μ g/mL, 40 μ g/mL and 20 μ g/mL, and to be measured after syringe-driven filter filters respectively；
Step 9ATP assay operating procedure；
1) open computer, detector and pump, install chromatographic column, open software, method group arranges sample size 20 μ L, flow velocity 1mL/min, retention time 10min, detect wavelength 254nm, store method group that setting completed；
2) with the methanol of 100% after step 6 processes, 10% methanol, distilled water cross chromatographic column 30min by methanol concentration order from big to small；
3) cross pillar with the mobile phase after step 6 processes, after baseline stability, start application of sample；
4) being separately added into each 20 μ L of ATP standard solution of 100 μ g/mL, 80 μ g/mL, 60 μ g/mL, 40 μ g/mL and 20 μ g/mL, in 10min, the retention time of separable ATP, ATP is 3min, calculates the peak area of the ATP standard substance of 5 kinds of variable concentrations；
5) add sample 20 μ L, detect the peak area of ATP at corresponding retention time place；
The calculating of step 10ATP content；
With standard concentration (μ g/mL) for abscissa, peak area is that vertical coordinate calculates ATP standard curve；Sample peak area is substituted into standard curve, calculates sample ATP content。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112161945A (en) * | 2020-10-27 | 2021-01-01 | 安徽农业大学 | Ultra-micro detection method for purine content of soybean grains |
| US20210170305A1 (en) * | 2018-02-23 | 2021-06-10 | Silcotek Corp. | Liquid chromatography technique |
-
2016
- 2016-01-21 CN CN201610039407.4A patent/CN105699507A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| M. T. VECIANA-NOGUES等: "Determination of ATP related compounds in fresh and canned tuna fish by HPLC", 《ANALYTICAL, NUTRITIONAL AND CLINICAL METHODS SECTION》 * |
| M.G. VOLONTÉ等: "Development of an HPLC method for determination of metabolic compounds in myocardial tissue", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| 谭善军等: "HPLC 法测定术后疲劳综合征大鼠骨骼肌 ATP，ADP，AMP 的含量", 《药物分析杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210170305A1 (en) * | 2018-02-23 | 2021-06-10 | Silcotek Corp. | Liquid chromatography technique |
| CN112161945A (en) * | 2020-10-27 | 2021-01-01 | 安徽农业大学 | Ultra-micro detection method for purine content of soybean grains |
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