CN101644679B - Kit and method for using same to detect ATP content, enzyme and medicament - Google Patents
Kit and method for using same to detect ATP content, enzyme and medicament Download PDFInfo
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- CN101644679B CN101644679B CN2008101177770A CN200810117777A CN101644679B CN 101644679 B CN101644679 B CN 101644679B CN 2008101177770 A CN2008101177770 A CN 2008101177770A CN 200810117777 A CN200810117777 A CN 200810117777A CN 101644679 B CN101644679 B CN 101644679B
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Abstract
The invention provides a kit. The kit comprises a mixed solution formed by silole quaternary ammonium salt and buffer solution and ATP, wherein the buffer solution may be Tris-HCL buffer solution, buffer solution of phosphate, Tris-EDTA buffer solution, Mes buffer solution or HEPES buffer solution; and the concentration of the silole quaternary ammonium salt in the mixing solution is 3*10<-5> to 1*10<-4>mol/l. The invention also provides a method for using the kit to detect the ATP content, the enzyme capable of hydrolyzing the ATP and the medicament, or using the ATP enzyme in a biochemical process. The kit has the advantages of simple preparation method, low cost, high sensitivity and strong specificity.
Description
Technical field
The present invention relates to a kind of kit and detect the method for ATP content, enzyme and medicine with this kit, described enzyme is that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process, and described medicine is that enzyme is had inhibiting medicine.
Background technology
Be subjected to various countries scientist's extensive concern for the detection of biomacromolecule, because content and the conformation of biomacromolecule in human body is closely bound up with human diseases.In numerous electronegative phosphate cpds, atriphos (ATP) is a kind of multi-functional nucleotide, is used for metabolic various reaction as the chemical energy in intracellular " energy currency " transitional cell: dna replication dna, transcribe, biosynthesizing in the cell division, cell, various enzymatic reactions etc.The ATP molecule can not store, and promptly is consumed in the synthetic back short time, and the undesired hydrolysis of ATP has very confidential relation in the generation of a lot of diseases and the body, such as local anemia, hypoglycemia, Parkinsonism etc.Just because of the ATP molecule has been played the part of important role like this in life process, so, develop the concern that new method that quick Sensitive Detection goes out ATP is subjected to scientist all the more.
Relatively the means of classical detection ATP are " luciferase-fluorescein " methods.Firefly has special luminescent substance---and luciferin and luciferase, fluorescein are easily oxidized, and it is under luciferase catalysis, activate by ATP, make it to combine with oxygen, be converted into oxyluciferin, oxyluciferin sends the visible light signal that is directly proportional with the ATP amount.Now, produce quick detection kit according to the principle of this kind method, still, the sensitivity of the method does not reach requirement sometimes.
1042-1043 page or leaf at 2007 the 129th phase J.AM.CHEM.SOC magazines discloses a kind of method that detects ATP based on electrochemica biological sensor that Chinese Academy of Sciences's Shanghai applied physics is developed, the researchist uses aptamer as the ATP molecular recognition elements: they are terminal modified on gold electrode with ATP aptamer one, the other end utilizes the means of chemical modification, ferrocene on the mark.In original state, ATP aptamer and its complementary strand form the duplex structure with rigidity, and ferrocene is away from electrode surface, and electron transfer process can't be carried out, so there is not electric signal output; When ATP existed, aptamer and ATP interaction of molecules formed tertiary structure, discharged its complementary strand, and at this moment, ferrocene and electrode surface are close, carry out electron transfer process, and electric signal output is arranged.Be that this sensor is to utilize aptamer its molecular conformation before and after identification ATP that marked change can take place, utilize electrochemical method can detect the electron transport capacity variation that this conformational change causes, thereby can be very sensitive detection ATP molecule, and selectivity is also very strong.But the testing process of this sensor need be carried out chemical labeling to ATP aptamer, thereby cost is uprised, and operation complicates.
So, develop high sensitivity, simple to operate, low-cost, be vital based on the detection method of the ATP content of chemical sensor and phosphatase for the early clinical diagnosis and the state of illness monitoring of some major diseases, especially under the physiological condition of simulation, the enzyme of hydrolytic action is arranged or in biochemical process, utilizes the detection of the enzyme of ATP to ATP content and to ATP.
Summary of the invention
The present invention's one purpose provides a kind of kit.
Another object of the present invention provides the method for utilizing this kit to detect ATP content.
A further object of the present invention provides the method for utilizing this kit to detect enzyme, and described enzyme is that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process.
A further object of the present invention has provided the method for utilizing this kit detection of drugs, and described medicine is that enzyme is had inhibiting medicine, and described enzyme is that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process.
One aspect of the present invention provides a kind of kit, this kit comprises by thiophene coughs up mixed solution and the ATP that quaternary ammonium salt and damping fluid are formed, described damping fluid is selected from a kind of in Tris-HCl (trihydroxy methyl amino aminomethane-hydrochloric acid) damping fluid, PBS (phosphate buffer), Tris-EDTA (the amino aminomethane-ethylenediamine tetraacetic acid of trihydroxy methyl) damping fluid, Mes (2-(N-morpholinyl) ethyl sulfonic acid) damping fluid and HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) damping fluid, and it is 3 * 10 that described thiophene is coughed up the concentration of quaternary ammonium salt in described mixed solution
-5-1 * 10
-4Mol/l.
Preferably, to cough up the concentration of quaternary ammonium salt in described mixed solution be 5 * 10 to described thiophene
-5-8 * 10
-5Mol/l.
More preferably, to cough up the concentration of quaternary ammonium salt in described mixed solution be 8 * 10 to described thiophene
-5Mol/l.
In the mixed solution of kit of the present invention, the concentration range that thiophene is coughed up quaternary ammonium salt is 3 * 10
-5-1 * 10
-4Mol/l, the binding ability that the thiophene in this concentration range is coughed up the ATP in quaternary ammonium salt and the determinand is strong, highly sensitive during detection.With concentration is 5 * 10
-6The ATP of mol/l is an example, is lower than 3 * 10 if the thiophene in the mixed solution of kit is coughed up quaternary ammonium salt concentration
-5The binding ability that mol/l, thiophene cough up quaternary ammonium salt and ATP weakens, and under the 370nm ultraviolet light, to cough up the fluorescence intensity of blank solution of quaternary ammonium salt approaching with only containing thiophene for fluorescence intensity, the sensitivity that influence detects.If thiophene is coughed up quaternary ammonium salt concentration and is higher than 1 * 10 in the mixed solution of kit
-4It is unstable in solution that mol/l, thiophene cough up quaternary ammonium salt, is easy to precipitation, makes test result insincere.
In the mixed solution of kit of the present invention, the concentration range that thiophene is coughed up quaternary ammonium salt is 5 * 10
-5-8 * 10
-5Mol/l, the binding ability that the thiophene in this concentration range is coughed up quaternary ammonium salt and ATP is stronger, and detection sensitivity is higher.With concentration is 5 * 10
-6The ATP of mol/l is an example, is 5 * 10 if the thiophene in the mixed solution of kit is coughed up quaternary ammonium salt concentration
-5Mol/l, under the 370nm ultraviolet light, fluorescence intensity is 50; If to cough up the concentration of quaternary ammonium salt be 8 * 10 to thiophene in the mixed solution of kit
-5Mol/l, under the 370nm ultraviolet light, fluorescence intensity is 500.
In the mixed solution of kit of the present invention, the concentration of coughing up quaternary ammonium salt when thiophene is 8 * 10
-5During mol/l, the binding ability that thiophene is coughed up quaternary ammonium salt and ATP is the strongest, and detection sensitivity is the highest.
Preferably, described thiophene is coughed up quaternary ammonium salt and is had as shown in the formula molecular formula shown in (I):
X is Cl, Br, I in the formula, and substituent R is C
1-C
10Straight chained alkyl.
Preferably, X is I in the formula (I), substituent R be C
1-C
5Straight chained alkyl.
More preferably, the substituent R in the formula (I) is a methyl.
Preferably, the mol ratio that thiophene is coughed up quaternary ammonium salt and described damping fluid in the described mixed solution is 1: 100-3500.
More preferably, the mol ratio that thiophene is coughed up quaternary ammonium salt and described Tris-HCl damping fluid in the described mixed solution is 1: 125.
Preferably, the pH value of damping fluid is 5-9 in the described mixed solution.
More preferably, the pH value of the damping fluid in the described mixed solution is 7-9.
Most preferably, the pH value of the damping fluid in the described mixed solution is 9.
The present invention provides the method for utilizing this kit to detect ATP content in the determinand on the other hand, and this method may further comprise the steps:
A. with coughing up the mixed solution that quaternary ammonium salt and described damping fluid form by thiophene and mix in the described kit, obtain ATP concentration 8.0 * 10 with ATP
-6Mol/l is with the ATP mixed solution of interior a plurality of concentration, and preferred concentration is 1.0 * 10
-6Mol/l-8.0 * 10
-6The ATP mixed solution of a plurality of concentration of mol/l;
B. the fluorescence intensity level of the ATP mixed solution of a plurality of concentration of obtaining of determination step a is drawn the standard relationship curve between ATP solution concentration and the fluorescence intensity;
C. extract the ATP in the determinand, the ATP that obtains is joined being coughed up in the mixed solution that quaternary ammonium salt and described damping fluid form by thiophene in the described kit, measure gained fluorescence intensity of solution value, obtain the content of ATP in the determinand according to the standard relationship curve of drawing among the step b.Because coughing up the mixed solution of quaternary ammonium salt and described damping fluid composition, thiophene not only can detect ATP, the effect of fluorescence developing is also arranged for other materials such as DNA simultaneously, so interference of other material in the determinand, ATP in the determinand can be extracted, the ATP after will extracting again detects.
Further aspect of the present invention also provides a kind of method of utilizing this kit to detect enzyme, and described enzyme is that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process, and this method may further comprise the steps:
A. with coughing up the mixed solution that quaternary ammonium salt and described damping fluid form by thiophene and mix in the described kit with ATP;
B. in the mixed solution that step a obtains, add enzyme to be measured, detect the fluorescence intensity level that adds a plurality of times of back;
If c. As time goes on fluorescence intensity level constantly reduces, this shows that the enzyme to be measured that adds among the step b is the enzyme that ATP is had the enzyme of hydrolytic action or utilize ATP in biochemical process.
Further aspect of the present invention also provides the method for utilizing this kit detection of drugs, and described medicine is that enzyme is had inhibiting medicine, and described enzyme is that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process, and this method may further comprise the steps:
A. with coughing up the mixed solution that quaternary ammonium salt and described damping fluid form by thiophene and mix in the described kit with ATP;
B. obtain adding simultaneously in the mixed solution medicine to be measured and described enzyme to step a, detect the fluorescence intensity level that adds a plurality of times of back;
If c. As time goes on fluorescence intensity level does not change, this shows that the medicine to be measured that adds among the step b is that described enzyme is had inhibiting medicine.
The invention has the beneficial effects as follows that the present invention has utilized positively charged thiophene to cough up the characteristic of aggregation inducing fluorescence of the organic molecule of quarternary ammonium salt compound, when its with after electronegative ATP molecule combines, utilize the interaction and the hydrophobic effect of positive and negative charge to make thiophene cough up the quarternary ammonium salt compound gathering, and then induce the generation hyperfluorescence, cough up in the mixed solution that quaternary ammonium salt and damping fluid of the present invention form thiophene at thiophene and cough up quaternary ammonium salt concentration 3 * 10
-5-1 * 10
-4In the mol/l scope, the concentration of ATP is in the scope of 0-8 μ M, and fluorescence intensity is directly proportional with ATP concentration, and the typical curve linear relationship optimum of the drafting in this proportional range, as Fig. 1, this moment, the excitation wavelength of fluorescence was 370nm, thereby had made up the kit that detects ATP content.Just because of this, the kit of the detection ATP content of the present invention's preparation is simple, only need comprise that thiophene coughs up mixed solution that quaternary ammonium salt and described damping fluid form and ATP solid or ATP solution and get final product, and carry out fluorometric assay with fluorescence spectrophotometer and promptly can obtain ATP content in the determinand, when containing ATP in the determinand, under the UV-irradiation of 370nm, be blue, and just can draw ATP content in the determinand by standard relationship curve and fluorescence intensity.Because thiophene is coughed up mixed solution that quaternary ammonium salt and described damping fluid form and is combined with multiple material and can send fluorescence, so the present invention need at first extract the ATP in the determinand when detection ATP content, checks the content of ATP again.And kit preparation method involved in the present invention is simple, with low cost, and has the characteristic of aggregation inducing fluorescence, does not therefore need it is carried out chemical labeling, has reduced a lot of chemical labeling trivial step.And the sensitivity of kit detection ATP of the present invention is very high, and specificity is stronger, and as can be seen from Figure 3, kit of the present invention can detect the content of ATP in the determinand, and is reactive poor for the ADP in the determinand, AMP and pyrophosphate.Kit of the present invention is highly sensitive, can reach to detect to contain tens nanomoles/other ATP of l concentration level.The calculating of detectability (LOD) is according to following equation: LOD=3S
0/ S, in this equation, S
0Definition be the degree that deviates from baseline, the definition of S is sensitivity; Referring to document: B.R.Eggins, Chemical Sensors andBiosensors, Wiley, Chichester, England, 2002.
Kit of the present invention is used for ATP is had the enzyme of hydrolytic action or utilizes enzyme when check of ATP in biochemical process, only the determinand of enzyme need be added to ATP solution and thiophene coughs up in the mixed solution of quaternary ammonium salt and described damping fluid composition, detection is a plurality of time fluorescence intensity levels after adding, if it is that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process that fluorescence intensity level, illustrates enzyme to be measured along with the increase of time constantly reduces.This method of inspection is simple, and the optimal reaction temperature that only needs to guarantee enzyme just can be finished.
Kit of the present invention can be applied to drug screening, and can filter out by the variation of check ATP content has inhibiting medicine to enzyme, and this kind of enzyme is that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process.In addition, we know most disease be with body in the undesired of ATP content cause, so this kit is expected to take place to disease by the change-detection of ATP content.Equally, many medicines also are the enzyme of hydrolytic action to be arranged or utilize the enzyme of ATP in biochemical process at ATP, so this kit also can detect the medicine with this type of character.And kit of the present invention has the advantage that at present a lot of detection meanss are not had: highly sensitive, simple to operate, naked eyes can be seen tangible fluorescence under the uviol lamp, can carry out original position monitors in real time, promptly directly add enzyme solutions in the mixed solution in kit, do not need to take out again the step of dilution, directly carry out the test of fluorescence spectrum at any time, carry out real-time follow-up in position.
Description of drawings
Below, describe embodiments of the invention in conjunction with the accompanying drawings in detail, wherein:
Fig. 1 represents the solution concentration of ATP and the standard relationship curve between the fluorescence intensity.
After Fig. 2 represents that the mixed solution in kit of the present invention adds ATP, ADP, AMP and pyrophosphate (Pyrophosphate) determinand respectively, the concentration of several determinands of this that obtains and the relation between the fluorescence intensity.
Fig. 3 represents to add calf intestinal alkaline phosphatase (CIAP) and ATP solution in the mixed solution of kit of the present invention, and along with the variation of joining day, thiophene is coughed up the variation of the fluorescence intensity that quarternary ammonium salt compound sends.
After Fig. 4 represented to add calf intestinal alkaline phosphatase (CIAP), sodium tungstate medicine and ATP solution simultaneously in the mixed solution of kit of the present invention, along with the variation of joining day, thiophene was coughed up the variation of the fluorescence intensity that quarternary ammonium salt compound sends.
Embodiment
Embodiment 1
Thiophene is coughed up the preparation of quarternary ammonium salt compound
1.1-methyl isophthalic acid-(3-amino) propyl group-2,3,4, the preparation of 5-silicon tetraphenyl heterocyclic pentylene
The methyl hydrogen thiophene is coughed up (1.0g, 2.5mmol), allylamine (0.36ml, 5.0mmol), H
2PtCl
6XH
2The toluene (10ml) that O (2.5mg, 5 μ mol) and distillation obtain places the Ar gaseous environment, refluxes 20 hours.With the solution cooling that obtains, by sintered glass filter, evaporation drying.The product that obtains is dissolved in the ether, and uses the hexane sedimentation and filtration.The yellow powder sediment that collection obtains totally 1.1 restrains yield 90%.Through the nuclear-magnetism check, the product of gained is 1-methyl isophthalic acid-(3-amino) propyl group-2,3,4,5-silicon tetraphenyl heterocyclic pentylene.1-methyl isophthalic acid-(3-amino) propyl group-2,3,4, the preparation method of 5-silicon tetraphenyl heterocyclic pentylene is referring to 2005 the 127th volumes of JACS, and exercise question is " Luminescent SiloleNanoparticles as Chemoselective Sensors for Cr (VI) (the fluorescence thiophene is coughed up nano particle is used for Cr (VI) ion as chemoreceptor detection) "
2. thiophene is coughed up the preparation of quarternary ammonium salt compound
Under the drying nitrogen protection; with 1-methyl isophthalic acid-(3-amino) propyl group-2; 3; 4; 5-silicon tetraphenyl heterocyclic pentylene (0.25g; 0.54mmol) be dissolved in the methanol solvate, in solution, add Anhydrous potassium carbonate (0.75g, 5.4mmol) and iodomethane (5mL; 80mmol); reaction mixture refluxed 6 hours under inert gas, removed solvent, and the reaction liquid behind the removal solvent is separated with silicagel column; the eluant, eluent of silicagel column is methylene chloride and methyl alcohol; the volume ratio of methylene chloride and methyl alcohol 15: 1 obtains the 260mg yellow solid behind the wash-out, 230 ℃ of this yellow solid fusing points.
This yellow solid is carried out nmr analysis, and nucleus magnetic hydrogen spectrum shows: 1H NMR (400MHz, DMSO-d
6) δ=6.81-7.16 (m, 20H, Ph), 3.21 (m, 2H), 2.93 (s, 9H), 1.64 (m, 2H), 0.93 (m, 2H), 0.50 (s, 3H); Nuclear-magnetism carbon spectrum shows:
13C NMR (100MHz, DMSO-d
6) δ=154.7,139.9,139.1,138.2,129.4,128.4,128.2,127.5,126.4,125.8,67.3,52.1,16.7,9.1 ,-6.0; C
35H
38Sin
+Molecular weight is: 500.2768; The molecular weight that high resolution mass spec is surveyed is 500.2786.The molecular formula that this thiophene is coughed up quaternary ammonium salt is as follows:
The preparation of kit
It is that 9 Tris-HCl damping fluid mixes that the thiophene that embodiment 1 is obtained is coughed up quarternary ammonium salt compound and pH value, and it is 8 * 10 that this thiophene is coughed up the concentration of quaternary ammonium salt in mixed solution
-5The mol ratio that mol/l, thiophene cough up quarternary ammonium salt compound and Tris-HCl damping fluid is 1: 125.It is 1 * 10 that the ATP standard items are mixed with concentration
-3The ATP solution of mol/l, the ATP standard items available from Sigma company, article number is 51963-61-2.
The preparation of kit
The preparation method of this kit is identical with the preparation method of the kit of embodiment 2, and different is, and thiophene is coughed up quarternary ammonium salt compound and pH value is that 5 Mes damping fluid mixes, and it is 1 * 10 that this thiophene is coughed up the concentration of quarternary ammonium salt compound in mixed solution
-4The mol ratio that mol/l, thiophene cough up quarternary ammonium salt compound and Mes damping fluid is 1: 3500, also comprises ATP solid standard items in this kit.
The preparation of kit
The preparation method of this kit is identical with the preparation method of the kit of embodiment 2, and different is, and thiophene is coughed up quarternary ammonium salt compound and pH value is that 8 phosphate buffer mixes, and it is 3 * 10 that this thiophene is coughed up the concentration of quarternary ammonium salt compound in mixed solution
-5Mol/l, thiophene cough up quarternary ammonium salt compound with and the mol ratio of phosphate buffer be 3: 1000, in this kit, also comprise ATP solid standard items.
Embodiment 5
The preparation of kit
The preparation method of this kit is identical with the preparation method of the kit of embodiment 4, and different is, it is that 7 HEPES damping fluid mixes that this thiophene is coughed up quarternary ammonium salt compound and pH value, and it is 5 * 10 that this thiophene is coughed up the concentration of quaternary ammonium salt in mixed solution
-5The mol ratio that mol/l, thiophene cough up quarternary ammonium salt compound and phosphate buffer is 5: 1000, also comprises ATP solid standard items in this kit.
Draw the standard relationship curve between ATP concentration and the fluorescence intensity
ATP solution among the embodiment 2 joined to cough up quarternary ammonium salt compound and pH value by thiophene be to mix in the mixed solution formed of 9 Tris-HCl damping fluid, mixed solution is 1ml altogether.And the ATP concentration in the adjusting mixed solution is respectively 1.0 * 10
-6Mol/l, 2 * 10
-6Mol/l, 3 * 10
-6Mol/l, 4 * 10
-6Mol/l, 5 * 10
-6Mol/l, 6 * 10
-6Mol/l and 7 * 10
-6These several concentration of mol/l, and the concentration that the thiophene in above several concentration is coughed up quaternary ammonium salt is 8 * 10
-5Mol/l.This mixed solution is carried out fluorescence spectrometry, draw the standard relationship curve between ATP solution concentration and the fluorescence intensity, see Fig. 1, each when detecting in the determinand ATP content, all need to draw the standard relationship curve between ATP solution concentration and the fluorescence intensity, eliminate the systematic error that checkout equipment brings.
Embodiment 7
The kit that utilizes embodiment 2 to provide detects and contains the ATP determinand.
The extracting method of cultivating zooblast or microbiological specimens ATP is as follows: at first prepare lysate, it is 8.0 1% Triton X-100 Triton X-100 (5ml) that this lysate contains pH value, 0.1% deoxysodium cholate (5ml), 100mM sodium chloride (2.93g), 0.1% sodium azide (1.5ml) and 10mM dithiothreitol (DTT) (770mg).Remove the cell culture fluid of cultivating in the red blood cell that obtains, on culture plate, add the phosphate buffer of capacity again, washed cell, cleansing solution inclines.Add the cell pyrolysis liquid of capacity again on culture plate, culture plate can shake 5-10min on microoscillator, scrapes cell on the culture plate with scraper, and cell suspension is moved into the 1.5ml centrifuge tube, fully suspendible concussion 30 seconds on the vortex oscillation device.Obtain containing the cell lysis suspension of the ATP of extraction.
Thiophene in embodiment 2 is coughed up the cell lysis suspension that adds the ATP that contains extraction in the mixed solution that quarternary ammonium salt compound and pH value are 9 Tris-HCl damping fluid preparation, represents to carry out its fluorescence intensity level of fluorescence spectrometry as Fig. 3.According to ATP solution concentration among Fig. 1 and the standard relationship curve between the fluorescence intensity, the ATP content that draws in this determinand red blood cell is 1.8 μ mol/l.
Cough up in cell lysis suspension from the mixed solution that quarternary ammonium salt compound and Tris-HCl damping fluid form to the ATP that contains extraction, thiophene again and add calf intestinal alkaline phosphatase, present embodiment is successively after adding calf intestinal alkaline phosphatase, carried out the mensuration of first order fluorescence intensity every 1 minute, the record thiophene is coughed up the variation of the fluorescence intensity that quarternary ammonium salt compound sends.
As can be seen from Figure 3, when being enzyme to be measured, when not adding calf intestinal alkaline phosphatase with calf intestinal alkaline phosphatase (CIAP), ATP concentration is very big, thiophene is coughed up the quarternary ammonium salt compound molecule and is sent intense fluorescence, even along with the variation of time, fluorescence intensity does not change yet; When adding calf intestinal alkaline phosphatase, along with the ATP hydrolytic enzyme joining day constantly increases, ATP is hydrolyzed, and fluorescence intensity reduces gradually, and the ATP hydrolysis is finished after 12 minutes, and the fluorescence intensity that thiophene is coughed up quarternary ammonium salt compound returns to the level of not assembling, and sees Fig. 3.By above experiment, calf intestinal alkaline phosphatase has hydrolytic action to ATP as can be seen.
Kit coughs up the cell lysis suspension that adds the ATP that contains extraction in the mixed solution that quaternary ammonium salt and phosphate forms by thiophene in embodiment 4, then add serum alkaline phosphatase again, present embodiment is successively after adding serum alkaline phosphatase, carried out the mensuration of first order fluorescence intensity every 1 minute, the result shows that along with the increase of joining day, fluorescence intensity level constantly diminishes, after 20 minutes, fluorescence intensity level reverts to and only contains the fluorescence intensity level that thiophene is coughed up the blank solution of quaternary ammonium salt.
Embodiment 9
Kit in embodiment 3 is coughed up by thiophene and is added the cell lysis suspension of the ATP that contains extraction in the mixed solution that quarternary ammonium salt compound and phosphate buffer form, then adds acid phosphatase again, present embodiment is successively after adding acid phosphatase, carried out the mensuration of first order fluorescence intensity every 1 minute, the result shows, increase along with the joining day, fluorescence intensity constantly diminishes, and after 20 minutes, fluorescence intensity level reverts to and only contains the fluorescence intensity level that thiophene is coughed up the blank solution of quaternary ammonium salt.
Sodium tungstate is that CIAP is had inhibiting micromolecule, if medicine to be measured is a sodium tungstate, the ability of CIAP hydrolysising ATP is limited, from the fluorescence spectrum as can be seen along with the growth that adds the CLAP time, fluorescence intensity does not change, therefore also can not react the variation of ATP amount, if but medicine to be measured is not that CIAP is had inhibiting medicine, from the fluorescence spectrum as can be seen along with the growth that adds the CLAP time, fluorescence intensity constantly reduces, and the ability of CIAP hydrolysising ATP is not limited, and reflects fluorescence intensity from fluorescence spectrum and constantly reduces, therefore the amount of ATP also constantly reduces, and sees Fig. 4.
Claims (13)
1. kit, this kit comprises by thiophene coughs up mixed solution and the ATP that quaternary ammonium salt and damping fluid are formed, described damping fluid is selected from a kind of in Tris-HCl damping fluid, phosphate buffer, Tris-EDTA damping fluid, Mes damping fluid and the HEPES damping fluid, and it is 3 * 10 that described thiophene is coughed up the concentration of quaternary ammonium salt in described mixed solution
-5-1 * 10
-4Mol/l, it has as shown in the formula molecular formula shown in (I):
X is Cl, Br, I in the formula, and substituent R is C
1-C
10Straight chained alkyl.
2. X is I in the kit according to claim 1, its Chinese style (I), and substituent R is C
1-C
5Straight chained alkyl.
3. kit according to claim 2, the substituent R in its Chinese style (I) is a methyl.
4. it is 5 * 10 that kit according to claim 1, thiophene are wherein coughed up the concentration of quaternary ammonium salt in described mixed solution
-5-8 * 10
-5Mol/l.
5. it is 8 * 10 that kit according to claim 4, thiophene are wherein coughed up the concentration of quaternary ammonium salt in described mixed solution
-5Mol/l.
6. according to each described kit among the claim 1-5, to cough up the mol ratio of quaternary ammonium salt and described damping fluid be 1 to thiophene in the wherein said mixed solution: 100-3500.
7. the mol ratio that kit according to claim 6, the thiophene in the wherein said mixed solution are coughed up quaternary ammonium salt and described Tris-HCl damping fluid is 1: 125.
8. according to each described kit among the claim 1-5, the pH value of damping fluid is 5-9 in the wherein said mixed solution.
9. kit according to claim 8, the pH value of damping fluid is 7-9 in the wherein said mixed solution.
10. kit according to claim 9, the pH value of damping fluid is 9 in the wherein said mixed solution.
11. a method of utilizing among the claim 1-5,7,9,10 each described kit to detect ATP content in determinand, this method may further comprise the steps:
A. with coughing up the mixed solution that quaternary ammonium salt and described damping fluid form by thiophene and mix in the described kit, obtain ATP concentration 8.0 * 10 with ATP
-6Mol/l is with the ATP mixed solution of interior a plurality of concentration;
B. the fluorescence intensity level of the ATP mixed solution of a plurality of concentration of obtaining of determination step a is drawn the standard relationship curve between ATP solution concentration and the fluorescence intensity;
C. extract the ATP in the determinand, the ATP that obtains is joined being coughed up in the mixed solution that quaternary ammonium salt and described damping fluid form by thiophene in the described kit, measure gained fluorescence intensity of solution value, obtain the content of ATP in the determinand according to the standard relationship curve of drawing among the step b.
12. a method of utilizing among the claim 1-5,7,9,10 each described kit to detect enzyme, described enzyme are that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process, this method may further comprise the steps:
A. with coughing up the mixed solution that quaternary ammonium salt and described damping fluid form by thiophene and mix in the described kit with ATP;
B. in the mixed solution that step a obtains, add enzyme to be measured, detect the fluorescence intensity level that adds a plurality of times of back;
If c. As time goes on fluorescence intensity level constantly reduces, this shows that the enzyme to be measured that adds among the step b is the enzyme that ATP is had the enzyme of hydrolytic action or utilize ATP in biochemical process.
13. method of utilizing each kit detection of drugs among the claim 1-5,7,9,10, described medicine is that enzyme is had inhibiting medicine, described enzyme is that ATP is had the enzyme of hydrolytic action or utilize the enzyme of ATP in biochemical process, and this method may further comprise the steps:
A. with coughing up the mixed solution that quaternary ammonium salt and described damping fluid form by thiophene and mix in the described kit with ATP;
B. in the mixed solution that step a obtains, add medicine to be measured and described enzyme simultaneously, detect the fluorescence intensity level that adds a plurality of times of back;
If c. As time goes on fluorescence intensity level does not change, this shows that the medicine to be measured that adds among the step b is that described enzyme is had inhibiting medicine.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101177770A CN101644679B (en) | 2008-08-05 | 2008-08-05 | Kit and method for using same to detect ATP content, enzyme and medicament |
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|---|---|---|---|
| CN2008101177770A CN101644679B (en) | 2008-08-05 | 2008-08-05 | Kit and method for using same to detect ATP content, enzyme and medicament |
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| CN101644679B true CN101644679B (en) | 2011-08-31 |
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| CN102621114B (en) * | 2012-02-27 | 2013-10-02 | 武汉大学 | Fluorescence detection method of five-position aldehyde-group deoxidizing uridine |
| CN102680442B (en) * | 2012-04-19 | 2014-02-26 | 太原理工大学 | Method for detecting trypsin using unmarked fluorescence |
| CN104655618B (en) * | 2015-02-10 | 2018-01-12 | 国家纳米科学中心 | A kind of method for detecting body fluid alkaline phosphatase |
| CN113564225B (en) * | 2021-09-23 | 2022-01-07 | 广州市雷德生物科技有限公司 | Cell lysate for ATP detection and application thereof |
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| CN1632134A (en) * | 2004-08-13 | 2005-06-29 | 湖南大学 | ATP and NAD and method for analysis of associated enzyme and substrate |
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| CN1632134A (en) * | 2004-08-13 | 2005-06-29 | 湖南大学 | ATP and NAD and method for analysis of associated enzyme and substrate |
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| Ming Wang et al..The convenient fluorescence turn-on detection of heparin with a silole derivative featuring an ammonium group.《chemical communications》.2008,4469-4471. * |
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