CN106932577B - A kind of kit and its detection method with aptamer detection ATP - Google Patents
A kind of kit and its detection method with aptamer detection ATP Download PDFInfo
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- CN106932577B CN106932577B CN201710347343.9A CN201710347343A CN106932577B CN 106932577 B CN106932577 B CN 106932577B CN 201710347343 A CN201710347343 A CN 201710347343A CN 106932577 B CN106932577 B CN 106932577B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
- G01N33/5735—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes co-enzymes or co-factors, e.g. NAD, ATP
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Abstract
The present invention provides a kind of kit and its detection method with aptamer detection ATP.The kit comprising the ATP of sequence aptamer as shown in SEQ ID NO.1 or SEQ ID NO.2 and tetramethylrhodamine (TMR) label.Realize and detect there is the advantages of easy to operate, detection is rapid, high sensitivity, accuracy are high and favorable reproducibility, not vulnerable to fluorescence intensity fluctuations, the influence of fluorescent bleach to ATP the present invention is based on the fluorescence polarization of aptamer (fluorescence anisotropy).Compared with the methods of chromatography, without expensive instrument and equipment, operating procedure is simple, it is only necessary to which simple sample mixing can be measured.
Description
Technical field
The invention belongs to technical field of biological, and in particular to it is a kind of with aptamer detection ATP kit and
Its detection method.
Background technique
Atriphos (ATP) is bioactive molecule important in organism, plays multiple functions.ATP is a kind of high energy
Phosphate cpd realizes energy storage and exoergic by the conversion with adenosine diphosphate (ADP) (ADP), provides energy for the vital movement of cell
Amount.In the physiological function of cell, ATP plays key effect in regulating cell metabolism and bioprocesses.ATP is also participated in
Construct the different physiological roles such as protein molecule, active transport ions and electroneurographic signal conduction.Monitoring ATP may be used to indicate carefully
The activity and cellular damage of born of the same parents.Moreover, the content of ATP is also frequently used to measure activity, the freshness of fish etc. of microorganism.
ATP also has critical function in signal transduction.Therefore, detection ATP molecule basic research and in terms of have it is important
Meaning also has various demands.The common detection method of ATP includes method, chromatography, bio-sensing method based on enzyme reaction
Deng.
Fluorescence polarization (fluorescence polarization) or fluorescence anisotropy (fluorescence
Anisotropy) analysis have the characteristics that favorable reproducibility, it is sensitive, easy to operate, be easy to high throughput analysis.Fluorescence polarization assay/
Fluorescence anisotropy assay excites fluorescent molecule using the exciting light of polarization, measures polarizing emission fluorescence in the glimmering of vertical direction
Luminous intensity I⊥With the fluorescence intensity I of horizontal direction∥, fluorescence polarization value or fluorescence are then calculated respectively to different according to corresponding formula
Property value.Fluorescence polarization value P is equal to (I∥-I⊥)/(I∥+I⊥), and fluorescence anisotropy value r is equal to (I∥-I⊥)/(I∥+2I⊥).It is glimmering
Light polarization and fluorescence anisotropy value are the ratio of not dimension, therefore are not influenced by fluorescence intensity fluctuations, and measurement has very
Good accuracy and reproducibility.It is generally required in fluorescence polarization assay/fluorescence anisotropy assay using fluorochrome label point
Son, fluorochrome label molecule can cause fluorescence polarization signal when molecule rotation changes in interaction of molecules or reaction
With the variation of fluorescence anisotropy signal.Fluorescence polarization assay/fluorescence anisotropy assay intermolecular interaction analyze, face
Bed drug test, drug screening, ligand screening etc. have important application.Common fluorescence polarization/fluorescence anisotropy point
Analysis method detection small molecule generally require using immune antiboidy as identification agent construct detection method, but immune antiboidy prepare,
There is limitations in terms of stability, cost.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of with the kit of aptamer detection ATP and its detection side
Method, to solve above-mentioned at least partly technical problem existing in the prior art.
To achieve the above object, the present invention provides a kind of kit with aptamer detection ATP comprising sequence is such as
The ATP of aptamer shown in SEQ ID NO.1 or SEQ ID NO.2 and tetramethylrhodamine (TMR) label.
The present invention also provides a kind of methods for detecting ATP, are by sequence such as SEQ ID NO.1 or SEQ ID NO.2 institute
The ATP of the aptamer, tetramethylrhodamine label that show is incubated after mixing with sample to be tested, measures the fluorescence of TMR respectively to different
Property changing value or fluorescence polarization changing value.
Compared with prior art, the positive effect of the present invention is that:
The present invention is based on the fluorescence polarization of aptamer (fluorescence anisotropy) to ATP realize detect, be different from
Past traditional method for needing the fluorescence polarization detection small molecule using immune antiboidy as affinity ligand.The system of immune antiboidy
Standby at high cost, stability is poor, poor reproducibility between batch, easy in inactivation.Nucleic acid aptamer sequence can by be chemically synthesized preparation,
Synthesis cost is low, has good thermal stability, and convenient for storage and transport, shelf life is long, not easy in inactivation.Fluorescence polarization assay
Method has the advantages of easy to operate, detection is rapid, high sensitivity, accuracy are high and favorable reproducibility, not vulnerable to fluorescence intensity wave
Dynamic, fluorescent bleach influence.Compared with the methods of chromatography, without expensive instrument and equipment, operating procedure is simple, it is only necessary to simple
Sample mixing can be measured.
Detailed description of the invention
Fig. 1 shows the structural schematic diagram of the ATP molecule of TMR label.
Fig. 2, which is shown in embodiment 1, is based on fluorescence using the ATP that aptamer (AD25 or AD25-10P) and TMR are marked
Anisotropy detection ATP's as a result, it shows the relationship between fluorescence anisotropy signal and ATP concentration, wherein abscissa is ATP
Concentration (unit is μM), ordinate is fluorescence anisotropy value r.
Fig. 3, which is shown in embodiment 1, is based on fluorescence using the ATP that aptamer (AD25 or AD25-10P) and TMR are marked
Polarization Detection ATP's as a result, it shows the relationship between fluorescence polarization signal and ATP concentration, wherein abscissa is that ATP concentration is (single
Position is μM), ordinate is fluorescence polarization value P.
Fig. 4, which is shown in embodiment 2, is based on Media by Fluorescence Anisotropy detection dilution using the ATP that AD25-10P and TMR is marked
ATP's as a result, it shows fluorescence anisotropy variation and the relationship of ATP concentration in urine sample, wherein abscissa is ATP concentration
(unit is μM), ordinate is fluorescence anisotropy value difference DELTA r (by fluorescence anisotropy value difference value corresponding in the presence of ATP
It subtracts the corresponding fluorescence anisotropy value of blank sample that ATP is not present to obtain).
Fig. 5 is shown in comparative example 1 and is detected using the ATP of aptamer AD25 and TMR label based on fluorescence anisotropy
The selective reference result of ATP method, wherein abscissa corresponds to different samples, and ordinate is measured fluorescence anisotropy
Value cuts the corresponding fluorescence anisotropy value of blank sample and obtains the changing value Δ r of fluorescence anisotropy.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in further detail.
Aptamer be from oligonucleotide library screening obtain can the highly selective identification target of high-affinity divide
The single stranded nucleic acid molecule of son.The appearance of aptamer provides new research means for detection ATP.As the affine of nucleic acid
Ligand, application of the aptamer in analysis detection field have lot of advantages, are such as synthetically prepared conveniently, thermal stability is high, easily
In introducing functional group such as fluorochrome label etc..Aptamer can be with immune antiboidy phase in terms of affinity and selectivity
Match in excellence or beauty, the analysis method of aptamer can overcome immune antiboidy in immunoassay stability and in terms of one
A little limitations show potentiality, and the application of aptamer is also widely paid close attention to, and show in analysis sensory field very big
Potentiality and application prospect.
Present inventor is based on the present state of the art, and practical proof by a large amount of Experimental Research and repeatedly is whole
Can have with ATP selectively acting in discovery sequence aptamer as shown in SEQ ID NO.1 or SEQ ID NO.2
Very strong affinity can develop the detection method of detection ATP using aptamer.
The principle of the present invention is: fluorescent dye tetramethylrhodamine (tetramethylrhodamine, TMR) label
ATP molecular volume is small, in the solution quick rotation, has low fluorescence anisotropy (fluorescence anisotropy)
With fluorescence polarization (fluorescence polarization) value, when the nucleic acid of the ATP and DNA of tetramethylrhodamine label are suitable
When ligand binding, become since molecular volume increases with intermolecular interaction, the rotation of the ATP of tetramethylrhodamine label
Slowly, high fluorescence polarization value (fluorescence anisotropy value) is generated.When ATP molecule to be measured is added in solution, ATP marks TMR
The ATP of note is competed from aptamer compound and is replaced, and fluorescence polarization (fluorescence anisotropy) value measured reduces, with
The increase of ATP concentration, fluorescence polarization signal (fluorescence anisotropy) be gradually reduced until reaching plateau, in solution TMR mark
The ATP molecule overwhelming majority of note becomes unbound state, and the detection to ATP may be implemented according to the variation of signal.Because of the class of ATP
Can also be in conjunction with aptamer like object (adenosine, AMP, ADP), this method can be also used for the analog gland of detection ATP
Glycosides, AMP and ADP.In the presence of other nucleic acid molecules such as GTP, CTP, TTP and its corresponding base molecule etc., letter cannot be generated
Number variation.Present inventor has found that the sequence by extending aptamers can make the molecular volume of aptamers under study for action
Become larger, if extending the sequence of each 10 bases at sequence aptamers sequence both ends as shown in SEQ ID NO.1, when TMR is marked
ATP it is in connection when can produce higher fluorescence anisotropy, can make in the presence of ATP ATP generate fluorescence anisotropy
Variation is bigger, and detection limit can reach 0.2 μM.
Kit provided by the invention with aptamer detection ATP comprising sequence such as SEQ ID NO.1 or SEQ
The ATP of aptamer shown in ID NO.2 and tetramethylrhodamine (TMR) label.
Wherein, the ATP that the TMR is marked refers to TMR dye marker to the phosphate terminal of ATP molecule.
Preferably, the kit further includes ATP molecule as positive control.
Preferably, the kit further includes the buffer that pH is 6~10, it is highly preferred that the pH of the buffer is
7.5 including 50mM Tris-HCl and 50mM MgCl2。
It is provided by the invention detection ATP method, be by sequence as shown in SEQ ID NO.1 or SEQ ID NO.2
The ATP that aptamer, tetramethylrhodamine mark is incubated after mixing with sample to be tested, and the fluorescence anisotropy for measuring TMR becomes
Change value or fluorescence polarization changing value.
Wherein it is preferred to which the use concentration of the aptamer is 5 μM.
Preferably, the use concentration of the ATP of the tetramethylrhodamine label is 10nM.
Preferably, the sample to be tested is pre-processed, i.e., uses pH dilute for 6~10 buffer progress sample to be tested
It releases.
Preferably, the temperature of the incubation is 20~30 DEG C, it is highly preferred that the temperature of the incubation is 25 DEG C.
Preferably, the incubation is carried out in the buffer that pH is 6~10, it is highly preferred that the pH of the buffer is
7.5, including 50mM Tris-HCl and 50mM MgCl2。
Preferably, the fluorescence anisotropy changing value of the measurement TMR or fluorescence polarization changing value are measured at 10 DEG C.
Preferably, the fluorescence anisotropy changing value of the measurement TMR or the testing conditions of fluorescence polarization changing value are: swashing
Hair wavelength is 555nm, launch wavelength 580nm.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
Some specific embodiments are set forth below, to be further described to implementation of the invention and technical effect.
In following specific embodiments, detecting excitation wavelength used is 555nm, launch wavelength 580nm, and narrow peak width is equal
For 5nm.
Nucleic acid aptamer probe is by Sangon Biotech's synthesizing and purifying.
Wherein the ATP of used TMR label refer to TMR dye marker to ATP molecule phosphate terminal, as shown in Figure 1.
Incubation be pH be 7.5 combination buffer in carry out, the combination buffer include 50mM Tris-HCl and
50mM MgCl2。
Embodiment 1: fluorescence anisotropy (fluorescence polarization) is based on using aptamer AD25 or AD25-10P and detects ATP
The aptamer of the present embodiment is sequence nucleic acid molecules (referred to as AD25) or sequence as shown in SEQ ID NO.1
Arrange the nucleic acid molecules (referred to as AD25-10P) as shown in SEQ ID NO.2.
ATP, 5 μM of aptamer AD25 (or AD25-10P) and the ATP of various concentration that 10nM TMR is marked are being tied
It closes and is incubated in buffer solution, (temperature is to survey at 10 DEG C to the fluorescence anisotropy value (r) or fluorescence polarization value (P) for then measuring TMR
It is fixed), as a result as shown in Figures 2 and 3.The sequence of aptamer AD25 is SEQ ID NO.1 (5'-CCT GGG GGA GTA
TTG CGG AGG AAG G-3').The sequence of aptamer AD25-10P is SEQ ID NO.2 (5'-ACT CAG CCG
GCC TGG GGG AGT ATT GCG GAG GAA GGC CGG CTG AGT-3')。
From figure 2 it can be seen that fluorescence anisotropy value (r) is gradually decreased with the increase of the concentration of ATP.ATP passes through
The variation of fluorescence anisotropy value and measure, detection limit reaches 0.5 μM.When using AD25-10P, the variation of fluorescence anisotropy
Amplitude is bigger, and detection limit is up to 0.2 μM.Fig. 3 shows the change that fluorescence polarization value (P) is gradually decreased as ATP concentration increases
Change situation.
Embodiment 2: the ATP diluted in urine sample is detected based on fluorescence anisotropy using aptamers AD25-10P
Buffer solution is combined to dilute in use with the ATP of various concentration ATP, 5 μM of AD25-10P that 10nM TMR is marked
It is incubated in 20 times of urine sample, then measures the fluorescence anisotropy value (r) (temperature is to measure at 10 DEG C) of TMR, cut blank sample
The corresponding fluorescence anisotropy value of product obtains the changing value Δ r of fluorescence anisotropy.The result shows that the ATP in dilution urine sample can
To be detected, the relationship that the changing value Δ r of fluorescence anisotropy changes with ATP concentration is referring to fig. 4.
Comparative example 1: it is investigated using aptamer AD25 based on the selectivity of fluorescence anisotropy detection ATP method
ATP, 5 μM of aptamer AD25 and 200 μM of the determinand that 10nM TMR is marked are in combining buffer solution
It incubates, then measures the fluorescence anisotropy value (r) (temperature is to measure at 10 DEG C) of TMR, cut the corresponding fluorescence of blank sample
Anisotropy value obtains the changing value Δ r of fluorescence anisotropy, as a result as shown in Figure 5.
Wherein, a is blank sample, and b is the sample added with ATP, and c is the sample added with guanosine, and d is added with uridine
Sample, e be the sample added with cytidine, f be the sample added with thymidine, g be the sample added with uridine triphosphate UTP, h
For the sample added with cytidine CTP, i is the sample added with guanosine triphosphate GTP, and j is the sample added with tryptophan
Product, k are the sample added with methionine.From fig. 5, it can be seen that only ATP can be such that fluorescence anisotropy signal is substantially reduced,
Remaining measured object can not make fluorescence anisotropy that significant change occur.
The result of above-described embodiment and comparative example explanation, the present invention detection aptamer and detection method used are to mesh
The response high specificity of molecule is marked, detection accuracy is high, and detection limit is low.
In conclusion the direct detection to ATP may be implemented in aptamer provided by the invention, it can be in detection body
It is specifically combined in system with ATP, dissociation constant has reached a μM level.It can be developed using aptamer and be adapted to based on nucleic acid
The method of physical examination survey ATP.Aptamer is readily synthesized that preparation, synthesis cost is low, thermal stability is high, has various excellent
Gesture has boundless application prospect in analysis detection field.
Particular embodiments described above has carried out further in detail the purpose of the present invention, technical scheme and beneficial effects
Describe in detail bright, it should be understood that the above is only a specific embodiment of the present invention, is not intended to restrict the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in protection of the invention
Within the scope of.
Sequence table
<110>Ecological Environment Research Center, Chinese Academy of Sciences
<120>a kind of kit and its detection method with aptamer detection ATP
<130> IB177075
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>artificial sequence
<220>
<223> AD25
<400> 1
cctgggggag tattgcggag gaagg 25
<210> 2
<211> 45
<212> DNA
<213>artificial sequence
<220>
<223> AD25-10P
<400> 2
actcagccgg cctgggggag tattgcggag gaaggccggc tgagt 45
Claims (13)
1. a kind of kit with aptamer detection ATP comprising sequence such as SEQ ID NO.1 or SEQ ID NO.2 institute
The ATP of aptamer and the tetramethylrhodamine label shown.
2. kit according to claim 1, which is characterized in that the kit further includes ATP molecule as positive right
According to.
3. kit according to claim 1, which is characterized in that the kit further includes the buffer that pH is 6~10.
4. kit according to claim 3, which is characterized in that the pH value of the buffer is 7.5, including 50mM
Tris-HCl and 50mM MgCl2。
5. it is a kind of detect ATP method, be by sequence aptamer as shown in SEQ ID NO.1 or SEQ ID NO.2,
The ATP of tetramethylrhodamine label is incubated after mixing with sample to be tested, and fluorescence anisotropy changing value or the fluorescence for measuring TMR are inclined
Shake changing value.
6. according to the method described in claim 5, it is characterized in that, the use concentration of the aptamer is 5 μM.
7. according to the method described in claim 5, it is characterized in that, the use concentration of the ATP of tetramethylrhodamine label
For 10nM.
8. according to the method described in claim 5, it is characterized in that, the sample to be tested is pre-processed, i.e., by sample to be tested
PH is used to be diluted for 6~10 buffer.
9. according to the method described in claim 5, it is characterized in that, the temperature of the incubation is 20~30 DEG C.
10. according to the method described in claim 9, it is characterized in that, the temperature of the incubation is 25 DEG C.
11. according to the method described in claim 5, it is characterized in that, it is described incubation be pH be 6~10 buffer in into
Row.
12. according to the method for claim 11, which is characterized in that the pH value of the buffer is 7.5, including
50mMTris-HCl and 50mM MgCl2。
13. according to the method described in claim 5, it is characterized in that, the fluorescence anisotropy changing value or glimmering of the measurement TMR
Light polarization changing value is measured at 10 DEG C；
The fluorescence anisotropy changing value of the measurement TMR or the testing conditions of fluorescence polarization changing value are: excitation wavelength is
555nm, launch wavelength 580nm.
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| CN116400066A (en) * | 2019-12-11 | 2023-07-07 | 北京聚树生物科技有限公司 | Method and kit for detecting concentration of target molecules in mixed system |
| CN113109305B (en) * | 2021-03-26 | 2023-04-25 | 南京邮电大学 | Method for detecting ATP (adenosine triphosphate) based on split type aptamer and thioflavin T |
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| CN103575713B (en) * | 2013-11-01 | 2016-01-20 | 山西大学 | The method of Ochratoxin A is detected based on aptamer fluorescence anisotropy |
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