CN106932577A - 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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- CN106932577A CN106932577A CN201710347343.9A CN201710347343A CN106932577A CN 106932577 A CN106932577 A CN 106932577A CN 201710347343 A CN201710347343 A CN 201710347343A CN 106932577 A CN106932577 A CN 106932577A
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- G—PHYSICS
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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.Described kit, its ATP for including aptamer and tetramethylrhodamine (TMR) mark of the sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.Fluorescence polarization (fluorescence anisotropy) of the present invention based on aptamer is realized detecting to ATP, and with simple to operate, detection is rapid, sensitivity is high, accuracy advantage high and favorable reproducibility, is difficult to be influenceed by fluorescence intensity fluctuations, fluorescent bleach.Compared with the methods such as chromatogram, 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 technology
Atriphos (ATP) is bioactive molecule important in organism, plays various functions.ATP is a kind of high energy
Phosphate cpd, energy storage and exoergic are realized by the conversion with adenosine diphosphate (ADP) (ADP), for the vital movement of cell provides energy
Amount.In the physiological function of cell, ATP plays key effect in regulating cell metabolism and bioprocesses.ATP is also participated in
Build 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.And, the content of ATP is also frequently used to determine activity, freshness of fish of microorganism etc..
ATP also has critical function in signal transduction.Therefore, detection ATP molecules have important at the aspect such as basic research and production practices
Meaning, also there is many demands.The conventional 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 favorable reproducibility, it is sensitive, simple to operate, the features such as be easy to high throughput analysis.Fluorescence polarization assay/
Fluorescence anisotropy assay excites fluorescence molecule using the exciting light of polarization, determines polarizing emission fluorescence in the glimmering of vertical direction
Luminous intensity I⊥With the fluorescence intensity I of horizontal direction∥, fluorescence polarization value is then calculated according to corresponding formula or fluorescence is each to different
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 without dimension, therefore are not influenceed by fluorescence intensity fluctuations, and determining has very
Good accuracy and reappearance.Generally required in fluorescence polarization assay/fluorescence anisotropy assay using fluorochrome label point
Son, fluorochrome label molecule molecule in interaction of molecules or reaction to be rotated and can cause fluorescence polarization signal when changing
With the change of fluorescence anisotropy signal.Fluorescence polarization assay/fluorescence anisotropy assay intermolecular interaction analyze, face
The aspects such as bed drug test, drug screening, part examination have important application.Conventional fluorescence polarization/fluorescence anisotropy point
Analysis method detection small molecule generally require using immune antiboidy as identification agent build detection method, but immune antiboidy prepare,
Stability, cost aspect have limitation.
The content of the invention
In view of this, it is an object of the invention to provide a kind of with the kit of aptamer detection ATP and its detection side
Method, to solve at least part of technical problem present in above-mentioned prior art.
To achieve the above object, the present invention provides a kind of kit that ATP is detected with aptamer, and it includes sequence such as
The ATP of aptamer and tetramethylrhodamine (TMR) mark shown in SEQ ID NO.1 or SEQ ID NO.2.
The present invention also provides a kind of method of detection ATP, and it is by sequence such as SEQ ID NO.1 or SEQ ID NO.2 institutes
The aptamer that shows, the ATP of tetramethylrhodamine mark are incubated after mix with sample to be tested, and the fluorescence of measure TMR is respectively to different
Property changing value or fluorescence polarization changing value.
Compared with prior art, positive effect of the invention is：
The present invention based on aptamer fluorescence polarization (fluorescence anisotropy) to ATP realize detect, its be different from
Toward tradition the need for using immune antiboidy as affinity ligand fluorescence polarization detect small molecule method.The system of immune antiboidy
Standby high cost, stability is poor, poor reproducibility, easy in inactivation between batch.Nucleic acid aptamer sequence can by be chemically synthesized preparation,
Synthesis low cost, with good heat endurance, be easy to store and transport, shelf life is long, not easy in inactivation.Fluorescence polarization assay
Method has simple to operate, and detection is rapid, sensitivity is high, accuracy advantage high and favorable reproducibility, is difficult by fluorescence intensity ripple
The dynamic, influence of fluorescent bleach.Compared with the methods such as chromatogram, without expensive instrument and equipment, operating procedure is simple, it is only necessary to simple
Sample mixing can be measured.
Brief description of the drawings
The structural representation of the ATP molecules of Fig. 1 display TMR marks.
The ATP marked using aptamer (AD25 or AD25-10P) and TMR in Fig. 2 display embodiments 1 is based on fluorescence
Anisotropy detects the result of ATP, and the relation between its display fluorescence anisotropy signal and ATP concentration, wherein abscissa are ATP
Concentration (unit for μM), ordinate is fluorescence anisotropy value r.
The ATP marked using aptamer (AD25 or AD25-10P) and TMR in Fig. 3 display embodiments 1 is based on fluorescence
The result of Polarization Detection ATP, the wherein relation between its display fluorescence polarization signal and ATP concentration, abscissa are (single ATP concentration
Position for μM), ordinate be fluorescence polarization value P.
The ATP marked using AD25-10P and TMR in Fig. 4 display embodiments 2 is based on Media by Fluorescence Anisotropy and detects dilution
The relation of the result of the ATP in urine sample, its display fluorescence anisotropy change and ATP concentration, wherein abscissa is ATP concentration
(unit for μM), ordinate is fluorescence anisotropy value difference DELTA r (by corresponding fluorescence anisotropy value difference value in the presence of ATP
Subtract the corresponding fluorescence anisotropy of the non-existent blank samples of ATP to be worth to).
Detected based on fluorescence anisotropy using the ATP of aptamer AD25 and TMR mark in Fig. 5 display comparisons example 1
The different sample of the selective reference result of ATP methods, wherein abscissa correspondence, ordinate is measured fluorescence anisotropy
Value cuts the changing value Δ r that the corresponding fluorescence anisotropy of blank sample is worth to fluorescence anisotropy.
Specific embodiment
To make the object, technical solutions and advantages of the present invention become more apparent, below in conjunction with specific embodiment, and reference
Accompanying drawing, the present invention is described in further detail.
Aptamer be from oligonucleotide library screening obtain can high-affinity high selectivity identification target point
The single stranded nucleic acid molecule of son.The detection ATP that appears as of aptamer provides new research meanses.As the affine of nucleic acid
Part, aptamer has lot of advantages in the application of analysis detection field, such as synthetically prepared convenience, and heat endurance 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 in stability and prepare etc. the one of aspect
A little limitations, show potentiality, and the application of aptamer is also widely paid close attention to, and are shown 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 substantial amounts of Experimental Research and repeatedly is whole
Can have with ATP selectively actings in aptamer of the discovery sequence as shown in SEQ ID NO.1 or SEQ ID NO.2
Very strong affinity, the detection method of detection ATP can be developed using aptamer.
Principle of the invention is：Fluorescent dye tetramethylrhodamine (tetramethylrhodamine, TMR) mark
ATP molecular volumes are small, in the solution quick rotation, with low fluorescence anisotropy (fluorescence anisotropy)
With fluorescence polarization (fluorescence polarization) value, when the nucleic acid of ATP and the DNA of tetramethylrhodamine mark is fitted
During ligand binding, due to molecular volume increase and intermolecular interaction, the rotation of the ATP of tetramethylrhodamine mark becomes
Slowly, fluorescence polarization value (fluorescence anisotropy value) high is produced.When ATP molecules to be measured are added in solution, ATP marks TMR
The ATP of note is competed from aptamer compound and replaced, fluorescence polarization (fluorescence anisotropy) value reduction for measuring, with
The increase of ATP concentration, fluorescence polarization signal (fluorescence anisotropy) is gradually reduced until reaching plateau, TMR marks in solution
The ATP molecule overwhelming majority of note is changed into unbound state, and the change according to signal can realize the detection to ATP.Because of the class of ATP
Can also be combined with aptamer like thing (adenosine, AMP, ADP), this method can be also used for detecting the analog gland of ATP
Glycosides, AMP and ADP.In the presence of other nucleic acid molecules such as GTP, CTP, TTP, and its corresponding base molecule etc., it is impossible to produce letter
Number change.Present inventor had found under study for action, and the molecular volume of aptamers can be made by the sequence for extending aptamers
Become big, if the aptamers sequence two ends in sequence as shown in SEQ ID NO.1 extend the sequence of each 10 bases, when TMR marks
ATP it is in connection when can produce fluorescence anisotropy higher, can make in the presence of ATP ATP produce fluorescence anisotropy
Change is bigger, and test limit can reach 0.2 μM.
The kit that ATP is detected with aptamer that the present invention is provided, it includes sequence such as SEQ ID NO.1 or SEQ
The ATP of aptamer and tetramethylrhodamine (TMR) mark shown in ID NO.2.
Wherein, the ATP of the TMR marks refers to phosphate terminal of the TMR dye markers to ATP molecules.
Preferably, the kit also includes ATP molecules as positive control.
Preferably, the kit is also including the buffer solution that pH is 6~10, it is highly preferred that the pH of the buffer solution is
7.5, including 50mM Tris-HCl and 50mM MgCl2。
The present invention provide detection ATP method, its be by sequence as shown in SEQ ID NO.1 or SEQ ID NO.2
Aptamer, the ATP of tetramethylrhodamine mark are incubated after mixing with sample to be tested, and the fluorescence anisotropy for determining TMR becomes
Change value or fluorescence polarization changing value.
Wherein it is preferred to, the concentration of the aptamer is 5 μM.
Preferably, the concentration of the ATP of the tetramethylrhodamine mark is 10nM.
Preferably, the sample to be tested is pre-processed, will sample to be tested to use pH to be carried out for 6~10 buffer solution dilute
Release.
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, described incubation is carried out in the buffer solution that pH is 6~10, it is highly preferred that the pH of the buffer solution is
7.5, including 50mM Tris-HCl and 50mM MgCl2。
Preferably, the fluorescence anisotropy changing value or fluorescence polarization changing value of the measure TMR are determined at 10 DEG C.
Preferably, the fluorescence anisotropy changing value of the measure TMR or the testing conditions of fluorescence polarization changing value are：Swash
Hair wavelength is 555nm, and launch wavelength is 580nm.
On the basis of common sense in the field is met, above-mentioned each optimum condition can be combined, and obtain final product each preferable reality of the present invention
Example.
Agents useful for same of the present invention and raw material are commercially available.
Some specific embodiments are set forth below, are further described with to implementation of the invention and technique effect.
In following specific embodiments, detection excitation wavelength used is 555nm, and launch wavelength is 580nm, and narrow peak width is equal
It is 5nm.
Nucleic acid aptamer probe is by Sangon Biotech's synthesizing and purifying.
The ATP of the TMR marks used in it refers to phosphate terminal of the TMR dye markers to ATP molecules, as shown in Figure 1.
Incubation is carried out in the combination buffer that pH is 7.5, 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 nucleic acid molecules (referred to as AD25) or sequence of the 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.
The ATP of ATP, 5 μM of aptamer AD25 (or AD25-10P) and various concentrations that 10nM TMR are marked is in knot
Incubation in cushioning liquid is closed, then (temperature is survey at 10 DEG C to fluorescence anisotropy value (r) or fluorescence polarization value (P) of measure 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 reduced with the increase of the concentration of ATP.ATP passes through
The change of fluorescence anisotropy value and determine, test limit reaches 0.5 μM.During using AD25-10P, the change of fluorescence anisotropy
Amplitude is bigger, and test limit is up to 0.2 μM.The change that Fig. 3 shows fluorescence polarization value (P) gradually to be reduced as ATP concentration increases
Change situation.
Embodiment 2：ATP in fluorescence anisotropy detection dilution urine sample is based on using aptamers AD25-10P
The ATP of ATP, 5 μM of AD25-10P and various concentrations that 10nM TMR are marked is using combination cushioning liquid dilution
Incubated in 20 times of urine sample, then determine fluorescence anisotropy value (r) (temperature is measure at 10 DEG C) of TMR, cut blank sample
The corresponding fluorescence anisotropy of product is worth to the changing value Δ r of fluorescence anisotropy.Result shows that the ATP in dilution urine sample can
To be detected, the changing value Δ r of fluorescence anisotropy is with the relation of ATP change in concentration referring to Fig. 4.
Comparative example 1：The selectivity for being based on fluorescence anisotropy detection ATP methods using aptamer AD25 is investigated
ATP, 5 μM of aptamer AD25 that 10nM TMR are marked and 200 μM of determinand are in cushioning liquid is combined
Incubate, then determine fluorescence anisotropy value (r) (temperature is measure at 10 DEG C) of TMR, cut the corresponding fluorescence of blank sample
Anisotropy is worth to 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 for being added with ATP, and c is the sample for being added with guanosine, and d is to be added with uridine
Sample, e is the sample for being added with cytidine, and f is the sample for being added with thymidine, and g is the sample for being added with uridine triphosphate UTP, h
To be added with the sample of cytidine CTP, i is the sample for being added with GTP GTP, and j is the sample for being added with tryptophan
Product, k is the sample for being added with methionine.From fig. 5, it can be seen that only ATP can be such that the fluorescence anisotropy signal substantially reduces, its
Remaining measured object can not make fluorescence anisotropy that significant change occurs.
The result explanation of above-described embodiment and comparative example, present invention detection aptamer used and detection method are to mesh
The response high specificity of molecule is marked, accuracy of detection is high, and test limit is low.
In sum, the aptamer that the present invention is provided can realize the direct detection to ATP, and it can be in detection body
Specifically combined with ATP in system, dissociation constant has reached a μM level.Can be developed using aptamer and be adapted to based on nucleic acid
The method that ATP is surveyed in physical examination.It is high that aptamer is readily synthesized preparation, synthesis low cost, heat endurance, with many excellent
Gesture, has boundless application prospect in analysis detection field.
Particular embodiments described above, has been carried out further in detail to the purpose of the present invention, technical scheme and beneficial effect
Describe in detail bright, it should be understood that the foregoing is only specific embodiment of the invention, be not intended to limit the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements done etc. 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 (10)
1. it is a kind of with aptamer detect ATP kit, it includes sequence such as SEQ ID NO.1 or SEQ ID NO.2 institutes
The ATP of aptamer and tetramethylrhodamine (TMR) mark for showing.
2. kit according to claim 1, it is characterised in that the kit also includes that ATP molecules are right as the positive
According to.
3. kit according to claim 1, it is characterised in that the kit also including the buffer solution that pH is 6~10,
It is highly preferred that the pH of the buffer solution is 7.5, including 50mM Tris-HCl and 50mM MgCl2。
4. a kind of method of detection ATP, its be aptamer by sequence as shown in SEQ ID NO.1 or SEQ ID NO.2,
The ATP of tetramethylrhodamine mark is incubated after mixing with sample to be tested, and the fluorescence anisotropy changing value or fluorescence for determining TMR are inclined
Shake changing value.
5. method according to claim 4, it is characterised in that the concentration of the aptamer is 5 μM.
6. method according to claim 4, it is characterised in that the concentration of the ATP of the tetramethylrhodamine mark
It is 10nM.
7. method according to claim 4, it is characterised in that the sample to be tested is pre-processed, will sample to be tested
PH is used to be diluted for 6~10 buffer solution.
8. method according to claim 4, it is characterised in that the temperature of the incubation is 20~30 DEG C, it is highly preferred that institute
The temperature for stating incubation is 25 DEG C.
9. method according to claim 4, it is characterised in that the incubation is carried out in the buffer solution that pH is 6~10,
It is highly preferred that the pH of the buffer solution is 7.5, including 50mM Tris-HCl and 50mM MgCl2。
10. method according to claim 4, it is characterised in that the fluorescence anisotropy changing value or glimmering of the measure TMR
Light polarization changing value is determined at 10 DEG C；
The fluorescence anisotropy changing value of the measure TMR or the testing conditions of fluorescence polarization changing value are：Excitation wavelength is
555nm, launch wavelength is 580nm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109709321A (en) * | 2019-01-08 | 2019-05-03 | 中国科学院生态环境研究中心 | A kind of method of enzyme-linked aptamer microwell plate optical analysis small molecule |
| CN112986551A (en) * | 2019-12-11 | 2021-06-18 | 清华大学 | Method and kit for detecting concentration of target molecules in mixed system |
| CN113109305A (en) * | 2021-03-26 | 2021-07-13 | 南京邮电大学 | Method for detecting ATP (adenosine triphosphate) based on split aptamer and thioflavin T |
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| WO2013140629A1 (en) * | 2012-03-23 | 2013-09-26 | Ｎｅｃソフト株式会社 | Atp or amp analysis device and analysis method |
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| WO2013140629A1 (en) * | 2012-03-23 | 2013-09-26 | Ｎｅｃソフト株式会社 | Atp or amp analysis device and analysis method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109709321A (en) * | 2019-01-08 | 2019-05-03 | 中国科学院生态环境研究中心 | A kind of method of enzyme-linked aptamer microwell plate optical analysis small molecule |
| CN112986551A (en) * | 2019-12-11 | 2021-06-18 | 清华大学 | Method and kit for detecting concentration of target molecules in mixed system |
| CN112986551B (en) * | 2019-12-11 | 2022-09-27 | 北京聚树生物科技有限公司 | Method and kit for detecting concentration of target molecules in mixed system |
| CN113109305A (en) * | 2021-03-26 | 2021-07-13 | 南京邮电大学 | Method for detecting ATP (adenosine triphosphate) based on split aptamer and thioflavin T |
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| CN106932577B (en) | 2019-03-15 |
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