CN109507168A - Active biosensor of a kind of detection ATP and the preparation method and application thereof - Google Patents
Active biosensor of a kind of detection ATP and the preparation method and application thereof Download PDFInfo
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- CN109507168A CN109507168A CN201811568677.XA CN201811568677A CN109507168A CN 109507168 A CN109507168 A CN 109507168A CN 201811568677 A CN201811568677 A CN 201811568677A CN 109507168 A CN109507168 A CN 109507168A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
Abstract
The present invention relates to biosensor technology fields, in particular to reunited based on nanogold particle and generate Surface enhanced Raman scattering detection ATP (atriphos) active biosensor, the characteristic that can be specifically bound with its aptamer (Aptamer) using ATP, the Walker Chain on nanogold particle surface is discharged, to Track Chain catalyzing hydrolysis using nuclease that nanometer gold surface is shorter, nanogold particle is caused to lose nucleic acid chains protection, reunite in high-salt buffer, generate surface plasmon resonance effect, greatly enhancing gold nano grain surface electromagnetic field intensity, make to mark and generates Surface enhanced Raman scattering (SERS) effect in the Raman dyestuff on nanogold particle surface, there is Raman spectrum in specific position；When ATP is not present in reaction solution, Protect Chain can not be replaced, so that the reaction of subsequent nanogold reunion can not be carried out, and then not have raman scattering spectrum generation；Nucleic acid chains of the present invention by modification in nanogold particle surface, which do substrate, realizes quick, sensitive, safe ATP Activity determination.
Description
Technical field
The present invention relates to biosensor technology fields, in particular to are reunited based on nanogold particle and generate surface enhanced drawing
Graceful scattering detection ATP (atriphos) active biosensor and the preparation method and application thereof.
Background technique
Atriphos (Adenosine Triphosphate, ATP) is made of adenine (A) and three phosphates, is changed
Formula is C10H16N5O13P3, molecular weight 507.184.It is present in from microorganism into all organisms of high animals and plants,
It is the direct sources of various vital movement energy.But the content of ATP in vivo itself is not high, in cell its mole it is dense
Degree is usually 1-10 mM.Main function is to provide energy to ATP in the cell, participates in the metabolism of fat, protein, sugar and nucleic acid,
Whether there is or not alternative effects in the normal function for maintaining organism.ATP is the significant energy component of living cells, dead cell
ATP cannot be measured, the content by measuring intracellular ATP can not only observe the state of cell, can also observe outside difference
Effect of boundary's factor to cell.ATP is quickly and accurately measured, for studying the physiological activity of cell or even body and being metabolized
Journey, progress drug sensitivity assay and food hygiene monitoring have very important meaning.In addition, ATP clinically also has extensively
General application, for treating the auxiliary enzyme drug of the diseases such as muscular atrophy, apoplexy sequela, myocardosis and hepatitis, ATP's
Detection is widely used in including the screening of tumour medicine sensibility, Apoptosis detection, a variety of important cells activity and microorganism
In terms of content analysis.Therefore, develop the novel detection method of ATP have for medical clinic applications and organism metabolism level it is important
Meaning.
Traditional ATP detection method has electrophoresis, high performance liquid chromatography, tracer method, biloluminescence method and change
Learn luminescence method.In electrophoresis technique determining ATP, sample need to be separated by electrophoresis, and ultraviolet specrophotometer is recycled to carry out colorimetric, operation
It is more complicated；Cumbersome separation process, instrument and expensive reagents are needed using high performance liquid chromatography, detection time is long, detection
Device is mostly ultraviolet detection, and sensitivity is limited, and micro ATP is difficult accurately to detect.Tracer method has simple, sensitive excellent
Point, but radioactive isotope influences the health of experimental implementation person, it is big using difficulty.
Although chemoluminescence method and biloluminescence method have very high measurement accuracy, the detection that can satisfy different range is wanted
It asks, but its stability and specificity are the problem of always existing.Develop performance to stablize, specificity and the preferable ATP of sensitivity
Detection method is of great significance.
In recent years, aptamer sensor because of its high stability, high sensitivity and is easy quantitative feature and has attracted people
Extensive interest, the ATP aptamers nucleic acid probe detection method largely based on fluorescence and colorimetric continue to bring out.But it is glimmering
Light method has the defect of anti-light bleaching, colorimetric method, and there are sensitivity differences.
Summary of the invention
The problems such as existing for above traditional approach detection ATP activity, the present invention provides a kind of using nucleic acid chains and
Nanogold particle is substrate, based on Surface enhanced Raman scattering (SERS) technology realize it is sensitive to ATP activity, accurately detect,
The problem of the problem of overcoming the photobleaching of fluorescence method, colorimetric method poor sensitivity.
What the present invention was obtained through the following steps:
A kind of active biosensor of detection ATP, including 3 DNA nucleic acid chains, gold nano grain, buffer, exonucleaseⅢ,
Object ATP；
The DNA nucleic acid chains, title and sequence are as follows:
Walker Chain: 5’---SH-TTTT TTTT TTTT TTTT TTTT TTTT ACCT TCCT TTTTTT CCCC
AGGT---3'；Sequence is as shown in SEQ No.1；
Track Chain: 5' ---SH—TTTTACCTGGGG---3'；Sequence is as shown in SEQ No.2；
Protect Chain: 5'---ACCTGGGGGAGTATTGCGGAGGAAGGT---3'；Sequence is as shown in SEQ No.3.
The HS of the end of the Track Chain nucleic acid chains 5 ' label.
A kind of preparation method of above-mentioned biosensor, comprising the following steps:
(1) by Walker Chain and Protect Chain anneal, double-spiral structure is formed, reaction solution is obtained；
(2) reaction solution and Track Chain for taking step (1) are put into togerther in nano-Au solution, by being constantly increasing salt
Concentration makes the HS- of the end of Track Chain nucleic acid chains 5 ' label form Au-S key with Au, so that the stable label of nucleic acid chains be existed
The nanogold particle solution of nucleic acid chains has been modified on the surface of gold nano grain；
(3) in 10 × Buffer buffer, the solution that step (2) obtain is added, adds exonucleaseⅢ, object is added
ATP；Sample mixing, which is placed in 37 DEG C of warm bath, reacts；
(4) after the reaction was completed, it extracts reaction solution and is placed in progress Raman spectrum detection and analysis above silicon wafer, obtain corresponding Raman and refer to
Line map.
The concentration ratio 1:1 of Walker Chain and Protect Chain in the step (1).
The annealing conditions of the step (1) are as follows: put 95 DEG C of water-baths 5 minutes after mixing both the above component, turn off electricity
Source, naturally cools to room temperature, takes out reaction solution, put -20 DEG C it is spare.
The concentration of reaction solution and Track Chain ratio are 20:1 in the step (2).
The concentration ratio of nanogold and Track Chain nucleic acid chains is 1:3000 in the step (2).
Salt is NaCl, final concentration of 0.3 M in (2).
Application of the above-mentioned biosensor in drug sensitivity assay.
According to the corresponding aptamer of ATP, the means of partial hybridization design Walker Chain；Then, exist
The aptamers (Aptamer) of 3 ' the end sections hybridization ATP of Walker Chain, i.e. Protect Chain；Finally, according to mesh
Mark object can be with partial hybridization and can be by excision enzyme by the 3 ' ends of Walker Chain for protecting chain to discharge after competing
III catalyzing hydrolysis devises Track Chain.
In order to reach the detection active purpose of ATP, the present invention can be with its aptamer (Aptamer) spy using ATP
The characteristic that the opposite sex combines discharges the Walker Chain on nanogold particle surface, thus using nuclease by nanometer gold surface
Shorter Track Chain catalyzing hydrolysis causes nanogold particle to lose nucleic acid chains protection, reunites in high-salt buffer,
Surface plasmon resonance effect is generated, gold nano grain surface electromagnetic field intensity is greatly enhanced, makes label in nanogold
The Raman dyestuff on grain surface generates Surface enhanced Raman scattering (SERS) effect, Raman spectrum occurs in specific position；When anti-
It answers in liquid there is no when ATP, Protect Chain can not be replaced, so that subsequent nanogold reunion can not be carried out
Reaction, and then do not have raman scattering spectrum generation.
Beneficial effects of the present invention:
Nucleic acid chains of the present invention by modification in nanogold particle surface, which do substrate, realizes quick, sensitive, safe ATP activity
Detection has the following advantages with ATP activity test method comparison before:
(1) high sensitivity: reuniting by nanogold enhances SERS signal, realizes super sensitivity detection ATP activity；
(2) detection is fast: without complicated sample pre-treatments step, substantially reducing detection time；Raman spectral signal can be straight
It connects to measure on Raman spectrometer and, be not necessarily to separation product；
(3) unglazed to drift: the problem of selection only has the dye marker of Raman fingerprint, avoids fluorescence photobleaching, it is effective to improve
Signal-to-background ratio.
Detailed description of the invention
Fig. 1 is the schematic diagram of the invention；
Fig. 2 is different ATP standard sample concentration and raman scattering intensity graph of relation；
Fig. 3 is sensitivity technique histogram of the invention.
Specific embodiment
The technical solution of the present invention is further described in detail with reference to the accompanying drawings and embodiments.Following instance is not constituted pair
Restriction of the invention.
Fig. 1 is the principle of the present invention figure.(nanogold particle surface modification nucleic acid chains and Raman dye as shown in Figure 1, substrate
Material), 37 DEG C of warm bath of exonucleaseⅢ and object ATP mixed liquor after sixty minutes, nanogold particle lose nucleic acid chains protection group
Poly-, generating Raman enhances signal.The concentration of object ATP is determined by the intensity of Raman signal.
The pure ATP Concentration Testing of embodiment 1
1. modifying Raman dyestuff and nucleic acid chains to gold nanoparticle surface:
A, it takes 1 mL nano-Au solution in centrifuge tube, is centrifuged 10 min, while it is spare to be centrifuged two pipes.Centrifugation to supernatant without
Color is transparent, removes supernatant, and 300 μ L aqua sterilisas, which are added, makes nano-Au solution be concentrated into 3 nM.It moves into 1 mL vial, uses
The Raman dyestuff (4-NTP) that 12 ul concentration are 0.25 nM is added in masking foil sealing.
B, after being placed at room temperature for 30 min, the hybridization solution that 150 μ L(Walker Chain/Protect Chain are added is same
Track Chain concentration ratio is 20:1) 30 μM of the modification nucleic acid chains of-SH place 24 h after mixing at 4 DEG C.
C, it is slowly added to 50 μ L PB buffers several times, magneton (the previous day is impregnated with chloroazotic acid) is added and stirs 10 min
Afterwards, 27 μ L PBS buffer solution are continuously added.Take out magneton, 4 DEG C of 48 h of placement.
D, the nano-Au solution marked is transferred in centrifuge tube, aqua sterilisa is added to 1 mL, is centrifuged 10 min, goes
Except supernatant.The centrifugation of 1 mL aqua sterilisa is added, this process is repeated twice (in order to elute the nucleic acid chains on unmarked).
2. reaction process in homogeneous phase solution:
A, it by the ATP solution of various concentration, is added containing 2 uL exonuclease III(2 U/mL), 2 uL10 × Buffer
(10 mM Tris-HCl,50 μM NaCl,10 mM MgCl2, 1 mM DTT, pH 7.9), 10 uL modified nucleic acid chains and
(add water polishing to the reaction system of 20 uL) in the nano-Au solution of Raman dyestuff, shakes 30 s, be put into 37 DEG C of water-bath
60 min of middle reaction, do the reaction system of 6 difference UDG concentration altogether；In addition it does a control tube: being added without in reaction system
Object ATP.
B, after 60 min, mixed solution is taken out from water-bath, observes color change, and detected with Raman spectrometer,
Obtain the SERS spectra of seven groups of positive samples and blank sample.According to a series of obtained spectrum, different spectrum peaks is obtained
It is as shown in Figure 2 to calculate matched curve for value.It is the corresponding Raman finger-print of ATP of various concentration in standard sample in Fig. 2 (A)；
It is Raman fingerprint image in 1327 cm in Fig. 2 (B)-1Locate intensity with the calibration curve between ATP concentration.
The sensitivity of 2 present invention detection ATP method of embodiment
1. modifying Raman dyestuff and nucleic acid chains to gold nanoparticle surface:
Same as Example 1, reaction substrate is still the nanogold particle solution of above-mentioned label end nucleic acid.
2. homogeneous phase solution medium sensitivity detects
Respectively by (ATP/ADP/GTP/CTP) solution of 2 uL, it is added containing 2 uL exonuclease III(2 U/mL), 2
uL10×Buffer (10 mM Tris-HCl,50 μM NaCl,10 mM MgCl2, 1 mM DTT, pH 7.9)、10 uL
It has modified in the nano-Au solution of nucleic acid chains and Raman dyestuff and (has added water polishing to the reaction system of 20 uL), in addition a blank pair
Product (blank) in the same old way;30 s are shaken, is put into 37 DEG C of water-bath and reacts 60 min.
After 60 min, mixed solution is taken out from water-bath, observes color change, and detected with Raman spectrometer, is obtained each
The SERS spectra of group sample.According to a series of obtained spectrum peaks, it is as shown in Figure 3 to draw peak strength.ATP is only added
When, stronger Raman signal can be measured, and in blank and other samples, measure very low Raman signal, it follows that,
The present invention has preferable sensitivity.
Sequence table
<110>University Of Ji'nan
<120>active biosensor of a kind of detection ATP and the preparation method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 46
<212> DNA
<213>artificial sequence (artiartificial sequence)
<400> 1
tttttttttt tttttttttt ttttaccttc cttttttcc ccaggt 45
<210> 2
<211> 12
<212> DNA
<213>artificial sequence (artiartificial sequence)
<400> 2
ttttacctgg gg 12
<210> 3
<211> 27
<212> DNA
<213>artificial sequence (artiartificial sequence)
<400> 3
acctggggga gtattgcgga ggaaggt 27
Claims (9)
1. a kind of active biosensor of detection ATP, which is characterized in that including 3 DNA nucleic acid chains, gold nano grain, bufferings
Liquid, exonucleaseⅢ, object ATP；
The DNA nucleic acid chains, title and sequence are as follows:
Walker Chain sequence is as shown in SEQ No.1；
Track Chain sequence is as shown in SEQ No.2；
Protect Chain sequence is as shown in SEQ No.3.
2. biosensor according to claim 1, which is characterized in that the Track Chain nucleic acid chains 5 ' hold mark
The HS of note.
3. a kind of preparation method of biosensor described in claim 1, which comprises the following steps:
(1) by Walker Chain and Protect Chain anneal, double-spiral structure is formed, reaction solution is obtained；
(2) reaction solution and Track Chain for taking step (1) are put into togerther in nano-Au solution, by being constantly increasing salt
Concentration makes the HS- of the end of Track Chain nucleic acid chains 5 ' label form Au-S key with Au, so that the stable label of nucleic acid chains be existed
The nanogold particle solution of nucleic acid chains has been modified on the surface of gold nano grain；
(3) in 10 × Buffer buffer, the solution that step (2) obtain is added, adds exonucleaseⅢ, object is added
ATP；Sample mixing, which is placed in 37 DEG C of warm bath, reacts；
(4) after the reaction was completed, it extracts reaction solution and is placed in progress Raman spectrum detection and analysis above silicon wafer, obtain corresponding Raman and refer to
Line map.
4. preparation method according to claim 3, which is characterized in that in the step (1) Walker Chain and
The concentration ratio 1:1 of Protect Chain.
5. preparation method according to claim 3, which is characterized in that the annealing conditions of the step (1) are as follows: will more than
Put 95 DEG C of water-baths 5 minutes after the mixing of two kinds of components, power off, naturally cool to room temperature, take out reaction solution, put -20 DEG C it is spare.
6. preparation method according to claim 3, which is characterized in that reaction solution and Track in the step (2)
The concentration ratio of Chain is 20:1.
7. preparation method according to claim 3, which is characterized in that nanogold and Track in the step (2)
The concentration ratio of Chain nucleic acid chains is 1:3000.
8. preparation method according to claim 3, which is characterized in that salt is NaCl in (2), final concentration of
0.3 M。
9. application of the biosensor described in claim 1 in drug sensitivity assay.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811568677.XA CN109507168A (en) | 2018-12-21 | 2018-12-21 | Active biosensor of a kind of detection ATP and the preparation method and application thereof |
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| CN201811568677.XA CN109507168A (en) | 2018-12-21 | 2018-12-21 | Active biosensor of a kind of detection ATP and the preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111440850A (en) * | 2020-04-09 | 2020-07-24 | 济南大学 | Chemiluminescent sensor for detecting acetamiprid and preparation method thereof |
| CN112557369A (en) * | 2020-11-30 | 2021-03-26 | 崔艳芳 | Biosensor for detecting microRNA-21 and preparation method and application thereof |
| CN113293197A (en) * | 2021-05-07 | 2021-08-24 | 南京邮电大学 | SPR-SERS dual-mode sensor for detecting disease nucleic acid marker, preparation method and application thereof |
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| CN105510420A (en) * | 2015-12-20 | 2016-04-20 | 青岛科技大学 | Method for determining ATP content on basis of magnetic bead separation and DNA marker gold nanoparticle probe |
| CN108169203A (en) * | 2017-12-14 | 2018-06-15 | 济南大学 | A kind of biosensor for detecting hOGG1 activity and its application |
| CN108872582A (en) * | 2018-03-20 | 2018-11-23 | 安徽师范大学 | A kind of aptamer sensor based on DNAWalker, preparation method and applications |
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2018
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| CN105510420A (en) * | 2015-12-20 | 2016-04-20 | 青岛科技大学 | Method for determining ATP content on basis of magnetic bead separation and DNA marker gold nanoparticle probe |
| CN108169203A (en) * | 2017-12-14 | 2018-06-15 | 济南大学 | A kind of biosensor for detecting hOGG1 activity and its application |
| CN108872582A (en) * | 2018-03-20 | 2018-11-23 | 安徽师范大学 | A kind of aptamer sensor based on DNAWalker, preparation method and applications |
Non-Patent Citations (2)
| Title |
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| SHASHA LU 等: "Enzyme-free aptamer/AuNPs-based fluorometric and colorimetric dual-mode detection for ATP", 《SENSORS AND ACTUATORS B: CHEMICAL》 * |
| XIAOLONG YANG 等: "Enzyme-Powered Three-Dimensional DNA Nanomachine for DNA Walking, Payload Release, and Biosensing", 《ACS NANO》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111440850A (en) * | 2020-04-09 | 2020-07-24 | 济南大学 | Chemiluminescent sensor for detecting acetamiprid and preparation method thereof |
| CN111440850B (en) * | 2020-04-09 | 2022-11-25 | 济南大学 | Chemiluminescence sensor for detecting acetamiprid and preparation method thereof |
| CN112557369A (en) * | 2020-11-30 | 2021-03-26 | 崔艳芳 | Biosensor for detecting microRNA-21 and preparation method and application thereof |
| CN113293197A (en) * | 2021-05-07 | 2021-08-24 | 南京邮电大学 | SPR-SERS dual-mode sensor for detecting disease nucleic acid marker, preparation method and application thereof |
| CN113293197B (en) * | 2021-05-07 | 2023-08-11 | 南京邮电大学 | SPR-SERS dual-mode sensor for detecting disease nucleic acid markers, preparation method and application thereof |
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Application publication date: 20190322 |