CN107976427A - A kind of biological sensor, preparation method and its detection application to copper ion, pyrophosphate and alkaline phosphatase - Google Patents
A kind of biological sensor, preparation method and its detection application to copper ion, pyrophosphate and alkaline phosphatase Download PDFInfo
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- CN107976427A CN107976427A CN201711090704.2A CN201711090704A CN107976427A CN 107976427 A CN107976427 A CN 107976427A CN 201711090704 A CN201711090704 A CN 201711090704A CN 107976427 A CN107976427 A CN 107976427A
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- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The present invention provides a kind of biological sensor, preparation method and its detection application to copper ion, pyrophosphate and alkaline phosphatase.The present invention is formed copper-amine complex, the fluorescence of system is quenched, realized and copper ion is detected based on the amido on copper ion and G tetrad thioflavins；Pyrophosphate is added, complex is formed with copper ion, deprives out copper ion, the fluorescence of system is recovered, realize and pyrophosphate is detected；Alkaline phosphatase is added, pyrophosphate can be hydrolyzed, it is lost complexing power so that system fluorescent quenching, realizes the Sensitive Detection to alkaline phosphatase.Compared with prior art, the preparation method of fluorescent optical sensor provided by the invention, uses unmarked DNA, and easy to operate, cost is very low, avoids any chemical labeling and modification.
Description
Technical field
The invention belongs to biological sensor technical field, and in particular to a kind of biological sensor, preparation method
And its detection application to copper ion, pyrophosphate and alkaline phosphatase., can based on DNA-G tetrad biological sensors
With realize to copper ion, pyrophosphate, alkaline phosphatase Sensitive Detection.
Background technology
Copper ion is the essential trace element of human and animal, and excessive copper ion can induce oxidative stress and cause neural change
Property disease, including Alzheimer disease, Parkinson's and hepatolenticular degeneration.Pyrophosphate is the Key Metabolic in various biochemical processes
Thing.Moreover, many researchs show that pyrophosphate may be used as some index diseases, such as arthritis and pia-arachnitis.As for alkalescence
Phosphatase, a kind of required hydrolase phosphate metabolism are responsible for protein, the dephosphorylation of the small molecule in nucleic acid or biology
Process, in addition ALP abnormal levels may also be related with some diseases, such as the dysfunctions such as dynamic osteopathy, liver, diabetes, breast
Gland cancer and prostate cancer.
It is complicated and time-consuming for being related to the detection in actual sample of copper ion, pyrophosphate, alkaline phosphatase at present more
Interminable detection process, multiple separated sensing strategies of analyte generally require cumbersome sampling, time-consuming pretreatment, and cause
Low accuracy.
Carrying out continuous detection using single sensor has the advantages that potential low cost and more effectively analysis, therefore open
Send out high selectivity, high sensitivity, simple unmarked biological sensor detection copper ion, pyrophosphate, alkaline phosphatase
It is most important.
The content of the invention
The purpose of the present invention is providing a kind of biological sensor and preparation method thereof, based on G- tetrads-thioflavin
(abbreviation ThT) fluorescence probe builds optical chemical biosensor.
Should present invention also offers a kind of detection of biological sensor to copper ion, pyrophosphate and alkaline phosphatase
With.The fluorescence signal of G tetrads-thioflavin is quenched using copper ion, adds pyrophosphate and recovers its fluorescence, add alkaline phosphorus
The fluorescence signal of system is quenched in sour enzyme, builds " off-on-off " sensing platform, copper ion, pyrophosphate, the alkali of various concentrations
Acid phosphatase fluorescence intensity is different, builds linear relationship, realize to copper ion, pyrophosphate, alkaline phosphatase continuity,
Sensitivity and specific detection.
A kind of preparation method of biological sensor provided by the invention, comprises the following steps：
1) DNA sequence dna is dissolved in Tris-HCl buffer solutions, obtains DNA buffer solutions, it is spare；
2), the buffer solution and K for preparing step 1)+Solution is mixed, and forms G- tetrads；
3) the G- tetrads and ThT hybrid reactions for, obtaining step 2), to obtain the final product.
DNA sequence dna is described in step 1)：AGGGTTAGGGTTAGGGTTAGGG；
Step 1) is specially：For the DNA sequence dna of the 2.5OD of purchase is dissolved in Tris-HCl buffer solutions, obtain dense
The DNA buffer solutions for 100 μM are spent, are saved backup at 4 DEG C；
The pH of Tris-HCl buffer solutions described in step 1) are 7.4, concentration 0.05M, magnesium chloride containing 0.05M.
K described in step 2)+Solution concentration is 10mM-50mM.
Step 2) is specially：2 μ L, 100 μM of DNA buffer solutions and 10 μ L 20mM potassium ions are mixed, culture, forms G-
Tetrad.
Mixed culture refers to cultivate 1h-3h at 20 DEG C -50 DEG C described in step 2).
Step 3) is specially：The G- tetrads and 10 μ L 100mM ThT that step 2) is obtained, hybrid reaction.
Hybrid reaction described in step 3) is to react 1min-15min at 20 DEG C -50 DEG C.
The concentration of G- tetrads described in step 3) is 1mM.
The preparation method of biological sensor is specially：
1) DNA sequence dna of purchase is dissolved in 0.05M Tris-HCl (pH 7.4) buffer solution, and is preserved at 4 DEG C
It is spare；DNA：AGGGTTAGGGTTAGGGTTAGGG；
2), by 2 μ L, 100 μM of DNA buffer solutions and 10 μ L 20mMK+Solution cultivates 2h at 37 DEG C, forms G- tetra- and connects
Body；
3) the G- tetrads for, obtaining step (2) and 10 μ L6 μM THT reaction 15min, to obtain the final product.
A kind of biological sensor provided by the invention, is prepared using the above method.
A kind of application of biological sensor detection copper ion using above-mentioned preparation provided by the invention.
Specifically the method for detection copper ion is：
By the copper ion solution of biological sensor and various concentrations in 20 DEG C of -50 DEG C of culture 1min-15min, measurement
Fluorescence signal；The linear relationship of copper ion and fluorescence intensity is built, realizes the detection to copper ion.
Detection copper ion principle be：Thioflavin dyestuff is combined with G- tetrads, can send strong fluorescence, add copper from
Son, can be quenched its fluorescence, and the concentration for adding copper ion is different, there is different degrees of quenching phenomenon, surveys system fluorescence intensity level,
Build concentration and the linear relationship of fluorescence intensity level；
A kind of application of biological sensor detection pyrophosphate using above-mentioned preparation provided by the invention.
Specifically the method for detection pyrophosphate is：
By biological sensor and copper ion solution after 20 DEG C of -50 DEG C of culture 1min-15min, various concentrations are added
Pyrophosphate solution in 20 DEG C -50 DEG C culture 10min-30min, measure fluorescence signal；Build pyrophosphate and fluorescence intensity
Linear relationship, realize the detection to pyrophosphate.
Detection pyrophosphate principle be：Pyrophosphate and copper ion have strong coordination ability, can by the copper in system from
Son is deprived, so as to recover the fluorescence of system, prepares a series of pyrophosphate of various concentrations, surveys system fluorescence intensity level,
Build concentration and the linear relationship of fluorescence intensity level；Realize the detection to pyrophosphate.
A kind of application of biological sensor detection of alkaline phosphatase using above-mentioned preparation provided by the invention.
Specifically the method for detection of alkaline phosphatase is：
By biological sensor and copper ion solution in 20 DEG C of -50 DEG C of culture 1min-15min, it is molten to add pyrophosphate
Then liquid adds the alkaline phosphatase enzyme solutions of various concentrations, 20 DEG C -50 DEG C are cultivated in 20 DEG C of -50 DEG C of culture 10min-30min
40min-80min, measures fluorescence signal；The linear relationship of alkaline phosphatase and fluorescence intensity is built, is realized to alkaline phosphatase
Detection.
The principle of detection of alkaline phosphatase is：Pyrophosphate in system can be hydrolyzed into phosphate radical by alkaline phosphatase, be made
It loses the coordination ability with copper ion, so that the fluorescence of system be quenched, prepares a series of alkaline phosphatase of various concentrations, surveys
System fluorescence intensity level, builds concentration and the linear relationship of fluorescence intensity level；Realize the detection to alkaline phosphatase.
A kind of biological sensor provided by the invention and its application, realize to copper ion, pyrophosphate, alkaline phosphorus
Sour enzyme sensitivity, specific detection." off-on-off " bioluminescence sensing platform is utilized, applied to copper ion, pyrophosphoric acid
The detection of root, alkaline phosphatase,
Compared with prior art, the preparation method of fluorescent optical sensor provided by the invention, uses unmarked DNA,
Easy to operate, cost is very low, avoids any chemical labeling and modification.It is used as fluorescence by G- tetrads and thioflavin dyestuff to visit
Pin, can prepare detection copper ion, pyrophosphate, alkaline phosphatase enzyme sensor.This sensor of the results show is to copper ion 2-
100nM has sensitive detection, has sensitive detection for 1-10 μM to pyrophosphate, has sensitively to alkaline phosphatase 0-0.04U/mL
Detection, and, easy to operate, high sensitivity simple with system, test limit are low.
Brief description of the drawings
Fig. 1 is to establish " off-on-off " system preparation principle figure based on G- tetrads-thioflavin fluorescence sense platform；
Fig. 2 is feasibility figure of the present invention, and a represents G- tetrads-thioflavin fluorogram, and d represents that adding copper ion fluorescence quenches
Go out figure, and b represents to add pyrophosphoric acid, and fluorescence recovers figure, and c represents to add alkaline phosphatase, fluorescent quenching figure；
Fig. 3 A add the fluorescence spectra that copper ion measures in G- tetrads-thioflavin system, and a to k copper ions are most
Final concentration is respectively (0,2,4,6,8,10,50,100,200,500,1000nM)；
Fig. 3 B are the relation (0-1000nM) of copper ion concentration and fluorescence intensity；
Fig. 3 C are the relation (2-100nM) of copper ion concentration and fluorescence intensity；
Fig. 3 D are copper ion selective figure；
Fig. 4 A are that the fluorescence spectra that pyrophosphate measures, a to h are added in G- tetrad-thioflavin-copper ion systems
Concentration is respectively 0,1,2,3,4,6,8,10 μM；
Fig. 4 B are copper ion concentration and the Linear Fit Chart of fluorescence intensity；
Fig. 4 C are pyrophosphate selective figure；
Fig. 5 A add the fluorescence light that alkaline phosphatase measures in G- tetrads-thioflavin-copper ion-pyrophosphate system
Spectrogram；A to j concentration is respectively 0,0.01,0.02,0.03,0.04,0.1,0.2,0.6,0.8,1U/mL；
Fig. 5 B are alkaline phosphatase concentration and the Linear Fit Chart (0-1U/mL) of fluorescence intensity；
Fig. 5 C are alkaline phosphatase concentration and the Linear Fit Chart (0-0.04U/mL) of fluorescence intensity；
Fig. 5 D are alkaline phosphatase selective figure；
Fig. 6 A are potassium concentration optimization figure；
Fig. 6 B are thioflavin concentration optimization figure；
Fig. 6 C are pH optimization figures.
Embodiment
Embodiment 1
A kind of preparation method of biological sensor, comprises the following steps：
1), the DNA sequence dna of purchase is dissolved in 0.05M Tris-HCl (pH 7.4) buffer solution, DNA buffer solutions,
And saved backup at 4 DEG C；DNA：AGGGTTAGGGTTAGGGTTAGGG；
2), 2 μ L, 100 μM of DNA buffer solutions and 10 μ L 20mMK for preparing step 1)+Solution is cultivated at 37 DEG C
2h, forms G- tetrads；
3), 6 μM of ThT of G- tetrads and 10 μ L that step 2) obtains are mixed, 15min is reacted at 37 DEG C, surveys its fluorescence,
Obtain biological sensor；
The biological sensor is built based on G- tetrads-ThT.Buffer solution pH used is that 7.4 concentration are
0.05M Tris-HCl (pH 7.4), magnesium chloride containing 0.05M in buffer solution.
Embodiment 2
A kind of biological sensor, is prepared using the above method.
Embodiment 3
A kind of biological sensor using above-mentioned preparation detects copper ion application.
Specifically detection method is：
By the DNA buffer solutions of 100 μM of 2 μ L and the 20mM K of 10 μ L+Solution is added to 178 μ L Tris-HCl (pH
7.4) in buffer solution, cultivate 2h in 37 DEG C and form G- tetrads, be then respectively adding the copper ion solution of 10 μ L concentration gradients,
Mixed solution is obtained, 37 DEG C of culture 5min, then survey its fluorescence；Build the linear relationship of copper ion and fluorescence intensity, realization pair
The detection of copper ion.
The preparation method of the copper ion solution of the concentration gradient is：The copper ion storing solution of configuration is gradually diluted with water
(1000nM).In detection architecture copper ion final concentration be respectively 0,2,4,6,8,10,50,100,200,500,1000nM it is molten
Liquid, then surveys its fluorescence；
Detect copper ion principle：Thioflavin does not have fluorescence signal in itself, when it is combined with G- tetrads, can send very strong
Fluorescence signal, thioflavin molecular structural formulaIn have amido, can with copper from
Son combines, and forms cupric ammine complex, therefore the fluorescence signal of system is quenched, based on this, it is possible to achieve the detection of copper ion.
Embodiment 4
A kind of biological sensor using above-mentioned preparation detects pyrophosphate application.
Specifically detection method is：
By the DNA buffer solutions of 100 μM of 2 μ L and the 20mM K of 10 μ L+Solution is added to 168 μ L Tris-HCl (pH
7.4) in buffer solution, cultivate 2h in 37 DEG C and form G- tetrads, then add the copper ion of 10 μ L200 μM, 37 DEG C of cultures
After 5min, the pyrophosphate solution of 10 μ L concentration gradients is then respectively adding, obtains mixed solution, 20min is cultivated in 37 DEG C,
Then its fluorescence is surveyed；The linear relationship of pyrophosphate and fluorescence intensity is built, realizes the detection to pyrophosphate.
The preparation method of the pyrophosphate solution of the concentration gradient is：The pyrophosphate deposit of configuration is gradually diluted with water
Liquid (10 μM), pyrophosphate final concentration is respectively 0,1,2,3,4,6,8,10 μM of solution in detection architecture.
Detect the principle of pyrophosphate：Pyrophosphate has very strong coordination ability with copper ion, therefore can deprive G- tetra-
Copper ion in disjunctor-thioflavin-copper ion system, so as to recover the fluorescence of system, based on this, it is possible to achieve pyrophosphate
Detection.
Embodiment 5
A kind of biological sensor detection of alkaline phosphatase application using above-mentioned preparation.
Specifically detection method is：
By the DNA buffer solutions of 100 μM of 2 μ L and the 20mM K of 10 μ L+Solution is added to 163 μ L Tris-HCl (pH
7.4) in buffer solution, cultivate 2h in 37 DEG C and form G- tetrads, add the copper ion solution of 10 μ L 200nM, in 37 DEG C of cultures
5min and then addition 10 μ L, 4 μM of pyrophosphate solution, 37 DEG C of culture 20min, are then respectively adding 5 μ L various concentrations gradients
Alkaline phosphatase enzyme solutions, obtain mixed solution, cultivate 50min in 37 DEG C, then survey its fluorescence；Build alkaline phosphatase and fluorescence
The linear relationship of intensity, realizes the detection to alkaline phosphatase.
The preparation method of the alkaline phosphatase enzyme solutions of the concentration gradient is：The alkaline phosphatase of configuration is gradually diluted with water
Storing solution (1U/mL).Detection architecture alkaline phosphatase final concentration is respectively 0,0.01,0.02,0.03,0.04,0.1,0.2,
0.6,0.8,1U/mL solution, then surveys its fluorescence；
The principle of detection of alkaline phosphatase：Alkaline phosphatase can hydrolyze pyrophosphate, pyrophosphate is lost and copper
The complexing power of ion, therefore, is quenched the fluorescence of system, based on this, it is possible to achieve the detection of alkaline phosphatase；
The present invention is formed copper-amine complex, the glimmering of system is quenched based on the amido on copper ion and G- tetrads-thioflavin
Light, realizes and copper ion is detected；Pyrophosphate is added, complex is formed with copper ion, deprives out copper ion, make the fluorescence of system
Recovered, realize and pyrophosphate is detected；Alkaline phosphatase is added, pyrophosphate can be hydrolyzed, it is lost complexing
Ability so that system fluorescent quenching, realizes the Sensitive Detection to alkaline phosphatase.
Claims (10)
1. a kind of preparation method of biological sensor, it is characterised in that the preparation method comprises the following steps：
1) DNA sequence dna is dissolved in Tris-HCl buffer solutions, obtains DNA buffer solutions, it is spare；
2), the buffer solution and K for preparing step 1)+Solution is mixed, and forms G- tetrads；
3) the G- tetrads and ThT hybrid reactions for, obtaining step 2), to obtain the final product.
2. preparation method according to claim 1 or 2, it is characterised in that the K described in step 2)+Solution concentration is
10mM-50mM。
3. according to claim 1 or 2 or the preparation method, it is characterised in that mixed culture refers to 20 described in step 2)
1h-3h is cultivated at DEG C -50 DEG C.
4. the preparation method according to claim 1 or 3, it is characterised in that hybrid reaction described in step 3) is 20 DEG C -50
1min-15min is reacted at DEG C.
A kind of 5. biological sensor prepared using claim 1-4 any one of them method.
A kind of 6. application of biological sensor detection copper ion using described in claim 5；Specific detection copper ion
Method is：
By the copper ion solution of biological sensor and various concentrations in 20 DEG C of -50 DEG C of culture 1min-15min, fluorescence is measured
Signal；The linear relationship of copper ion and fluorescence intensity is built, realizes the detection to copper ion.
A kind of 7. application of biological sensor detection pyrophosphate using described in claim 5.
8. application according to claim 7, it is characterised in that specifically the method for detection pyrophosphate is：
By biological sensor and copper ion solution after 20 DEG C of -50 DEG C of culture 1min-15min, Jiao of various concentrations is added
Phosphate radical solution measures fluorescence signal in 20 DEG C of -50 DEG C of culture 10min-30min；Build pyrophosphate and the line of fluorescence intensity
Sexual intercourse, realizes the detection to pyrophosphate.
A kind of 9. application of biological sensor detection of alkaline phosphatase using described in claim 5.
10. application according to claim 9, it is characterised in that specifically the method for detection of alkaline phosphatase is：Fluorescence is given birth to
Thing sensor, in 20 DEG C of -50 DEG C of culture 1min-15min, adds pyrophosphate solution in 20 DEG C of -50 DEG C of cultures with copper ion solution
10min-30min, then adds the alkaline phosphatase enzyme solutions of various concentrations, 20 DEG C of -50 DEG C of culture 40min-80min, measurement
Fluorescence signal；The linear relationship of alkaline phosphatase and fluorescence intensity is built, realizes the detection to alkaline phosphatase.
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| CN108707644A (en) * | 2018-05-28 | 2018-10-26 | 南京工业大学 | Method for detecting pyrophosphate and alkaline phosphohydrolase based on DNA templated copper cluster probe |
| CN109406467A (en) * | 2018-10-16 | 2019-03-01 | 商丘师范学院 | Division aptamer sensor and its application for ATP detection |
| CN109724957A (en) * | 2018-12-28 | 2019-05-07 | 南京工业大学 | Method for detecting aluminum ions based on aluminum ion induced phosphorescence copper nano cluster aggregation enhanced fluorescence and application thereof |
| CN110057879A (en) * | 2019-04-18 | 2019-07-26 | 桂林理工大学 | A kind of instant detection method of alkaline phosphatase activities |
| CN110982873A (en) * | 2019-11-27 | 2020-04-10 | 山西大学 | Room-temperature phosphorescence detection method of alkaline phosphatase and application |
| CN113433103A (en) * | 2021-06-29 | 2021-09-24 | 中国农业大学 | Method for detecting 2,4-dichlorophenoxyacetic acid |
| CN113758909A (en) * | 2021-09-27 | 2021-12-07 | 长江大学 | Detection method of alkaline phosphatase |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108707644A (en) * | 2018-05-28 | 2018-10-26 | 南京工业大学 | Method for detecting pyrophosphate and alkaline phosphohydrolase based on DNA templated copper cluster probe |
| CN108707644B (en) * | 2018-05-28 | 2021-06-15 | 南京工业大学 | Method for detecting pyrophosphate and alkaline phosphohydrolase based on DNA templated copper cluster probe |
| CN109406467A (en) * | 2018-10-16 | 2019-03-01 | 商丘师范学院 | Division aptamer sensor and its application for ATP detection |
| CN109724957A (en) * | 2018-12-28 | 2019-05-07 | 南京工业大学 | Method for detecting aluminum ions based on aluminum ion induced phosphorescence copper nano cluster aggregation enhanced fluorescence and application thereof |
| CN110057879A (en) * | 2019-04-18 | 2019-07-26 | 桂林理工大学 | A kind of instant detection method of alkaline phosphatase activities |
| CN110982873A (en) * | 2019-11-27 | 2020-04-10 | 山西大学 | Room-temperature phosphorescence detection method of alkaline phosphatase and application |
| CN113433103A (en) * | 2021-06-29 | 2021-09-24 | 中国农业大学 | Method for detecting 2,4-dichlorophenoxyacetic acid |
| CN113758909A (en) * | 2021-09-27 | 2021-12-07 | 长江大学 | Detection method of alkaline phosphatase |
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