CN105548167B - Manganese dioxide thin slice simulates the detection method of enzyme sensor and preparation method and T4PNK - Google Patents
Manganese dioxide thin slice simulates the detection method of enzyme sensor and preparation method and T4PNK Download PDFInfo
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- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 title claims abstract description 126
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 46
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 46
- 238000001514 detection method Methods 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 239000000243 solution Substances 0.000 claims abstract description 70
- 238000004088 simulation Methods 0.000 claims abstract description 27
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 claims abstract description 23
- 239000011259 mixed solution Substances 0.000 claims abstract description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 238000002835 absorbance Methods 0.000 claims description 13
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 claims description 9
- 239000011572 manganese Substances 0.000 claims description 9
- 229910052748 manganese Inorganic materials 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 238000011088 calibration curve Methods 0.000 claims description 6
- WGTYBPLFGIVFAS-UHFFFAOYSA-M tetramethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)C WGTYBPLFGIVFAS-UHFFFAOYSA-M 0.000 claims description 6
- 238000010348 incorporation Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 3
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 3
- 239000011565 manganese chloride Substances 0.000 claims description 3
- -1 tetramethyl amine salt Chemical class 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 235000002867 manganese chloride Nutrition 0.000 claims description 2
- 229940099607 manganese chloride Drugs 0.000 claims description 2
- IPJKJLXEVHOKSE-UHFFFAOYSA-L manganese dihydroxide Chemical compound [OH-].[OH-].[Mn+2] IPJKJLXEVHOKSE-UHFFFAOYSA-L 0.000 claims description 2
- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 claims 2
- 229940099596 manganese sulfate Drugs 0.000 claims 1
- 235000007079 manganese sulphate Nutrition 0.000 claims 1
- 239000011702 manganese sulphate Substances 0.000 claims 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 abstract description 16
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 108020004414 DNA Proteins 0.000 description 23
- 238000000862 absorption spectrum Methods 0.000 description 12
- 230000026731 phosphorylation Effects 0.000 description 6
- 238000006366 phosphorylation reaction Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 4
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000002055 nanoplate Substances 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 229910052698 phosphorus Inorganic materials 0.000 description 3
- 239000011574 phosphorus Substances 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 2
- 230000005778 DNA damage Effects 0.000 description 2
- 231100000277 DNA damage Toxicity 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 238000009739 binding Methods 0.000 description 2
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- 239000002904 solvent Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 108020005124 DNA Adducts Proteins 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 201000011032 Werner Syndrome Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- GOPYZMJAIPBUGX-UHFFFAOYSA-N [O-2].[O-2].[Mn+4] Chemical class [O-2].[O-2].[Mn+4] GOPYZMJAIPBUGX-UHFFFAOYSA-N 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004523 catalytic cracking Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002060 nanoflake Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940048102 triphosphoric acid Drugs 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
Classifications
-
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of manganese dioxide thin slice simulation enzyme sensor and the detection method of preparation method and T4PNK, the preparation methods, comprising: 1) by MnO2Nanometer sheet solution and TMB (3,3', 5,5'- tetramethyl benzidine) solution are mixed so that MnO is made2- TMB mixed solution；2) hair clip DNA is dissolved in Tris-HCl buffer solution, ATP (atriphos) and λ EXO (λ excision enzyme) is added then to be mixed to prepare DNA solution；3) by MnO2- TMB mixed solution is mixed with DNA solution manganese dioxide thin slice is made and simulates enzyme sensor.The preparation method is easy to operate, while sensor obtained has the characteristics that high sensitivity, detection limit are low and easy to operate for the detection of T4PNK.
Description
Technical field
The present invention relates to simulation enzyme sensors, and in particular, to a kind of manganese dioxide thin slice simulation enzyme sensor and system
The detection method of Preparation Method and T4PNK.
Background technique
T4 polynucleotide kinase (PNK), a kind of 5 ' kinases, since it 1965 infects even number in e. coli protein extract
Bacteriophage is found for the first time, up to the present, it has also become most common molecular biology enzyme.It can be catalyzed γ-triphosphoric acid
The nucleotide of the 5 ' end of phosphoric acid salt residual quantity of adenosine (ATP) or 5 '-hydroxyl bearings of nucleotide.This is to cellular nucleic acid metabolism
Very important, especially in cell to DNA damage reaction, this and many human diseases, such as adult progeria, Bloom are comprehensive
Simulator sickness and Rothmund-Thomson-Thomson syndrome etc. suffer from close ties.T4PNK is also widely used for detecting
DNA adduct or oligonucleotides and DNA damage reparation.Therefore, one it is sensitive, it is accurate for detect T4PNK it is active and
Detect the experimental method of its potential inhibitor development be very it is necessary to.
Traditional method describes phosphorylation detection and the measurement of DNA kinase activity, including active isotope32P label, gathers
Acrylamide gel electrophoresis (PAGE), the methods of autoradiograph, fluorescence.However, these methods suffer from varying degrees
It is time-consuming, it is arduously, insensitive, or the shortcomings that require radiolabeled substrate.
Summary of the invention
The object of the present invention is to provide the inspections of a kind of manganese dioxide thin slice simulation enzyme sensor and preparation method and T4PNK
Survey method, the preparation method is easy to operate, while sensor obtained there is high sensitivity, detection to limit the detection of T4PNK
Low and easy to operate feature.
To achieve the goals above, the present invention provides a kind of manganese dioxide thin slice simulation enzyme sensor preparation method,
Include:
1) by MnO2Nanometer sheet solution and TMB (3,3', 5,5'- tetramethyl benzidine) solution are mixed so that MnO is made2-TMB
Mixed solution；
2) hair clip DNA is dissolved in Tris-HCl buffer solution, ATP (atriphos) and λ EXO is then added (outside λ
Enzyme cutting) to be mixed to prepare DNA solution；
3) by MnO2- TMB mixed solution is mixed with DNA solution manganese dioxide thin slice is made and simulates enzyme sensor.
The present invention also provides a kind of manganese dioxide thin slices to simulate enzyme sensor, which simulates enzyme sensor
It is prepared by above-mentioned method.
Present invention provides the detection methods of T4PNK a kind of, comprising:
1) the T4PNK solution of different known concentrations contact with above-mentioned manganese dioxide thin slice simulation enzyme sensor anti-
It answers；
2) after the colour stable of reaction system, the absorbance of reaction system is detected, then using absorbance as ordinate,
The concentration of T4PNK is that abscissa draws standard curve or obtains calibration curve equation；
3) the T4PNK solution of unknown concentration and above-mentioned manganese dioxide thin slice simulation enzyme sensor are subjected to haptoreaction,
Then the absorbance for detecting reaction system, then according to standard curve or calibration curve equation to learn unknown concentration
The concentration of T4PNK solution.
Through the above technical solutions, reaction mechanism such as Fig. 1 of manganese dioxide thin slice simulation enzyme sensor provided by the invention
It is shown: the effect of manganese dioxide thin slice is simulated in enzyme sensor, and manganese dioxide nano-plates can play analogue enztme, in analogue enztme
In the presence of, TMB can be navy blue by catalyzed coloration, and can not be cut by λ EXO without the hair clip DNA of phosphorus acylation reaction
It is segmented into single-stranded, the presence still in the form of double-strand；Once T4PNK is added in manganese dioxide thin slice simulation enzyme sensor, due to
T4PNK can hold phosphorus acylation reactions with the 5 ' of hair clip DNA so that DNA can be cut into the λ EXO of specificity it is single-stranded, this
When single stranded DNA have inhibiting effect for the activity of manganese dioxide analogue enztme so that the color of TMB is gradually faded away；Entirely
The logarithm of the concentration of the depth and T4PNK of the color of system is in a linear relationship, so that manganese dioxide provided by the invention is thin
Piece, which simulates enzyme sensor, there is excellent sensitivity and detection to limit the detection of T4PNK.In addition, colorimetric mould provided by the invention
It is unmarked DNA used in the preparation process of quasi- enzyme sensor, easy to operate, cost is very low, avoids any chemical labeling
And modification, so that the sensor is convenient for use on a large scale.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the working principle diagram of manganese dioxide thin slice simulation enzyme sensor provided by the invention；
Fig. 2 is MnO in detection example 12The low power SEM of nanometer sheet schemes；
Fig. 3 is MnO in detection example 12The high power SEM of nanometer sheet schemes；
Fig. 4 is uv absorption spectra in detection example 2；
Fig. 5 is uv absorption spectra in detection example 3；
Fig. 6 is uv absorption spectra in application examples 1；
Fig. 7 is highest photon absorbing intensity result statistical chart in Fig. 6；
Fig. 8 is the uv absorption spectra of the λ EXO of various concentration in application examples 2；
Fig. 9 is the uv absorption spectra of the ATP of various concentration in application examples 2；
Figure 10 is the uv absorption spectra of different incubation times in application examples 3.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of preparation methods of manganese dioxide thin slice simulation enzyme sensor, comprising:
1) by MnO2Nanometer sheet solution and TMB (3,3', 5,5'- tetramethyl benzidine) solution are mixed so that MnO is made2-TMB
Mixed solution；
2) hair clip DNA is dissolved in Tris-HCl buffer solution, ATP (atriphos) and λ EXO is then added (outside λ
Enzyme cutting) to be mixed to prepare DNA solution；
3) by MnO2- TMB mixed solution is mixed with DNA solution manganese dioxide thin slice is made and simulates enzyme sensor.
In the step 1) of above-mentioned preparation method, the dosage of each material can select in a wide range, but in order to
So that manganese dioxide thin slice simulation enzyme sensor obtained has superior sensitivity and detection limit, it is preferable that in step 1)
In, MnO2MnO in nanometer sheet solution2Concentration be 3-15mmol/L, the concentration of TMB solution is 30-50mmol/L；And it is opposite
In the MnO of 1mL2Nanometer sheet solution, the dosage of TMB solution are 1-2mL.
In the step 1) of above-mentioned preparation method, mixing condition can select in a wide range, but in order to enable
Manganese dioxide thin slice simulation enzyme sensor obtained has superior sensitivity and detection limit, it is preferable that in step 1), mixes
Be bonded to and meet the following conditions less: mixing temperature is 15-30 DEG C, incorporation time 4-10min.
In the step 2) of above-mentioned preparation method, the dosage of each material can select in a wide range, but in order to
So that manganese dioxide thin slice simulation enzyme sensor obtained has superior sensitivity and detection limit, it is preferable that in step 2)
In, the dosage of hair clip DNA, ATP relative to 100nmol are 10-15nmol, and the dosage of λ EXO is 10-15U, and Tris-HCl is slow
The dosage for rushing solution is 20-30 μ L；
In the step 1) of above-mentioned preparation method, the type of Tris-HCl buffer solution and DNA can be in wide models
Interior selection is enclosed, but in order to enable manganese dioxide thin slice obtained simulation enzyme sensor has superior sensitivity and detection
Limit, it is preferable that the pH of Tris-HCl buffer solution is 7.1-7.5, the Serial No. 5'-GGCCTTGGATTGAA of hair clip DNA
GGGAGCTCTACGGCC-3'。
In the step 3) of above-mentioned preparation method, the dosage of each material can select in a wide range, but in order to
So that manganese dioxide thin slice simulation enzyme sensor obtained has superior sensitivity and detection limit, it is preferable that in step 3)
In, relative to the DNA solution of 1 parts by volume, MnO2The dosage of-TMB mixed solution is 3-5 parts by volume.
In the step 3) of above-mentioned preparation method, mixing condition can select in a wide range, but in order to enable
Manganese dioxide thin slice simulation enzyme sensor obtained has superior sensitivity and detection limit, it is preferable that in step 3), mixes
Be bonded to and meet the following conditions less: mixing temperature is 15-30 DEG C, incorporation time 15-25min.
In the present invention, MnO2The preparation method of nanometer sheet solution can be any conventional preparation side in this field
Method, but in order to enable manganese dioxide thin slice obtained simulation enzyme sensor has superior sensitivity and detection limit, preferably
Ground, before step 1), the preparation method further include: by TMA (tetramethyl amine salt) solution, H2O2Solution is contacted with manganese source
Reaction is to be made MnO2Nanometer sheet solution.
In above-mentioned MnO2In the preparation method of nanometer sheet solution, the dosage of each material can be selected in wide range, still
In order to enable manganese dioxide thin slice simulation enzyme sensor obtained has superior sensitivity and detection limit, it is preferable that TMA is molten
The concentration of liquid is 1-1.5mmol/L, H2O2The concentration of solution is 20-40 weight %；Also, relative to the manganese source of 1g, the use of TMA
Amount is 15-25mL, H2O2The dosage of solution is 2-4mL.
In above-mentioned MnO2In the preparation method of nanometer sheet solution, the specific type of TMA and manganese source can be in wide range
Selection, but taking cost into account, it is preferable that TMA is tetramethylammonium hydroxide, and manganese source is four chloride hydrate manganese manganese sulfates, oxygen
Change one of manganese and manganous hydroxide or a variety of.
In above-mentioned MnO2In the preparation method of nanometer sheet solution, catalytic condition can be selected in wide range, but
It is the yield in order to improve nano-manganese dioxide, it is preferable that haptoreaction at least meets the following conditions: reaction temperature 15-25
DEG C, reaction time 10-20s.
The present invention also provides a kind of manganese dioxide thin slices to simulate enzyme sensor, which simulates enzyme sensor
It is prepared by above-mentioned method.
Present invention provides the detection methods of T4PNK a kind of, comprising:
1) the T4PNK solution of different known concentrations contact with above-mentioned manganese dioxide thin slice simulation enzyme sensor anti-
It answers；
2) after the colour stable of reaction system, the absorbance of reaction system is detected, then using absorbance as ordinate,
The concentration of T4PNK is that abscissa draws standard curve or obtains calibration curve equation；
3) the T4PNK solution of unknown concentration and above-mentioned manganese dioxide thin slice simulation enzyme sensor are subjected to haptoreaction,
Then the absorbance for detecting reaction system, then according to standard curve or calibration curve equation to learn unknown concentration
The concentration of T4PNK solution.
In above-mentioned detection method, for the ease of can fully be reacted between T4PNK, hair clip DNA and λ EXO, preferably
Ground, in step 1), haptoreaction reacts 25-35min at 30-40 DEG C first, then reacts 5-15min at 70-80 DEG C.
The present invention will be described in detail by way of examples below.In following embodiment, the hair clip DNA (sequence of trade mark A40
Row number: 5'-GGCCTTGGATTGAA GGGAGCTCTACGGCC-3'), ATP and T4PNK (10U/uL) be Shanghai Sangon Biotech Company
Commercially available product, λ EXO (10U/uL) are the commercially available product for matching Mo Feishier company, H2O2(30wt%), MnCl2·4H2O (99.99%)
For the commercially available product of Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Ultra-violet absorption spectrum is the inspection by the ultraviolet-visible spectrophotometer that the Hitachi, Japan trade mark is UV-3010
It surveys and obtains, SEM sensing chamber is obtained by the detection for the electronic scanner microscope that the Hitachi, Japan trade mark is S-4800.
Preparation example 1
MnO2The preparation of nanometer sheet solution:
Utilize " K.Kai, Y.Yoshida, H.Kageyama, G.Saito, T.Ishigaki, Y.Furukawa and
Prepared by the method for J.Kawamata, J.Am.Chem.Soc., 2008,130,15938-15943. " report: in 25 DEG C open
Under the conditions of mouthful, by the tetramethyl ammonium hydroxide solution (1.0M, solvent are water) of 12mL, the H of 2mL2O2(30wt%, solvent are solution
Water), (MnCl containing 0.593g into 10mL manganese chloride solution is added2·4H2O) reaction 15s is to be made MnO2Nanometer sheet is molten
Liquid.
Embodiment 1
1) at 25 DEG C, by the above-mentioned MnO of 1mL2Nanometer sheet solution (10mM) is mixed with TMB (40mM) solution of 1mL
5min is to be made MnO2- TMB mixed solution；
2) the hair clip DNA (100nM) of trade mark A40 is dissolved in Tris-HCl buffer solution (pH 7.4), is then added
ATP (10mM) and λ EXO (10U) are to be mixed to prepare DNA solution；
3) at 25 DEG C, by the MnO of 75 μ L2- TMB mixed solution is with the DNA solution mixing 15min of 25 μ L dioxy is made
Change manganese thin slice analogue enztme sensors A 1.
Embodiment 2
It carries out that manganese dioxide thin slice analogue enztme sensors A 2 is made according to the method for embodiment 1, the difference is that the use of λ EXO
Amount is 2U.
Embodiment 3
It carries out that manganese dioxide thin slice analogue enztme sensors A 3 is made according to the method for embodiment 1, the difference is that the use of λ EXO
Amount is 6U.
Embodiment 4
It carries out that manganese dioxide thin slice analogue enztme sensors A 4 is made according to the method for embodiment 1, the difference is that the use of λ EXO
Amount is 12U.
Embodiment 5
It carries out that manganese dioxide thin slice analogue enztme sensors A 5 is made according to the method for embodiment 1, the difference is that the use of λ EXO
Amount is 14U.
Embodiment 6
It carries out that manganese dioxide thin slice analogue enztme sensors A 6 is made according to the method for embodiment 1, the difference is that the dosage of ATP
For 2nM.
Embodiment 7
It carries out that manganese dioxide thin slice analogue enztme sensors A 7 is made according to the method for embodiment 1, the difference is that the dosage of ATP
For 4nM.
Embodiment 8
It carries out that manganese dioxide thin slice analogue enztme sensors A 8 is made according to the method for embodiment 1, the difference is that the dosage of ATP
For 6nM.
Embodiment 9
It carries out that manganese dioxide thin slice analogue enztme sensors A 9 is made according to the method for embodiment 1, the difference is that the dosage of ATP
For 8nM.
Embodiment 10
It carries out that manganese dioxide thin slice analogue enztme sensors A 9 is made according to the method for embodiment 1, the difference is that the dosage of ATP
For 12nM.
Comparative example 1
It carries out that manganese dioxide thin slice simulation enzyme sensor B1 is made according to the method for embodiment 1, the difference is that being not used
ATP。
Comparative example 2
It carries out that manganese dioxide thin slice simulation enzyme sensor B2 is made according to the method for embodiment 1, the difference is that λ is not used
EXO。
Detect example 1
By SEM to MnO2Nanometer sheet solution is detected, and concrete outcome is shown in Fig. 2 and Fig. 3, by Fig. 2 and Fig. 3 it is found that MnO2
Manganese dioxide in nanometer sheet solution is nano flake shape, having a size of 200nm or so.
Detect example 2
By ultraviolet absorption spectrum instrument to MnO2MnO in nanometer sheet solution and embodiment 12- TMB mixed solution carries out
Detection, concrete outcome is shown in Fig. 4, as seen from the figure, the catalysis oxidation of substrate TMB be by ultra-violet absorption spectrum and the photo of coloration into
Row test, it can be seen that MnO is added in the TMB solution in 652nm2(curve a) is much high for absorbance after nanometer sheet solution
In absorbance (the curve b) of no manganese dioxide nano-plates solution.Correspondingly, the colourless TMB of manganese dioxide nano-plates catalysis oxidation is extremely
The oxidation TMB of blue (see illustration in Fig. 4).It has been indicated above the property of the analogue enztme of MnO2 nanometer sheet.
Embodiment 3
T4PNK is added into A1 so that T4PNK initial concentration be 10U/mL, then pass through ultraviolet absorption spectrum instrument
The system of detection addition T4PNK and the system for being not added with T4PNK, concrete outcome are shown in Fig. 5, as seen from the figure, as shown, are not having
When having T4PNK, at 652nm wavelength, UV absorption intensity is very high, in the Tris-HCl buffer of pH 7.4 (curve b), this
It may be because the catalytic cracking reaction of Phosphorylation events and λ EXO do not trigger.And in the presence of 10U/mLT4PNK, it inhales
It receives intensity and is decreased obviously that (curve a), which demonstrate Phosphorylation events to help to cause DNA to inhibit to simulate oxidase active and split
Solution reaction.Correspondingly the result is that solution is blue when without T4PNK, and solution is close to colourless when have T4PNK
(see illustration in Fig. 5).Therefore, one it is simple and effectively the detection without T4PNK labelling strategies and separation process can be built
It is vertical.
Application examples 1
Different amounts of T4PNK is added to A1 so that the initial concentration of T4PNK be followed successively by 0U/mL, 0.005U/mL,
0.01U/mL, 0.05U/mL, 0.1U/mL, 0.5U/mL, 1U/mL, 5U/mL and 10U/mL then cultivate system at 37 DEG C
Then 30min heats 10min at 75 DEG C, finally detect ultra-violet absorption spectrum, and concrete outcome is shown in Fig. 6 and Fig. 7, as seen from the figure,
As shown in fig. 6, it shows the increase in T4PNK U/mL concentration not equal from 0 to 10, the TMB absorption peak of ultraviolet visible absorption spectra
It is gradually reduced.In addition, the variation of the manganese dioxide nano-plates-TMB system color of various concentration T4PNK is by using digital phase
Machine records (see illustration in Fig. 6), can significantly observe with the naked eye.Shown as the result is shown in Fig. 7 accordingly, signal and
Relationship between T4PNK concentration, and shown in the illustration of Fig. 7, a relatively good range of linearity is in 0-10U/mL.Related side
Journey can be described as A=-0.013977X (U/mL)+0.04217, coefficient R2=0.9993, wherein A is UV absorption, and
X is T4PNK concentration.It can be observed T4PNK concentration is down to 0.005U/mL signal.
Application examples 2
According to application 1 method detected, unlike, T4PNK is sequentially added into A2-A9, B1 and B2 so that
Initial concentration be 10U/mL, then detect maximum absorbance, concrete outcome is shown in Fig. 8 and Fig. 9, as seen from the figure, absorption it is single-stranded
DNA may will affect the activity of analogue enztme with λ EXO cracking digestion, and determine detection sensitivity.At this point, to different
The concentration of λ EXO is assessed.It was found that absorption peak is reduced with the increase of λ EXO amount, when the amount of λ EXO is 10U, almost
Reach (Fig. 8) of minimum saturation value.
ATP plays an important role in Phosphorylation events, the influence of concentration and then is detected, as a result shown in the figure.
As shown in figure 9, the ultraviolet false positive signal of decrease, which develops the color, to be observed with the increase of ATP concentration.When concentration is > 10mM, have
It slightly decreases.This slight inhibiting effect may be the phosphorus due to DNA and high concentration in Phosphorylation events
Competitive binding reaction in acidization.Therefore, when the concentration of ATP is relatively high, the binding site of T4PNK is partially blocked up
Plug.Therefore, 10mM is as optimal concentration, to obtain high sensitivity.
Application examples 3
According to application 1 method detected, unlike, incubation time is successively changed to 5min, 15min, 18min,
Then 20min and 25min detects maximum absorbance, concrete outcome is shown in Figure 10, as seen from the figure in current research, when reaction
Between be PNK catalytic phosphatase an important parameter.The excessive reaction time may cause the ultraviolet light signal of false positive.Such as
Shown in Figure 10, with the increase in reaction time, it is then rapidly achieved balance in 30min, prompts complete phosphorylation.
Through the foregoing embodiment, comparative example, detection example and application examples are it is found that manganese dioxide thin slice provided by the invention is simulated
Enzyme sensor there is excellent sensitivity and detection to limit the detection of T4PNK, be 20-100mM's in particular for concentration
T4PNK solution.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (11)
1. a kind of preparation method of manganese dioxide thin slice simulation enzyme sensor characterized by comprising
1) by MnO2Nanometer sheet solution and 3,3', the mixing of 5,5'- tetramethyl benzidine (TMB) solution is to be made MnO2- TMB mixing
Solution；
2) hair clip DNA is dissolved in Tris-HCl buffer solution, atriphos (ATP) and λ excision enzyme (λ is then added
EXO it) mixes so that DNA solution is made；
3) by the MnO2- TMB mixed solution is mixed with the DNA solution the manganese dioxide thin slice analogue enztme sensing is made
Device；
Wherein, in step 1), the MnO2MnO in nanometer sheet solution2Concentration be 3-15mmol/L, the TMB solution it is dense
Degree is 30-50mmol/L；And the MnO relative to 1mL2Nanometer sheet solution, the dosage of the TMB solution are 1-2mL；?
In step 2), the dosage of hair clip DNA, the ATP relative to 100nmol are 10-15nmol, and the dosage of the λ EXO is 10-
15U, the dosage of the Tris-HCl buffer solution are 20-30 μ L；In step 3), the DNA relative to 1 parts by volume is molten
Liquid, the MnO2The dosage of-TMB mixed solution is 3-5 parts by volume；The Serial No. 5'- of the hair clip DNA
GGCCTTGGATTGAAGGGAGCTCTACGGCC-3'。
2. preparation method according to claim 1, wherein in step 1), the mixing at least meets the following conditions: mixed
Closing temperature is 15-30 DEG C, incorporation time 4-10min.
3. preparation method according to claim 1, wherein the pH of the Tris-HCl buffer solution is 7.1-7.5.
4. preparation method according to claim 1, wherein in step 3), the mixing at least meets the following conditions: mixed
Closing temperature is 15-30 DEG C, incorporation time 15-25min.
5. preparation method described in any one of -3 according to claim 1, wherein before step 1), the preparation method
Further include: by tetramethyl amine salt (TMA) solution, H2O2Solution and manganese source carry out haptoreaction so that the MnO is made2Nanometer sheet is molten
Liquid.
6. preparation method according to claim 5, wherein the concentration of the TMA solution is 1-1.5mmol/L, the H2O2
The concentration of solution is 20-40 weight %；Also, relative to the manganese source of 1g, the dosage of the TMA is 15-25mL, described
H2O2The dosage of solution is 2-4mL.
7. preparation method according to claim 5, wherein the TMA is tetramethylammonium hydroxide, and the manganese source is four water
Close one of manganese chloride, manganese sulfate, manganese oxide and manganous hydroxide or a variety of.
8. preparation method according to claim 5, wherein the haptoreaction at least meets the following conditions: reaction temperature
It is 15-25 DEG C, reaction time 10-20s.
9. a kind of manganese dioxide thin slice simulates enzyme sensor, which is characterized in that the manganese dioxide thin slice simulation enzyme sensor is logical
Method described in any one of claim 1-8 is crossed to be prepared.
10. a kind of detection method of T4PNK characterized by comprising
1) the T4PNK solution of different known concentrations and manganese dioxide thin slice as claimed in claim 9 simulation enzyme sensor are carried out
Haptoreaction；
2) after the colour stable of reaction system, the absorbance of reaction system is detected, then using absorbance as ordinate, T4PNK
Concentration be abscissa draw standard curve or obtain calibration curve equation；
3) the T4PNK solution of unknown concentration is contacted with manganese dioxide thin slice as claimed in claim 9 simulation enzyme sensor
Reaction then detects the absorbance of reaction system, then described to learn according to the standard curve or calibration curve equation
The concentration of the T4PNK solution of unknown concentration.
11. detection method according to claim 10, wherein in step 1), the haptoreaction is first at 30-40 DEG C
Lower reaction 25-35min, then reacts 5-15min at 70-80 DEG C.
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| CN105838365B (en) * | 2016-05-06 | 2018-08-10 | 曲阜师范大学 | Fluorescent carbon point CDs solution, CDs-MnO2Composite material and preparation method and application |
| CN106093272B (en) * | 2016-06-01 | 2018-08-07 | 西安交通大学 | A kind of method of manganese dioxide nano-plates simulation oxide enzyme detection reproducibility biomolecule |
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| CN109295168B (en) * | 2018-10-18 | 2022-02-15 | 山东大学 | Manganese dioxide nanosheet-mediated ratio fluorescence biosensor for detecting and imaging intracellular microRNA |
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