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Manganese dioxide thin sheet mimic enzyme sensor and its preparation method and detection method of t4pnk

A technology of manganese dioxide and detection method, which is applied in the direction of material analysis, color/spectral property measurement, and chemical reaction of materials by observing the effect on chemical indicators, etc., can solve problems such as laborious and time-consuming

Active Publication Date: 2019-05-14
ANHUI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are time-consuming, labor-intensive, insensitive, or require radiolabeled substrates to varying degrees.

Method used

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  • Manganese dioxide thin sheet mimic enzyme sensor and its preparation method and detection method of t4pnk
  • Manganese dioxide thin sheet mimic enzyme sensor and its preparation method and detection method of t4pnk
  • Manganese dioxide thin sheet mimic enzyme sensor and its preparation method and detection method of t4pnk

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preparation example Construction

[0028] The present invention provides a kind of preparation method of manganese dioxide flake imitation enzyme sensor, comprising:

[0029] 1) MnO 2 Nanosheet solution mixed with TMB (3,3',5,5'-tetramethylbenzidine) solution to make MnO 2 - TMB mixed solution;

[0030] 2) Dissolving the hairpin DNA in a Tris-HCl buffer solution, and then adding ATP (adenosine triphosphate) and λEXO (λ exonuclease) to mix to prepare a DNA solution;

[0031] 3) MnO 2 - The TMB mixed solution was mixed with the DNA solution to prepare a manganese dioxide flake-like enzyme sensor.

[0032] In step 1) of the above-mentioned preparation method, the consumption of each material can be selected in a wide range, but in order to make the manganese dioxide thin sheet imitation enzyme sensor have more excellent sensitivity and detection limit, preferably, in step 1), MnO 2 MnO in nanosheet solution 2 The concentration of 3-15mmol / L, the concentration of TMB solution is 30-50mmol / L; And with respect ...

preparation example 1

[0051] MnO 2 Preparation of nanosheet solution:

[0052] Using the method reported in "K.Kai, Y.Yoshida, H.Kageyama, G.Saito, T.Ishigaki, Y.Furukawa and J.Kawamata, J.Am.Chem.Soc., 2008, 130, 15938-15943." Preparation: Under open conditions at 25°C, mix 12mL of tetramethylammonium hydroxide solution (1.0M, solvent is water), 2mL of H 2 o 2 Solution (30wt%, solvent is water), join in the 10mL manganese chloride solution (the MnCl that contains 0.593g 2 4H 2 O) react 15s to make MnO 2 nanosheet solution.

Embodiment 1

[0054] 1) At 25°C, 1 mL of the above MnO 2 Nanosheet solution (10 mM) was mixed with 1 mL of TMB (40 mM) solution for 5 min to prepare MnO 2 - TMB mixed solution;

[0055] 2) Dissolve hairpin DNA (100nM) of grade A40 in Tris-HCl buffer solution (pH 7.4), then add ATP (10mM) and λEXO (10U) to mix to prepare a DNA solution;

[0056] 3) At 25°C, 75 μL of MnO 2 - The TMB mixed solution was mixed with 25 μL of the DNA solution for 15 min to prepare the manganese dioxide flake-mimicking enzyme sensor A1.

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Abstract

The invention discloses a manganese dioxide sheet mimic enzyme sensor and a preparation method thereof as well as a T4PNK detection method. The preparation method comprises the following steps: (1) mixing a MnO2 nanosheet solution with a TMB (3,3',5,5'-tetramethyl benzidine) solution to obtain a MnO2-TMB mixed solution; (2) dissolving hairpin DNA in a Tris-HCl buffer solution, and adding ATP (adenosine triphosphate) and lambda EXO (lambda exonuclease) for mixing to obtain a DNA solution; and (3) mixing the MnO2-TMB mixed solution and the DNA solution to obtain the manganese dioxide sheet mimic enzyme sensor. The preparation method is simple in operation, and the sensor has the characteristics of high sensitivity, low detection limit and simple operation when used for detecting T4PNK.

Description

technical field [0001] The invention relates to a simulated enzyme sensor, in particular to a manganese dioxide thin slice simulated enzyme sensor, a preparation method and a T4PNK detection method. Background technique [0002] T4 polynucleotide kinase (PNK), a 5' kinase, has been the most commonly used enzyme in molecular biology since it was first discovered in 1965 in E. coli protein extracts infected with even-numbered phages. It can catalyze the 5'-hydroxyl bearing of nucleotides or nucleotides at the 5' end of the phosphate residue of γ-adenosine triphosphate (ATP). This is very important for cellular nucleic acid metabolism, especially in the cellular response to DNA damage, which is closely related to many human diseases, such as Werner syndrome, Bloom syndrome and Rothmund-Thomson–Thomson syndrome. T4PNK is also widely used to detect DNA adducts or oligonucleotides and DNA damage repair. Therefore, the development of a sensitive and accurate assay for detecting T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78G01N21/33
CPCG01N21/33G01N21/78
Inventor 林琳
Owner ANHUI NORMAL UNIV
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