Fluorescent probe for determining acetylcholin esterase as well as preparation method and application of fluorescent probe
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A technology of acetylcholinesterase and fluorescent probe, which is applied in the field of enzyme activity detection, can solve the problems of complicated instruments, limited practical application, etc., and achieves the effect that the preparation method is simple and feasible, and the organic synthesis step is omitted.
Active Publication Date: 2019-07-19
QINGDAO UNIV OF SCI & TECH
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Nowadays, the detection of acetylcholinesterase mainly relies on Ellman method, electrochemical method, surface-enhanced resonance Raman scattering method, colorimetric method and fluorescence method. Most of the above methods require complex instruments, which limits their practical application.
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[0043] A method for preparing a fluorescent probe for measuring acetylcholinesterase, the specific steps of the method are as follows:
[0044] Add probe master solution and CuCl in PBS buffer solution at room temperature 2 Solution, stirring and coordination reaction to obtain the target product HBTP-Cu 2+ Complex fluorescent probes.
[0045] Preferably, the pH value of the PBS buffer solution is controlled between 7.5 and 8.5.
[0046] Preferably, the probe mother liquor and the CuCl 2 The molar ratio of the solution is 2:1, and the concentration of the probe mother solution is 2.0~3.0mmol / L, the CuCl 2 The concentration of the solution is 0.05-0.20 mol / L.
[0047] A HBTP-Cu 2+ Application of complex fluorescent probe to selectively recognize acetylcholinesterase in solvent system.
[0048] In order to further realize the technical effect of the present invention, in the solvent system, the volume percentage of water is 95-100%.
[0049] A HBTP-Cu 2+ The application of...
Embodiment 1
[0052] HBTP-Cu 2+ The preparation method of the complex: Add the probe mother solution (8 μL, 2.0 mmol·L -1 ) and CuCl 2 Solution (0.1μL, 0.05mol L -1 , 0.5equ.), that is, mixed according to the molar ratio of 2:1, coordination occurs, and HBTP-Cu is obtained 2+ Aqueous solution of the complex.
Embodiment 2
[0054] HBTP-Cu 2+ The preparation method of the complex: add the probe mother solution (8 μL, 2.5 mmol L to 2 mL PBS buffer solution (pH=7.8) -1 ) and CuCl 2 Solution (0.1μL, 0.1mol L -1 , 0.5equ.), that is, mixed according to the molar ratio of 2:1, coordination occurs, and HBTP-Cu is obtained 2+ Aqueous solution of the complex.
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Abstract
The invention discloses a fluorescent probe for determining acetylcholin esterase as well as a preparation method and application of the fluorescent probe. The fluorescent probe is an HBTP-Cu<2+> compound, and the HBTP-Cu<2+> compound disclosed by the invention takes competitive combination of HBTP and thiocholine (generated by specifically catalyzing acetylcholine through acetylcholin esterase) and Cu<2+> as a basic action mechanism, and takes HBTP luminescence as a fluorescent signal reporting group. When acetylcholin esterase is added into the system, thiocholine can be coordinated and combined with Cu<2+> in the HBTP-Cu<2+> compound structure, the compound structure is disintegrated, the existing form of HBTP is converted from ordered aggregation to monomer dispersion in a bulk phase solution, and color and fluorescence change of the solution is generated. The recognition effect of the HBTP-Cu<2+> compound on acetylcholin esterase is studied through a nuclear magnetic resonance method, an ultraviolet-visible absorption spectrum method and a fluorescence spectrum method, results show that the HBTP-Cu<2+> compound can efficiently and selectively recognize acetylcholin esterase ina pure water solvent system, and the HBTP-Cu<2+> compound has very high sensitivity on acetylcholin esterase.
Description
technical field [0001] The invention belongs to the technical field of enzyme activity detection, and relates to a method strategy for detecting endogenous acetylcholinesterase in cells. More specifically, it relates to a fluorescent probe, its preparation method and its application in detecting acetylcholinesterase. Background technique [0002] Acetylcholinesterase (AChE), a cholinesterase that controls carboxypeptidase and aminopeptidase functions, selectively catalyzes the hydrolysis of the substrate acetylcholine (ATh), a neurotransmitter for nerve signaling. AChE helps promote neuronal development and nerve regeneration, and is mainly distributed in tissues in the human body consisting of the brain, liver, and spinal cord. In addition, there is growing evidence that abnormally elevated alkaline phosphatase levels in the blood are associated with various diseases such as cancer, heart disease, bone disease, and liver disease. Therefore, it is of great significance to ...
Claims
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