Preparation method and application of enzyme-integrated rare earth coordination polymer

A technology of coordination polymers and rare earths, which is applied in the preparation and application of fluorescent probes, can solve the problems of nano-probes in their infancy, improve detection sensitivity and selectivity, improve catalytic activity and stability, and eliminate interference Effect

Pending Publication Date: 2021-11-09
JIANGXI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Even so, most of the current research focuses on using coordination polymers as carriers to enhance the catalytic performance of enzymes, and the use of enzyme-integrated coordination polymers as nanoprobes is still in its infancy.

Method used

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  • Preparation method and application of enzyme-integrated rare earth coordination polymer
  • Preparation method and application of enzyme-integrated rare earth coordination polymer
  • Preparation method and application of enzyme-integrated rare earth coordination polymer

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preparation example Construction

[0027] A method for preparing an enzyme-integrated rare-earth coordination polymer, combining an enzyme with a rare-earth coordination polymer with fluorescent properties, specifically comprising the following steps:

[0028] (1) Metal salt terbium nitrate (Tb(NO 3 ) 3 ) aqueous solution and 20 μg / mL~25 μg / mL superoxide dismutase (SOD) are pre-stirred for 30 minutes;

[0029] (2) Add an aqueous solution of the ligand adenosine triphosphate (ATP), react for 3 hours, centrifuge at 13,000 rpm for 10 minutes, and separate to obtain a solid product, wherein the metal salt terbium nitrate (Tb(NO 3 ) 3 ) and the molar ratio of ligand adenosine triphosphate (ATP) is 8:3;

[0030] (3) Wash the solid product separated in step (2) with water for 3 times, then mix and react with the antenna molecule carboxyphenylboronic acid (CPBA) for 12 hours, and then separate the final solid product, which is the enzyme-integrated rare earth Coordination polymers.

Embodiment 1

[0033] Follow the steps below to prepare SOD@ATP / Tb-CPBA:

[0034] (1) 5.5mg of terbium nitrate was dissolved in 4mL water to obtain an aqueous solution of terbium nitrate;

[0035] (2) 0.1mg SOD was dissolved in 0.1mL water to obtain SOD aqueous solution;

[0036] (3) 6.1 mg of ATP was dissolved in 4 mL of water to obtain an aqueous ATP solution;

[0037] (4) After mixing 4 mL of terbium nitrate aqueous solution and 0.1 mL of SOD aqueous solution, stir for 30 min; then add ATP aqueous solution, and continue stirring for 3 h; wash with water for 3 times to obtain SOD@ATP / Tb;

[0038] (5) 13.3mg CPBA was dissolved in 0.4mL water to obtain CPBA aqueous solution;

[0039] (6) Take the 3.6mL SOD@ATP / Tb (0.1mg / mL) aqueous solution prepared in the above step (4) and step (5) and mix it with 0.4mL CPBA aqueous solution, stir for 12h and centrifuge (13000rpm, 10min), and Washed 3 times with water to obtain SOD@ATP / Tb-CPBA.

Embodiment 2

[0041] Combined with water-soluble tetrazolium salt reagent (WST-1) to detect superoxide anion, and record the absorbance corresponding to its maximum absorption peak with a UV spectrophotometer to detect the catalytic activity of superoxide anion of SOD. Its method steps are:

[0042] (1) 4-Hydroxyethylpiperazineethanesulfonic acid (HEPES) with pH = 7.4 was used as a buffer solution with a total volume of 200 μL and a reaction temperature of 37° C.;

[0043] (2) The superoxide anion is formed by KO dissolved in dimethyl sulfoxide (DMSO) solution 2 get;

[0044] (3) Use a 96-well plate, and number the reaction wells as a, b, c, and d respectively; where,

[0045]The preparation method of the solution in the reaction well a is: HEPBS buffer solution with pH=7.4 containing 10 μM WST-1 reagent, react in a water bath at 37°C for 20 minutes;

[0046] The preparation method of the solution in reaction well b is: HEPBS buffer solution with pH=7.4 containing 10 μM WST-1 reagent and...

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Abstract

The invention relates to the field of preparation and application of fluorescent probes, in particular to a preparation method and application of an enzyme-integrated rare earth coordination polymer. According to the invention, an enzyme is combined with a rare earth coordination polymer with fluorescence characteristics to construct the enzyme-integrated rare earth coordination polymer fluorescent probe; and the enzyme-integrated rare earth coordination polymer fluorescent probe is applied to time-resolved fluorescence detection of superoxide anions and overcomes the problems that existing fluorescent probes are insoluble in water, poor in light stability and serious in background interference in actual detection. The rare earth coordination polymer can protect an enzyme structure from being damaged by environmental factors, and the catalytic activity and stability of the enzyme are greatly improved. In a superoxide anion detection process, the enzyme can specifically recognize superoxide anions and quench fluorescence of the rare earth coordination polymer through an intramolecular charge transfer process. The interference of background fluorescence can be eliminated by the long fluorescence lifetime of the rare earth fluorescence, so the time-resolved fluorescence detection is realized. The method has the advantages of simplicity and convenience in operation, low cost, high efficiency and the like.

Description

technical field [0001] The invention relates to the field of preparation and application of fluorescent probes, in particular to a method for combining rare earth coordination polymers with enzymes to form complex probes. Background technique [0002] Superoxide anion (O 2 ·- ) is a type of reactive oxygen species (ROS), which play an important role in cell growth and metabolism. However, O 2 ·- The overexpression of can cause oxidative stress in the organism, leading to serious diseases such as rheumatoid arthritis, cardiovascular disease and cancer. Therefore, for O 2 ·- The highly sensitive and selective detection of methionine is of great significance for evaluating its role in physiological and pathological processes. Currently, a variety of O 2 ·- Detection techniques such as electron spin resonance, electrochemistry, chemiluminescence, and fluorescence. Among them, fluorescence analysis is often considered as an ideal analysis method because of its advantage...

Claims

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Application Information

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IPC IPC(8): C08G83/00G01N21/64
CPCC08G83/008G01N21/643
Inventor 谭宏亮翁宇豪朱巧玉
Owner JIANGXI NORMAL UNIVERSITY
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