An organic molecule mimicking enzyme and its application

Abstract

An organic molecular mimetic enzyme capable of catalyzing the oxidation of TMB by H2O2, wherein the organic molecular mimetic enzyme is PQQ. The organic molecular mimetic enzyme is used to catalyze a color change reaction caused by the oxidation of TMB by H2O2. The organic molecular mimetic enzyme is used to detect alkaline phosphatase by catalyzing a color change reaction caused by the oxidation of TMB by H2O2. The detection steps for alkaline phosphatase by catalyzing a color change reaction caused by the oxidation of TMB by H2O2 are as follows: PQQ, TMB, H2O2, and acetate buffer solution are uniformly mixed and incubated for 30 minutes; alkaline phosphatase ALP and phosphorylated ascorbic acid AAP are uniformly mixed and incubated for 30 minutes. Finally, the two solutions are mixed and the color change of the solution is observed visually or the absorbance of the solution at 650 nm is measured using a spectrophotometer. The present invention has the advantages of being simple, rapid, and requiring no special equipment.

Description

Technical Field

[0001] The present invention relates to the field of organic molecule enzyme mimics, in particular to pyrroloquinoline quinone (PQQ) as a mimic of enzyme activity and application, and belongs to the field of chemistry. Background Art

[0002] Natural enzymes play an important role in organisms and have demonstrated widespread applications in disease diagnosis, environmental protection, and biosensor development. Due to their advantages such as high catalytic efficiency, substrate specificity, and mild reaction conditions, catalytic amplification techniques based on natural enzymes have become one of the most widely used analytical tools. However, natural enzymes are expensive to prepare, require demanding storage conditions, and have low recycling and reuse rates. With the advancement of materials chemistry and biotechnology, a variety of enzyme mimics (including organic compounds and nanomaterials) have been reported to exhibit enzyme-like catalytic activity, offering unique advantages such as low cost, high stability, and tunable catalytic activity. Among them, small organic molecules have become a key research focus in the field of enzyme mimics due to their outstanding catalytic activity, high stability, convenient storage, and simple structure. Although research on organic molecular enzyme mimics has made considerable progress, most suffer from poor catalytic activity, susceptibility to oxidative self-destruction, and autoaggregation. Therefore, the design and discovery of novel organic molecular enzyme mimics with high catalytic activity, thermal stability, and environmental stability is highly desirable. Quinone compounds are important redox-active organic molecules that can act as catalysts to promote electron transfer reactions. They have attracted widespread attention in biochemical processes, pollutant degradation, analytical determination, and organic synthesis. Summary of the Invention

[0003] The purpose of the present invention is to discover an organic molecule mimetic enzyme capable of catalyzing TMB oxidation and its application.

[0004] To achieve the purpose of the present invention, the following technical solution is adopted: an organic molecule mimicking enzyme, which can catalyze the oxidation of TMB by H2O2, and the organic molecule mimicking enzyme is PQQ.

[0005] The invention discloses an application of an organic molecule mimicking enzyme, wherein the organic molecule mimicking enzyme is PQQ, and the organic molecule mimicking enzyme is used for catalyzing the color change reaction of TMB oxidized by H2O2.

[0006] The invention discloses an application of an organic molecule mimicking enzyme, wherein the organic molecule mimicking enzyme is PQQ, and the organic molecule mimicking enzyme is used for detecting alkaline phosphatase by catalyzing the color change reaction of TMB oxidized by H2O2.

[0007] Furthermore, the organic molecular mimetic enzyme is used to detect alkaline phosphatase by catalyzing the color change reaction of TMB oxidized by H₂O₂ as follows: PQQ, TMB, H₂O₂, and acetate buffer are mixed uniformly and incubated for 30 minutes; alkaline phosphatase (ALP) and phosphorylated ascorbic acid (AAP) are mixed uniformly and incubated for 30 minutes. Finally, the two solutions are mixed and the color change of the solution is observed visually or the absorbance of the solution at 650 nm is measured using a spectrophotometer.

[0008] The positive and beneficial technical effects of the present invention are as follows: (1) PQQ has the advantages of high catalytic efficiency, good water solubility, and high thermal and chemical stability. (2) The detection of alkaline phosphatase based on the oxidation reaction of TMB catalyzed by PQQ is simple, rapid, and does not require special equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the absorption spectrum of TMB oxidation catalyzed by PQQ.

[0010] Figure 2 It is a comparison of the performance of different substances in catalyzing TMB oxidation.

[0011] Figure 3 This is the curve of TMB oxidation catalyzed by different concentrations of PQQ.

[0012] Figure 4 It is based on the feasibility of detecting alkaline phosphatase ALP using the PQQ-catalyzed TMB oxidation system.

[0013] Figure 5 This is the curve for detecting alkaline phosphatase ALP based on the PQQ-catalyzed TMB oxidation system.

[0014] Figure 6 This is the curve for detecting alkaline phosphatase (ALP) inhibitors based on the PQQ-catalyzed TMB oxidation system. DETAILED DESCRIPTION

[0015] In order to more fully explain the implementation of the present invention, the following examples are provided. These examples are merely elaborations of the process and do not limit the scope of the present invention. The present invention is described in the following examples, but is not limited to the following examples. Any variations are included within the technical scope of the present invention.

[0016] The following examples are specifically described using alkaline phosphatase ALP as an example. Figure 1 This is the absorption spectrum of TMB oxidation catalyzed by PQQ (pyrroloquinoline quinone). Figure 1 It can be seen that in the presence of H2O2, PQQ can catalyze the oxidation of TMB (3,3',5,5'-tetramethylbenzidine). Figure 2The catalytic effects of different substances on TMB oxidation are as follows from 1 to 14: PQQ, phenanthrenequinone, anthraquinone, 1,2-naphthoquinone, anthraquinone-2-carboxylic acid, 1,2-naphthoquinone-4-sulfonic acid sodium salt, 2,3-dichloro-1,4-naphthoquinone, coenzyme Q, rhein, 9-mesityl-10-methylacridine perchlorate, fluorescein, 5-carboxyfluorescein, ATP, GTP. Figure 2 It can be seen that PPQ has the best catalytic effect. Figure 3 This is the relationship curve of TMB oxidation catalyzed by different concentrations of PQQ. Figure 3 It can be seen that the higher the PQQ concentration, the more TMB was oxidized.

[0017] Figure 4 It is based on the feasibility of detecting alkaline phosphatase ALP by the PQQ-catalyzed TMB oxidation system. As can be seen from the figure, ascorbic acid AA can reduce oxidized TMB, resulting in a decrease in absorption intensity and a change in the solution color from blue to colorless; while phosphorylated ascorbic acid AAP cannot cause a significant change in absorption intensity and solution color; however, alkaline phosphatase ALP catalyzes the hydrolysis of AAP to produce AA, resulting in a decrease in absorption intensity; and once the activity of alkaline phosphatase ALP is inhibited by the inhibitor Na3VO4, its ability to catalyze the hydrolysis of AAP to produce AA becomes weak, and the solution of the PQQ-catalyzed TMB oxidation system remains blue with a large absorption intensity. Figure 5 This is the curve for detecting alkaline phosphatase (ALP) based on the PQQ-catalyzed TMB oxidation system. The concentrations of alkaline phosphatase (ALP) are 0, 1, 10, 25, 50, 100, and 250 mU / mL, respectively. Figure 6 This is the curve based on the PQQ-catalyzed TMB oxidation system to detect the alkaline phosphatase (ALP) inhibitor Na3VO4. The Na3VO4 concentrations are 0, 0.01, 0.05, 0.1, 0.2, 0.25, 0.5, 1, and 2 mM, respectively.

[0018] Example 1: PQQ-catalyzed TMB oxidation

[0019] Mix 100 μL of 5 μM PQQ with 100 μL of 50 mM acetate buffer (pH 4.0) containing 1 mM TMB and 50 mM H₂O₂ and incubate for 30 minutes. Finally, observe the color change of the solution visually or measure the absorbance of the solution at 650 nm using a spectrophotometer. Figure 1 This is a feasibility verification of PQQ-catalyzed TMB oxidation. It can be seen that in the presence of H2O2, PQQ can catalyze TMB oxidation. Figure 2This figure compares the performance of different substances substituted for PQQ for the catalytic oxidation of TMB: PQQ, phenanthrenequinone, anthraquinone, 1,2-naphthoquinone, anthraquinone-2-carboxylic acid, 1,2-naphthoquinone-4-sulfonic acid sodium salt, 2,3-dichloro-1,4-naphthoquinone, coenzyme Q, rhein, 9-mesityl-10-methylacridine perchlorate, fluorescein, 5-carboxyfluorescein, ATP, and GTP. As can be seen in the figure, PQQ has the highest efficiency in catalyzing the oxidation of TMB. Figure 3 This is the relationship curve of TMB oxidation catalyzed by different concentrations of PQQ. It can be seen that the higher the PQQ concentration, the more TMB is oxidized.

[0020] Example 2: PQQ-TMB system for detecting alkaline phosphatase ALP

[0021] An application of an organic molecular enzyme mimicking PQQ, wherein the organic molecular enzyme is PQQ. This organic molecular enzyme catalyzes the color change reaction of TMB oxidized by H₂O₂, which is used to detect alkaline phosphatase (ALP). The steps for alkaline phosphatase detection are as follows: Solution A: PQQ, TMB, H₂O₂, and acetate buffer are mixed evenly and incubated for 30 minutes; Solution B: Alkaline phosphatase (ALP) and phosphorylated ascorbic acid (AAP) are mixed evenly and incubated for 30 minutes. Finally, solutions A and B are mixed, and the color change of the solutions is observed visually or the absorbance at 650 nm is measured using a spectrophotometer.

[0022] Solution A: Mix 100 μL of 2 μM PQQ with 100 μL of 50 mM acetate buffer (pH 3.5) containing 1 mM TMB and 50 mM H₂O₂, then incubate for 30 minutes. Solution B: Add 10 μL of various alkaline phosphatase (ALP) concentrations to 40 μL of 10 mM Tris-HCl buffer (pH 7.4) containing 10 mM phosphoascorbic acid (AAP), then incubate for 30 minutes. Finally, mix Solution A and Solution B, and observe the color change visually or measure the absorbance at 650 nm using a spectrophotometer. Figure 5 The figure shows a curve of the absorbance value of the solution at 650 nm and the concentration of alkaline phosphatase (ALP). As can be seen from the figure, as the concentration of alkaline phosphatase (ALP) increases, the absorbance value of the solution decreases, indicating that the absorption intensity of TMB oxide changes due to ascorbic acid AA produced by ALP catalysis. The concentration of alkaline phosphatase (ALP) can be measured, and 1 mU / mL of alkaline phosphatase can be detected using a spectrophotometer.

[0023] Example 3: PQQ-TMB system for detecting alkaline phosphatase inhibitors

[0024] An application of an organic molecule mimicking enzyme, wherein the organic molecule mimicking enzyme is PQQ: the organic molecule mimicking enzyme is used to detect alkaline phosphatase inhibitors by catalyzing the color change reaction of TMB oxidized by H2O2.

[0025] The steps for detecting alkaline phosphatase inhibitors are as follows: Solution A: Mix PQQ, TMB, H2O2, and acetate buffer, then incubate for 30 minutes. Solution C: Mix a mixture of alkaline phosphatase (ALP) and its inhibitor with phosphorylated ascorbic acid (AAP), then incubate for 30 minutes. Finally, mix Solutions A and C, and observe the color change visually or measure the absorbance at 650 nm using a spectrophotometer. The steps for visual analysis of alkaline phosphatase inhibitors are as follows: Solution A: Mix PQQ, TMB, H2O2, and acetate buffer, then incubate for 30 minutes. Solution C: Mix a mixture of alkaline phosphatase (ALP) and its inhibitor with phosphorylated ascorbic acid (AAP), then incubate for 30 minutes. Finally, mix Solutions A and C, and observe the color change visually or measure the absorbance at 650 nm using a spectrophotometer.

[0026] Solution A: Mix 100 μL of 2 μM PQQ with 100 μL of acetate buffer (50 mM, pH 3.5) containing 1 mM TMB and 50 mM H2O2, and incubate for 30 minutes. Solution C: Add 10 μL of a mixture containing 100 mU / mL alkaline phosphatase (ALP) and different concentrations of the inhibitor Na3VO4 to 40 μL of Tris-HCl buffer (10 mM, pH 7.4) containing 10 mM phosphoascorbic acid (AAP) and incubate for 30 minutes. Finally, mix Solution A and Solution C, and observe the color change of the solution with the naked eye or measure the absorbance of the solution at 650 nm using a spectrophotometer. The experimental results are shown in Figure 2. Figure 6 As shown in the figure, as the concentration of the inhibitor Na3VO4 increases, the absorption value of the solution increases, indicating that high concentrations of Na3VO4 are more conducive to inhibiting the activity of ALP. The calculated half-inhibitory concentration of Na3VO4 is approximately 263 μM.

[0027] The mechanism of this invention is discussed below: PQQ catalyzes the decomposition of H₂O₂ to produce reactive oxygen species, which in turn oxidize colorless TMB to its blue oxidized form. ALP catalyzes the hydrolysis of phosphorylated ascorbic acid (AAP). The resulting ascorbic acid (AA) has reducing properties and can reduce the blue oxidized TMB to colorless TMB, thereby lightening the solution color. This process can be monitored visually or with a spectrophotometer.

[0028] After describing the embodiments of the present invention in detail, people familiar with the technology can clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above-mentioned patent application. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are within the scope of the technical solution of the present invention, and the present invention is not limited to the embodiments of the examples given in the specification.

Claims

1. An application of an organic molecule mimicking an enzyme, characterized in that: The organic molecule mimicking enzyme is pyrroloquinoline quinone, and the organic molecule mimicking enzyme is used for catalyzing the color change reaction of TMB oxidized by H2O2.

2. The use of an organic molecule mimicking enzyme according to claim 1, characterized in that: The organic molecule mimic enzyme is used for detecting alkaline phosphatase by catalyzing the color change reaction of TMB oxidized by H2O2.

3. The use of an organic molecule mimicking enzyme according to claim 2, characterized in that: The organic molecular mimic enzyme is used to detect alkaline phosphatase by catalyzing the color change reaction of TMB oxidized by H2O2. The steps are as follows: pyrroloquinoline quinone, TMB, H2O2 and acetate buffer solution are mixed evenly and incubated for 30 minutes; alkaline phosphatase ALP and phosphorylated ascorbic acid AAP are mixed evenly and incubated for 30 minutes; finally, the two solutions are mixed and the color change of the solution is observed with the naked eye or the absorbance of the solution at 650 nm is measured using a spectrophotometer.

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