Anal. Chem. | A general analysis method for catalase and catalase-like nanoenzyme based on dopamine
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*This article was first published on the "Nanozymes" public account, September 12, 2022
* Edited by Yu Jiyuan
catalase (Catalase, CAT) is an important part of the cellular antioxidant system and can be used as an biomarker for many diseases or drugs-induced toxicity. Therefore, the determination of CAT activity is crucial for the precise evaluation of the physiological and pathological conditions of an organism and the rational and efficient design of CAT nanoenzymes. Recently, Professor Wei Hui from , School of Modern Engineering and Applied Sciences, Nanjing University, published a research work titled "A Dopamine-Enabled Universal Assay for Catalase and Catalase-Like Nanozymes" on Analytical Chemistry. The first author of the article is doctoral student Lin Anqi. The authors designed a universal detection method based on Dopamine (DA) to determine the level of oxygen produced by decomposition to determine CAT activity. As a sensitive molecular probe, DA is inhibited in low oxygen conditions or the presence of H2O2; while in the presence of CAT, the O2 explanatory H2O2 promotes the polymerization of DA. Under optimized conditions, the authors achieved CAT activity assays for a variety of typical samples.
Figure 1 Application of DA-based CAT analysis method
First, the author conducted a series of tests on natural CAT. As shown in Figure 2a, under the conditions of oxygen-rich , the aqueous DA solution can undergo spontaneous polymerization, but the process is slow. In an oxygen-deficient environment, DA polymerization is strongly inhibited. In addition, the presence of H2O2 also inhibits the polymerization of DA under aerobic conditions. As shown in Figure 2b, after 30 minutes of incubation under aerobic conditions, DA showed a wide absorption peak at about 405 nm compared to hypoxic conditions, which resulted from the coloring product polydopamine (PDA), and the higher absorbance (O.D.405 nm) observed at 405 nm when CAT is present in the detection system, confirming the feasibility of this method for CAT. As shown in Figure 2c, the presence of CAT and H2O2 results in darkening the solution color and a higher O.D.405 nm value. In addition, as shown in Figure 2d, the O.D.405 nm of CAT is linearly related to its concentration (within the range of 0.5~5U/ml).
Figure 2 Sensing mechanism and detection method of natural CAT. (a) Schematic diagram of CAT-enhanced DA autopolymerization in hypoxia; (b) UV-visible light absorption spectrum of DA polymerization under different conditions; (c) Quantitative bar graph of DA activation analysis of CAT; (d) Linear relationship between CAT concentration and O.D.405 nm
Then, the authors selected 12 nanoenzymes with obvious CAT-like activity for standardized comparison. In order to establish a comparison of effective CAT nanoenzymes, ΔO.D.405 nm was defined to eliminate the effect of OXD activity of nanoenzymes, as shown in eq 1:
where x1 is O.D.405 nm (DA), x2 is O.D.405 nm (DA+H2O2), x3 is O.D.405 nm (DA+H2O2+ sample), and x4 is O.D.405 nm (DA+ sample).
As shown in Figure 3a, under the same conditions, ΔO.D.405 nm is conducive to objective and standardized comparison of different nanoenzymes. In addition, the authors used A405 nm/A480 nm as a measure to determine the selectivity of the nanoenzyme CAT/POD. As shown in Figure 3b, the various nanoenzymes tested above can be classified well according to the following criteria. Based on the above results, this detection method can not only detect the activity of CAT-like nanoenzymes, but also detect the selectivity of nanoenzymes on CAT/POD activity (Fig. 3c).
Next, in order to evaluate the feasibility of this method in practical application, different animal tissues were selected for testing. As shown in Figure 3d, the height of O.D.405 nm can reflect the CAT content in different tissues, the CAT content in the liver is the highest and the CAT content in the muscle is the lowest. This result is consistent with the literature reports.
In addition, the authors also collected non-stimulating saliva from 10 healthy subjects at different times, calculated ΔO.D.405 nm, and summarized it as a heat map shown in Figure 3e. ΔO.D.405 nm can reflect saliva CAT activity and change within a reasonable range over time.
According to the results of animal tissue and human saliva, this detection method is versatile among different types of samples and is suitable for ex vivo analysis of biological samples (Figure 3f).
Figure 3 CAT analysis of CAT-like nanoenzymes, animal tissues and human saliva. (a) Comparison of the activities of different CAT nanoenzymes; (b) Classification of different materials according to the A405 nm/A480 nm value; (c) Schematic diagram of in vitro analysis; (d) Testing of different tissue homogenates prepared by ICR mice; (e) Testing of saliva of healthy subjects collected at different times; (f) Schematic diagram of biological samples ex vivo analysis. Compared with common methods, the test methods mentioned in this article have great advantages in terms of sensitivity, specificity and universality, which helps to formulate measurement standards for CAT and CAT-like nanoenzymes.
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Written by: Zhang Hanjie
Reviewed by: Liu Quanyi
Edited by: Wang Jiazheng
