Angelw: biocatalysts turn waste into treasure! Simple and rapid detection of halogenated / nitrophenol pollutants!

With the continuous development of city and industry, more and more chemical substances are needed, followed by more and more chemical pollutants Among them, halogenated phenols (HPS) and nitrophenols (NPS) are widely used in home and industrial production The leakage of these two compounds and the degradation of pesticides and herbicides lead to their continuous enrichment in the environment, which is considered to bring great harm to the natural environment and human health As a powerful, sustainable and economical catalyst, biocatalyst may be an effective way to solve these pollution crises Based on this, a flavin dependent monooxygenase (hada) based biocatalysis system (Figure 1) has been developed by pimchaiyen research group of vidyasirimedhi Institute of science and technology in Thailand, which can convert HPS and NPs into benzoquinone (BQ), while benzoquinone and D-cysteine (d-cystein), D-luciferin is produced by d-cys reaction The latter can be used as the substrate of luciferin enzyme to emit fluorescence and realize rapid and simple quantitative / qualitative detection of related pollutants Relevant research results are published on angew Chem Int ed (DOI: 10.1002 / anie 201904923) Firstly, the author examined the efficiency of hada to catalyze the formation of BQ from a series of phenol derivatives (4-nitrophenol (4-NP), 4-Fluorophenol (4-FP), 4-chlorophenol (4-CP), 4-bromophenol (4-brp) and 4-iodophenol (4-ip)) It was found that hada can convert all raw materials into BQ (conversion rate 100%) within 3 hours When d-cys is present in the reaction solution, it is proved that d-fluorescein can be produced by mass spectrometry and 1H NMR When the author added fluorescein enzyme to the reaction solution, the solution would emit yellow green light, and its bioluminescence peak was about 560nm Since l-fluorescein was not the substrate of fluorescein enzyme, it was proved that the product was indeed d-fluorescein The intermediate product of d-fluorescein synthesis (Figure 2, table 1) was also detected by mass spectrometry (picture source: angel Chem Int ed.) in order to improve the yield of d-fluorescein, the author has carried out a series of optimization (table 1:1) to adjust the concentration of the reaction system: the results show that the optimal concentration is d-cys: 4-NP = 10 / 1, when the concentration of NAD +, fad, hada is 10, 4, 50 μ m, the yield can reach 4.6% 2) Increase the stability of BQ: the addition of ROS scavenger such as catalase or superoxide dismutase can remove the hydrogen peroxide produced by the reaction, thus increasing the stability of BQ, at this time, the highest yield can reach 15.8% 3) Adjusting the pH value of solution: the yield can reach 21% when the pH value is equal to 8, which is 70 times of the yield of the condensation reaction between BQ and d-cys At the same time, the author also studied the effect of adding other reagents on the bioluminescence signal, and finally found that adding 0.05 mm coenzyme A, 4 mm DTT, 0.5% (V / V) glycerin, 62 μ m EDTA can increase the fluorescence signal by two times We also found that the signal of bioluminescence is linearly related to the concentration of ppb, which is exactly the range of detection required by the U.S Environmental Protection Agency and other departments, so this method can be used to detect 4-NP / HPS in the environment and food (image source: angelw Chem Int ed.) due to NP, HP It is the main metabolite of Nitrophenols and Halophenols in vivo, so it is often used as a biomarker in urine and blood to detect the health risks of contacting these substances However, the traditional methods relying on mass spectrometry need pre purification and advanced operation technology, which to some extent hinders the timely detection of these compounds For this reason, the authors have tried whether the method developed by them can detect related substances with bioluminescence (Figure 2, table 1) The authors added 4-NP and 4-CP to blood and synthetic urine samples to simulate samples containing these substances, and then tested them with their developed methods The results showed that the detection limit and quantitative limit of the methods they developed were as follows: the quantitative limit of 4-NP and 4-CP were 0.1 and 0.4 ppb respectively, and the detection limit of 4-NP and 4-CP were 0.04 and 0.1 ppb respectively Therefore, the detection ability of this method meets the detection requirements of the U.S Environmental Protection Agency and other departments At the same time, this method does not need any purification of the sample (photo source: angelw Chem Int ed.) in summary, the author developed a chemical enzyme coupling reaction system, which can convert 4-nps and 4-hps into BQ, and finally synthesize d-fluorescein Through a series of conditions, the best reaction conditions are selected D-fluorescein can be synthesized efficiently and fluorescein can be produced under the action of fluorescein enzyme Based on this method, the author can directly detect the related substances in biological samples qualitatively and quantitatively, without the need of purification of the samples and superb operation skills Therefore, it is a convenient and cheap method for the detection of chemical pollutants, which can be used to prevent food and consumer goods pollution.