Rapid Detection of Phenol Using a Membrane Containing Laccase Nanoflowers

Abstract

With the rapid development of nanoscience and nanotechnology, nanostructured biocatalysts that take the advantage of nanomaterials in terms of both functional and structural availability have offered new opportunities for improving biological functions of enzymes and expanding applications in areas such as biosensors, bioanalytical devices, and industrial biocatalysis. Recently, we reported a method of preparing protein–inorganic hybrid nanostructures with flower-like shapes, which have shown much greater activities than free enzymes and most of the reported immobilized enzymes. To bring this appealing catalyst into practical use, however, an effective accommodation of these high-performance enzyme catalysts is required. One way is to weakly attach these enzyme nanoflowers to porous materials by physical adsorption. Recently, Krieg et al. reported the fabrication of a supramolecular membrane by noncovalent modification of a commercial membrane, which suggests the possibility of fabricating functional filtration membranes by a simple post-modification procedure, thus enabling many new and interesting applications. It thus came to our mind to fabricate a membrane incorporating enzyme nanoflowers for the rapid detection of hazardous compounds through visualization of the catalyzed product. Owing to their high toxicity even at a low concentration, phenols are listed as major toxic pollutants by the Environmental Protection Agency of the USA and other countries. Sensitive detection of phenolic compounds has been well established using instrumental analysis such as liquid chromatography. However, these methods usually require sophisticated instrumentation and a multistep procedure, making them less convenient for rapid and on-site detection. The present study started by the fabrication of an enzyme nanoflower incorporated into a membrane. As shown in Figure 1, a suspension of laccase–inorganic hybrid nanoflowers, which have a high activity (ca. 200% that of free laccase) for phenol oxidization, as we observed previously, was injected into a commercial disposable syringe filter equipped with a cellulose acetate membrane (pore size 0.2 mm). This procedure thus deposited enzyme nanoflowers with an average size of 4 mm onto the membrane. Then the aqueous sample containing phenol was mixed with an aqueous solution of 4-aminoantipyrine and was passed through the membrane with incorporated laccase nanoflowers, causing oxidative coupling of phenol with 4-aminoantipyrine to form an antipyrine dye that has an absorption maximum at 495 nm. This procedure allowed rapid analysis by a UV/ Vis spectrophotometer or by the naked eye. Finally, pure water was injected into the filter to remove unreacted reagents and the reaction products, followed by drying the membrane in air for the next use. For the preparation of laccase–copper phosphate nanoflowers, typically, 0.8 mm aqueous CuSO4 was added to phosphate buffered saline (PBS) containing 0.1 mgmL 1 laccase at pH 7.4 and 25 8C. After three days, the precipitate of laccase nanoflowers appeared with porous, flower-like structures. Scanning electron microscopy (SEM) images of the nanoflowers are presented in Figure 2a,b, from which the average diameter of the laccase nanoflowers was determined [a] L. Zhu, L. Gong, Y. Zhang, R. Wang, Prof. J. Ge, Prof. Z. Liu Department of Chemical Engineering, Tsinghua University Beijing 100084 (China) E-mail : junge@mail.tsinghua.edu.cn liuzheng@mail.tsinghua.edu.cn [b] Prof. R. N. Zare Department of Chemistry, Stanford University Stanford, CA 94305-5080 (USA) E-mail : zare@stanford.edu [] These authors contributed equally to this work. Figure 1. Fabrication, use, washing, and reuse of the membrane with incorporated laccase nanoflowers. Phenol and ortho-, meta-, and para-substituted phenols carrying carboxy, halogen, methoxy, or sulfonic acid groups react with 4-aminoantipyrine to form colored compounds, which can then be readily detected.