Label-Free Surface Enhanced Raman Scattering Approach for High-Throughput Screening of Biocatalysts.
Label-Free Surface Enhanced Raman Scattering Approach for High-Throughput Screening of Biocatalysts.
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DOI：
10.1021/acs.analchem.6b00813
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发表时间：
2016-05
影响因子：
7.4
通讯作者：
C. Westley;Yun Xu;A. Carnell;N. Turner;R. Goodacre
中科院分区：
文献类型：
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作者：
C. Westley;Yun Xu;A. Carnell;N. Turner;R. Goodacre
生物催化剂发现和定向进化是许多药物研究项目的核心，但缺乏对生成的大型酶变体库（通常为 10(6)-10(8)）的强大的高通量筛选方法，阻碍了进展并减慢了酶优化。我们开发了一种基于拉曼光谱的无标记通用方法，可显着缩短采集时间（>30 倍）。采用表面增强拉曼散射 (SERS) 来监测次黄嘌呤到黄嘌呤到尿酸的黄嘌呤氧化酶的酶催化转化。该方法直接测量底物和产物，不需要显色底物或冗长的色谱法，成功地针对 HPLC 进行了基准测试，并显示出高水平的准确性和再现性。此外，我们证明这种 SERS 方法可用于监测酶抑制，说明这种高通量筛选方法具有额外的医学意义。
Biocatalyst discovery and directed evolution are central to many pharmaceutical research programs, yet the lack of robust high-throughput screening methods for large libraries of enzyme variants generated (typically 10(6)-10(8)) has hampered progress and slowed enzyme optimization. We have developed a label-free generally applicable approach based on Raman spectroscopy which results in significant reductions in acquisition times (>30-fold). Surface enhanced Raman scattering (SERS) is employed to monitor the enzyme-catalyzed conversion by xanthine oxidase of hypoxanthine to xanthine to uric acid. This approach measures the substrates and products directly and does not require chromogenic substrates or lengthy chromatography, was successfully benchmarked against HPLC, and shows high levels of accuracy and reproducibility. Furthermore, we demonstrate that this SERS approach has utility in monitoring enzyme inhibition illustrating additional medical significance to this high-throughput screening method.