Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201, USA.
Departments of Chemistry and Biological Chemistry, University of Michigan, 930 N. University Ave., Ann Arbor, MI 48109, USA.
Int J Mol Sci. 2022 Nov 29;23(23):14936. doi: 10.3390/ijms232314936.
Determination of metal ions such as zinc in solution remains an important task in analytical and biological chemistry. We describe a novel zinc ion biosensing approach using a carbonic anhydrase- luciferase fusion protein that employs bioluminescence resonance energy transfer (BRET) to transduce the level of free zinc as a ratio of emission intensities in the blue and orange portions of the spectrum. In addition to high sensitivity (below nanomolar levels) and selectivity, this approach allows both quantitative determination of "free" zinc ion (also termed "mobile" or "labile") using bioluminescence ratios and determination of the presence of the ion above a threshold simply by the change in color of bioluminescence, without an instrument. The carbonic anhydrase metal ion sensing platform offers well-established flexibility in sensitivity, selectivity, and response kinetics. Finally, bioluminescence labeling has proven an effective approach for molecular imaging in vivo since no exciting light is required; the expressible nature of this sensor offers the prospect of imaging zinc fluxes in vivo.
测定溶液中的金属离子，如锌，仍然是分析化学和生物化学中的一项重要任务。我们描述了一种新的锌离子生物传感方法，该方法使用碳酸酐酶-荧光素融合蛋白，利用生物发光共振能量转移（BRET）将游离锌的水平转化为光谱蓝区和橙区发射强度的比值。除了高灵敏度（低于纳摩尔水平）和选择性外，该方法还允许使用生物发光比定量测定“游离”锌离子（也称为“可动”或“易变”），并且无需仪器仅通过生物发光颜色的变化即可确定离子是否超过阈值。碳酸酐酶金属离子传感平台在灵敏度、选择性和响应动力学方面具有很好的灵活性。最后，由于不需要激发光，生物发光标记已被证明是一种有效的体内分子成像方法；这种传感器的可表达性质为体内锌通量的成像提供了前景。
