Precise pH measurements in the immediate environment of receptors is essential for elucidating the mechanisms through which local pH changes associated with diseased phenotypes manifest into aberrant receptor function. However, current pH sensors lack the ability to localize and target specific receptor molecules required to make these measurements. Herein we present the Litmus-body, our recombinant protein-based pH sensor, which through fusion to an anti-IgG nanobody is capable of piggybacking on IgG antibodies for molecular targeting to specific proteins on the cell surface. By normalizing a pH-dependent green fluorescent protein to a long Stokes shift red fluorophore or fluorescent protein, we readily report pH independent of sensor concentration using a single 488 nm excitation. Our Litmus-body showed excellent responsiveness in solution, with a greater than 50-fold change across the regime of physiological pH. The sensor was further validated for use on live cells and shown to be specific to the protein of interest. In complex with our Litmus-body, cetuximab therapeutic antibody retained its functionality in binding and inhibiting ligand interaction of its target epidermal growth factor receptor (EGFR), triggering receptor-mediated endocytosis that allowed tracking of local pH from the cell surface through the endocytic pathway.

中文翻译：

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Litmus-Body：用于细胞表面 pH 测量的分子靶向传感器。

在受体的直接环境中进行精确的 pH 测量对于阐明与患病表型相关的局部 pH 变化表现为异常受体功能的机制至关重要。然而，当前的 pH 传感器缺乏定位和靶向进行这些测量所需的特定受体分子的能力。在这里，我们展示了 Litmus-body，这是我们基于重组蛋白的 pH 传感器，它通过与抗 IgG 纳米抗体融合，能够搭载 IgG 抗体，以分子靶向细胞表面的特定蛋白质。通过将依赖于 pH 值的绿色荧光蛋白标准化为长斯托克斯位移红色荧光团或荧光蛋白，我们可以使用单个 488 nm 激发轻松报告与传感器浓度无关的 pH 值。我们的石蕊试体在溶液中表现出出色的响应能力，在生理 pH 值范围内变化超过 50 倍。该传感器经过进一步验证，可用于活细胞，并显示出对感兴趣的蛋白质具有特异性。与我们的 Litmus-body 复合，西妥昔单抗治疗性抗体保留了其结合和抑制其靶表皮生长因子受体 (EGFR) 配体相互作用的功能，触发受体介导的内吞作用，允许通过内吞途径从细胞表面跟踪局部 pH 值.