Intra Q-body: an antibody-based fluorogenic probe for intracellular proteins that allows live cell imaging and sorting.
Intra Q-body: an antibody-based fluorogenic probe for intracellular proteins that allows live cell imaging and sorting.
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DOI：
10.1039/d2sc02355e
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发表时间：
2022-08-24
期刊：
影响因子：
8.4
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中科院分区：
文献类型：
--
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尽管细胞内生物标志物可以用荧光染料标记的抗体成像，但是由于来自未结合探针的背景噪声，使用这种探针对活细胞中的细胞内生物标志物进行精确成像仍然具有挑战性。在此，我们描述了一种条件活性Fab型淬灭体（Q体）探针的开发，该探针来源于单克隆抗体（DO-1），具有以低背景信号在活细胞中靶向和时空可视化细胞内p53的能力。p53是用于癌症诊断和治疗的关键肿瘤抑制因子和经验证的生物标志物。Q体显示高达27倍的p53水平依赖性荧光增强在体外与检测限为0.72 nM。在固定和活细胞中，分别观察到8.3倍和8.4倍的增强。此外，我们展示了基于p53表达的活细胞分选。这项研究提供了第一个证据的可行性和适用性的Q体探针的活细胞成像的内在细胞内的蛋白质，并打开了一个新的途径，研究和诊断应用细胞内的目标为基础的活细胞分选。基于Q-body技术，开发了一种能够照亮活细胞中肿瘤生物标志物p53的荧光免疫传感器。该技术进一步应用于p53水平的活细胞监测和基于p53表达的活细胞分选。
Although intracellular biomarkers can be imaged with fluorescent dye(s)-labeled antibodies, the use of such probes for precise imaging of intracellular biomarkers in living cells remains challenging due to background noise from unbound probes. Herein, we describe the development of a conditionally active Fab-type Quenchbody (Q-body) probe derived from a monoclonal antibody (DO-1) with the ability to both target and spatiotemporally visualize intracellular p53 in living cells with low background signal. p53 is a key tumor suppressor and validated biomarker for cancer diagnostics and therapeutics. The Q-body displayed up to 27-fold p53 level-dependent fluorescence enhancement in vitro with a limit of detection of 0.72 nM. In fixed and live cells, 8.3- and 8.4-fold enhancement was respectively observed. Furthermore, we demonstrate live-cell sorting based on p53 expression. This study provides the first evidence of the feasibility and applicability of Q-body probes for the live-cell imaging of intrinsically intracellular proteins and opens a novel avenue for research and diagnostic applications on intracellular target-based live-cell sorting. A fluorescent immunosensor that lights up tumor biomarker p53 in living cells was developed based on the Q-body technology. The technology was further applied to the live cell monitoring of p53 levels, and live cell sorting based on p53 expression.
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