Detection of specific biomarkers in cell membranes is critical for cell biology and disease theranostics. Here we develop a versatile terminal protection assay strategy for wash-free quantification and imaging of cell surface proteins using small molecule-linked DNA with programmable signal sequences. DNA probes are designed to link to a small molecule ligand at 3′ end for specific recognition of the cell surface protein and a programmable signal sequence at 5′ terminal for delivering detectable responses. Binding of the small molecule ligand to target protein enables protection of the DNA probes from exonuclease I mediated degradation, leaving the surface-binding probes intact while the non-binding probes degraded. This strategy thus allows wash-free detection of the cell surface protein via the selectively protected signal sequence. By programming the signal sequences as peroxidase-like DNAzyme, quantitative polymerase chain reaction (qPCR) targeting DNA and Ag nanoclusters (AgNCs) forming DNA template based on our new finding that the exonuclease I is able to quench the fluorescence of AgNCs, we can develop this strategy into a versatile platform for colorimetric detection, qPCR quantification and fluorescence imaging of the cell surface protein. This platform is demonstrated using a folate-linked DNA probe for folate receptor detection on tumor cell surface. The results revealed that this strategy enables highly selective and sensitive detection of the tumor cells as well as quantification and localization of the membrane protein on the cells, implying its potential in membrane protein based biomedical and clinical applications.

细胞膜中特定生物标志物的检测对于细胞生物学和疾病诊断学至关重要。在这里，我们开发了一种多功能的末端保护测定策略，用于使用具有可编程信号序列的小分子连接的DNA进行细胞表面蛋白的免洗定量和成像。DNA探针设计为在3'末端连接至小分子配体，以特异性识别细胞表面蛋白，并在5'末端连接可编程信号序列，以传递可检测的反应。小分子配体与靶蛋白的结合能够保护DNA探针免受核酸外切酶I介导的降解，使表面结合探针完整无损，而非结合探针降解。因此，该策略允许通过选择性保护的信号序列对细胞表面蛋白进行免洗检测。通过将信号序列编程为过氧化物酶样DNA酶，针对DNA和Ag纳米团簇（AgNCs）的定量聚合酶链反应（qPCR）形成DNA模板，基于我们的新发现，即核酸外切酶I能够淬灭AgNCs的荧光，我们可以开发出该策略可用于细胞表面蛋白的比色检测，qPCR定量和荧光成像的多功能平台。使用叶酸连接的DNA探针在肿瘤细胞表面检测叶酸受体，证明了该平台的有效性。结果表明，该策略能够对肿瘤细胞进行高度选择性和灵敏的检测，以及对细胞膜蛋白的定量和定位，这暗示了其在基于膜蛋白的生物医学和临床应用中的潜力。