A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses Self-Induced Back-Action (SIBA) for optical trapping to enable SIBA-Actuated Nanopore Electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A Natural Killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1bQdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1bGroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (KD). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (koff)for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. Thekoffvalue of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cellsin vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.

中文翻译：

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使用自诱导反作用驱动纳米孔电泳 (SANE) 传感器量化自然杀伤细胞抑制受体和靶向配体之间的低亲和力结合相互作用

制造了一种能够检测双峰光学和电学分子特征的等离子体纳米孔传感器，并测试了其表征低亲和力配体-受体相互作用的能力。这种等离子体纳米传感器使用自诱导反作用 (SIBA) 进行光捕获，使 SIBA 驱动的纳米孔电泳 (SANE) 通过位于光阱体积正下方的纳米孔。合成了自然杀伤 (NK) 细胞抑制性受体异二聚体分子 CD94/NKG2A，以靶向特定的呈递肽的 Qa-1bQdm 配体作为免疫细胞与呈递肽的癌细胞之间低亲和力相互作用的简化模型，该模型发生在癌症免疫治疗期间. 与癌症无关的 Qa-1bGroEL 配体也被与对照实验相同的受体靶向，以测试非特异性结合。对不同对双峰 SANE 传感器特征的分析能够区分配体、受体及其复合物，并能够区分特异性和非特异性配体相互作用。我们能够检测到浓度比游离溶液平衡结合常数 (KD) 低 500 多倍的配体-受体复合物结合。此外，SANE 传感器测量能够估计这种低亲和力特异性配体 - 受体系统的快速解离率 (koff)，以前证明用商业技术量化具有挑战性。已知靶向肽呈递配体的 koff 值与体内免疫细胞的后续激活相关，这表明 SANE 传感器作为癌症免疫治疗中的筛选工具的潜在用途。