A Strategy for Accessing Nanobody-Based Electrochemical Sensors for Analyte Detection in Complex Media.
A Strategy for Accessing Nanobody-Based Electrochemical Sensors for Analyte Detection in Complex Media.
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DOI：
10.1149/2754-2726/ac5b2e
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发表时间：
2022-03-01
期刊：
影响因子：
--
通讯作者：
Andrew, Trisha L
中科院分区：
文献类型：
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作者：
Fan, Ruolan;Li, Yanfeng;Andrew, Trisha L
纳米抗体是从骆驼科动物分离的单可变结构域抗体，并且由于其相对稳定性、易于生产和分离以及高结合亲和力而迅速将其自身区分为生物传感器中的理想识别元件。然而，通过纳米抗体在真实的时间中转导分析物结合是具有挑战性的，因为大多数纳米抗体在靶识别时不直接产生光学或电信号。在这里，我们报告了一种制造灵敏且选择性的电化学传感器的一般策略，该传感器包含纳米抗体，用于检测异质介质（例如细胞溶解产物）中的目标分析物。石墨毡可以用重组HaloTag修饰的纳米抗体共价官能化。随后使用气相沉积工艺用水凝胶薄层包封提供了包封的电极，其在抗原结合时直接显示电流降低，而不添加氧化还原介体。对于特定抗原浓度，微分脉冲伏安法提供了跨多个电极样品的电极电流的明确和一致的降低。观察到的电流相对于增加的抗原浓度的变化符合Langmuir结合特征，如预期的那样。重要的是，在未纯化的细胞裂解物中的选择性和可重复的靶结合仅由封装的电极证明，抗原检测限约为100 μ g。30 pmol，而在对照实验中缺乏封装的裸电极产生许多假阳性信号。
Nanobodies are single variable domain antibodies isolated from camelids and are rapidly distinguishing themselves as ideal recognition elements in biosensors due to their comparative stability, ease of production and isolation, and high binding affinities. However, transducing analyte binding by nanobodies in real time is challenging, as most nanobodies do not directly produce an optical or electrical signal upon target recognition. Here, we report a general strategy to fabricate sensitive and selective electrochemical sensors incorporating nanobodies for detecting target analytes in heterogeneous media, such as cell lysate. Graphite felt can be covalently functionalized with recombinant HaloTag-modified nanobodies. Subsequent encapsulation with a thin layer of a hydrogel using a vapor deposition process affords encapsulated electrodes that directly display a decrease in current upon antigen binding, without added redox mediators. Differential pulse voltammetry affords clear and consistent decreases in electrode current across multiple electrode samples for specific antigen concentrations. The change in observed current vs increasing antigen concentration follows Langmuir binding characteristics, as expected. Importantly, selective and repeatable target binding in unpurified cell lysate is only demonstrated by the encapsulated electrode, with an antigen detection limit of ca. 30 pmol, whereas bare electrodes lacking encapsulation produce numerous false positive signals in control experiments.