《Nano Lett》基于“纳米抗体镊”二价探针的晶体管传感器件

【1】研究背景

晶体管表面具有电/化学的双向场效应的特性，一方面能够高灵敏监测其表面的生物化学反应，另一方面可以对其表面的电化学反应进行电学调控。因此，建立起生物体系和电子器件之间的多价作用界面对于模拟不同生物体系之间的相互作用，从而构建智能生物电子系统至关重要。由于表面蛋白是生物体系间相互作用的主要部分，本工作旨在建立晶体管与蛋白的多价结合体系，有助于类器官智能芯片的建立。

【2】研究工作简介

如何在纳米尺度上实现具有空间自适应的多价结合仍然具有挑战, 本工作报告了一种“抗体纳米镊”。该“抗体纳米镊”由DNA框架构建，通过控制两个纳米抗体之间的距离使得其与蛋白表面两个不同表位的距离匹配且每个纳米抗体局部有空间自由度，因此，两个纳米抗体可以精准地结合蛋白上两个不同表位且无应力，从而增强结合亲和力（Figure 1）。

Figure 1. Antibody nanotweezer on transistor. (a) Target epitopes on viral protein. (b) Antibody nanotweezer with spatial tolerance. (c)   Schematics of g-FET functionalized with antibody nanotweezers. (d) Micrograph of graphene channel. (e) Photo of packaged device. (f) AFM image of TDD structures in 1× TM buffer. (g) Fluorescence images of antibody nanotweezer with DNA linker 1 and linker 2 modified with Cy3 and Alexa 488, excited by 561 and 488 nm lasers, respectively. Scale bar: 30 µm.

“抗体纳米镊”构筑在晶体管沟道后，其与蛋白的结合会对晶体管进行电学掺杂，从而产生电学信号。该工作用以冠病毒刺突蛋白为例，测试了“抗体纳米镊”对不同浓度蛋白的电学响应。为了验证二价结合的优势，分别测量了修饰有单个纳米抗体以及纳米抗体混合修饰下对刺突蛋白的响应。结果显示，“抗体纳米镊”对刺突蛋白的电学响应明显高于其他的几种情况，也说明了“抗体纳米镊”较单价纳米抗体对刺突蛋白有更高的结合亲和力（Figure 2）。此外，作者将微流控系统与晶体管相结合，测试了“抗体纳米镊”以及单价纳米抗体与刺突蛋白之间的结合亲和力，证明了“抗体纳米镊”较单价纳米抗体高约一个数量级（Figure 3）。

Figure 2. Performance of antibody nanotweezer. (a) Transport curves of g-FETmodified with antibody nanotweezer upon addition of SARS-CoV-2 spike protein atdifferent concentrations. (b)∆V_Diracas a function ofspike protein concentration. (c)Real-time ∆I_ds/I_ds,0 measurement upon consecutiveaddition of SARS-CoV-2 spike protein at different concentrations. (d) ∆I_ds/I_ds,0 response upon spike protein of g-FET functionalized with n3088, mixture of n3088 and n3021, rigid bivalent nanobody, respectively. (e) Gel electrophoresis of TDD with pendent DNA and biotin on top vertexes. (f) ∆I_ds/I_ds,0 response comparison between different receptors upon spike protein. Error bars are determined by the standard deviation of three measurements.

Figure 3. Mechanism study of enhanced performance byantibody nanotweezer. (a) Schematics of microfluidic g-FET for binding kineticsassay. Measured association. (b) and dissociation (c) profilesbetween SARS-CoV-2 spike protein and antibody nanotweezer, n3021, n3088. (d)Binding schematics between protein and nanobodies randomly functionalized ongraphene. (e) AFM image of graphene randomly functionalized with n3021 andn3088 nanobodies. Scale bar: 100 nm. (f) Inter-nanobodydistance histogram for random functionalization of nanobodies on graphene andGaussian fit.

“抗体纳米镊”能实现与蛋白的无应力地二价结合，增强结合亲和力。为了验证该方法的普适性，作者针对寨卡病毒制备了“抗体纳米镊”，并对其相互作用进行了与上述相同的电学验证。与新冠刺突蛋白类似，“抗体纳米镊”能够显著提高对寨卡病毒膜蛋白的电学响应。通过上述“抗体纳米镊”与新冠病毒刺突蛋白和寨卡病毒膜蛋白二价结合的电学验证，证明了“抗体纳米镊”这一个策略的普适性。

Figure 4. Universality theantibody nanotweezer. (a) Envelope protein domain III of Zika virus and itscustomized antibody nanotweezer based on Z1-G8 and Z1-F6 nanobodies. (b)Real-time ∆I_ds/I_ds,0 response of g-FET functionalized with antibody nanotweezer upon consecutive addition of Zikaenvelope protein at different concentrations. (c) ∆I_ds/I_ds,0measurements upon Zika envelope protein of g-FET functionalized with Z1-G8, Z1-F6, mixture of Z1-G8 and Z1-F6, respectively. (d) ∆I_ds/I_ds,0 response comparison between different receptors upon Zika envelope protein. Error bars aredetermined by the standard deviation of three measurements.

与单价抗体相比，“抗体纳米镊”的有效结合亲和力增强了约一个数量级。因此，“抗体纳米镊“修饰的晶体管对病原体蛋白的结合具有更高的信号转导效率，可用于临床病原体的检测。作为展示，作者测试了新冠病毒临床咽拭子样本、人鼻病毒、健康人的临床样本。结果显示，“抗体纳米镊”能有效区分新冠病毒阳性样本和阴性样本，对新冠病毒变异株没有明显的免疫逃逸。

Figure 5. Application of antibodynanotweezer in clinical pathogen testing. (a) ∆V_Dirac response of the bare g-FET and antibodynanotweezer g-FET when exposed to full serum. (b) Real-time ∆I_ds/I_ds,0 response upon sequentialaddition of clinical sample N1, H1, P1 and estimated diagnosetime. (c) |∆I_ds/I_ds,0| response upon clinicalsamples P1-P7, H1-H5, N1-N4. (d) Statistical significance analysis betweenresponses of bivalent nanobody g-FET to positive and negative clinical COVID-19samples, determined by one-way ANOVA followed by a t test (ap: p-value >0.05, **: p-value < 0.01, ***: p-value < 0.001). (e) Real-time ∆I_ds/I_ds,0 response to P2 sample with serial dilution. (f) |∆I_ds/I_ds,0| response to original SARS-CoV-2 strain and itsdelta variant. Error bars are determined by the standard deviation of three measurement.

【3】工作的亮点、新颖性和意义

针对晶体管器件与生物体系的多价作用界面，该工作提出了“纳米抗体镊“的策略，通过DNA框架结构在纳米尺度上控制纳米抗体的间距和局域空间自由度，实现了具有空间自适应无应力地二价结合，为智能生物电子系统构建以及生物分子的精准检提供了一个新的思路。

【4】论文信息

Xuejun Wang, HuaKang, Keke Huang, Mingquan Guo, Yanling Wu, Tianlei Ying, Yunqi Liu, DachengWei, Antibody Nanotweezer Constructing Bivalent Transistor–BiomoleculeInterface with Spatial Tolerance, NanoLett. 2024, 10.1021/acs.nanolett.3c05140

https://pubs.acs.org/doi/10.1021/acs.nanolett.3c05140