Solid-state nanochannels (SSNs) provide a promising approach for biosensing due to the confinement of molecules inside, their great mechanical strength and diversified surface chemical properties; however, until now, their sensitivity and specificity have not satisfied the practical requirements of sensing applications, especially in complex matrices, i.e., media of diverse constitutions. Here, we report a protocol to achieve explicit regional and functional division of functional elements at the outer surface (FE_OS) and inner wall (FE_IW) of SSNs, which offers a nanochannel-based sensing platform with enhanced specificity and sensitivity. The protocol starts with the fabrication and characterization of the distribution of FE_OS and FE_IW. Then, the evaluation of the contributions of FE_OS and FE_IW to ionic gating is described; the FE_IW mainly regulate ionic gating, and the FE_OS can produce a synergistic effect. Finally, hydrophobic or highly charged FE_OS are applied to ward off interference molecules, non-target molecules that may affect the ionic signal of nanochannels, which decreases false signals and helps to achieve the highly specific ionic output in complex matrices. Compared with other methods currently available, this method will contribute to the fundamental understanding of substance transport in SSNs and provide high specificity and sensitivity in SSN-based analyses. The procedure takes 3–6 d to complete.

固态纳米通道（SSNs）由于分子被限制在内部、机械强度高和表面化学性质多样化，为生物传感提供了一种有前途的方法；然而，到目前为止，它们的灵敏度和特异性还不能满足传感应用的实际要求，特别是在复杂的矩阵中，即不同组成的介质中。在这里，我们报告了一种协议，用于在SSN的外表面 (FE _OS ) 和内壁 (FE _IW ) 上实现功能元素的明确区域和功能划分，该协议提供了一个基于纳米通道的传感平台，具有增强的特异性和灵敏度。该协议从 FE _OS和 FE _IW分布的制造和表征开始. _{然后，描述了 FE OS}和 FE _IW对离子门控的贡献的评估；FE _IW主要调节离子门控，FE _OS可以产生协同作用。最后，疏水或高电荷的 FE _OS被用于抵御可能影响纳米通道离子信号的干扰分子、非目标分子，从而减少错误信号并有助于在复杂基质中实现高度特异性的离子输出。与目前可用的其他方法相比，该方法将有助于从根本上理解 SSN 中的物质转运，并在基于 SSN 的分析中提供高特异性和灵敏度。该过程需要 3-6 天才能完成。