Xu Lilan, Chen Guanyu, Wu Jiayan, Chen Mingzhu, Wang Wenlu, Chen Zhuhua, Lin Lifang, Sun Weiming, Yao Xu, Zhang Jianzhong, Chen Jinghua, Zhang Xi
Department of Pharmaceutical Analysis, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province, 350122, PR China; Fujian Key Laboratory of Drug Target Discovery and Structural and Functional Research, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province, 350122, PR China; Innovative Drug Research Institute, Fujian Medical University, Fuzhou, Fujian Province, 350122, PR China.
Fujian Key Laboratory of Drug Target Discovery and Structural and Functional Research, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province, 350122, PR China; Innovative Drug Research Institute, Fujian Medical University, Fuzhou, Fujian Province, 350122, PR China.
Biosens Bioelectron. 2025 Sep 15;284:117540. doi: 10.1016/j.bios.2025.117540. Epub 2025 May 2.
It remains a huge challenge to integrate the stability, reproducibility, and sensitivity of electrochemical DNA biosensors (E-sensors) for practical applications in a simplistic yet cost-effective way. In this work, we present a versatile and inclusive hexanethiol self-assembled monolayer (HT SAM) platform that strategically recruits cholesterol and methylene blue (MB) through double-stranded DNA (dsDNA) coordination, incorporating immobilization and reference functionalities onto the HT SAM. Systematically augmented anchoring sites substantially enhanced interfacial DNA probe immobilization stability and efficiency. Additionally, co-immobilized MB functions as an intrinsic reference signal, effectively mitigating the precision limitations arising from reproducibility issues inherent in conventional E-sensors. The upright dsDNA and the coaxial base-stacking promote the target-probe interactions and improve both hybridization efficiency and rate for the interface DNA probes. The tightly packed hydrophobic HT SAM facilitates [Fe(CN)]-mediated cascade electrocatalytic amplification, further increasing E-sensor sensitivity. As a proof-of-concept, the designed base-stacking-driven ratiometric E-sensor using dsDNA-mediated MB-and-cholesterol co-immobilization successfully detected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene-related fragments, demonstrates a wide dynamic range (10 fM to 10 nM) with a low detection limit of 1.32 fM, exhibiting excellent reproducibility and selectivity. With its high detection performance, ease of operation and low cost, this E-sensor is well-suited for point-of-care testing in large-scale disease screening. Above all, the hydrophobic HT SAM as a versatile and inclusive platform combined with the ease of modification of DNA structures to recruit functional molecules and maximize their contributions is key to synergistically enhancing the overall performance of E-sensors.
以一种简单且经济高效的方式将电化学DNA生物传感器（E传感器）的稳定性、可重复性和灵敏度整合到实际应用中仍然是一个巨大的挑战。在这项工作中，我们提出了一个通用且包容的己硫醇自组装单分子层（HT SAM）平台，该平台通过双链DNA（dsDNA）配位策略性地引入胆固醇和亚甲基蓝（MB），将固定和参考功能整合到HT SAM上。系统增加的锚定位点显著提高了界面DNA探针固定的稳定性和效率。此外，共固定的MB作为一个内在的参考信号，有效减轻了传统E传感器中因可重复性问题而产生的精度限制。直立的dsDNA和同轴碱基堆积促进了靶标与探针的相互作用，提高了界面DNA探针的杂交效率和速率。紧密堆积的疏水HT SAM促进了[Fe(CN)]介导的级联电催化放大，进一步提高了E传感器的灵敏度。作为概念验证，使用dsDNA介导的MB和胆固醇共固定设计的碱基堆积驱动的比率型E传感器成功检测到严重急性呼吸综合征冠状病毒2（SARS-CoV-2）N基因相关片段，显示出宽动态范围（10 fM至10 nM），检测限低至1.32 fM，具有出色的可重复性和选择性。凭借其高检测性能、易于操作和低成本，这种E传感器非常适合大规模疾病筛查中的即时检测。最重要的是，疏水HT SAM作为一个通用且包容的平台，结合DNA结构易于修饰以引入功能分子并最大化其贡献，是协同提高E传感器整体性能的关键。
