构象转换策略驱动固态纳米孔实现高灵敏生物标志物的定量检测

doi:10.6023/A25040134

摘要/Abstract

miRNAs的浓度检测在肺癌早期精准诊断中具有重要的应用价值, 然而, 由于miRNAs具有低丰度、短链长等特性, 使得快速、低成本且对浓度变化敏感的miRNA检测平台的开发面临实际挑战性. 本研究以肺癌特异性miRNA-21的替代物(22nt单链DNA)为研究对象, 揭示了固态纳米孔在低浓度检测中存在的捕获率非线性饱和现象. 基于此, 提出了孔口竞争理论模型, 认为生物标志物分子在孔口附近的竞争性引发的空间位阻效应是限制检测灵敏度的关键因素. 为克服这一限制, 本研究采用构象转换策略, 将单链DNA (ssDNA)转化为双链DNA (dsDNA), 从而显著降低了孔口的位阻效应和穿孔能垒. 通过数值模拟, 进一步验证了电泳力、电渗力以及位阻效应对分子易位动力学的影响. 实验结果表明, 双链化策略极大提高了相同条件下的捕获率, 并使捕获率与浓度之间的响应曲线转变为线性关系. 同时, 双链化还改变了生物标志物的易位方向, 为基于固态纳米孔技术的复杂生物样本中低丰度生物标志物的超灵敏检测提供了重要的理论基础.

关键词: 生物标志物, 固态纳米孔, MicroRNA, 浓度检测, 构象转换策略, 单链DNA, 双链DNA

The quantitative detection of microRNA (miRNA) is of critical importance for the early diagnosis of cancers, particularly lung cancer, where specific miRNAs such as miRNA-21 have been identified as key biomarkers. However, the low abundance, short chain length, and high sequence homology of miRNAs present significant challenges in developing a rapid, low-cost, and concentration-sensitive detection platform. In this study, we investigate the detection of 22 nt single-stranded DNA (ssDNA), a surrogate for miRNA-21, using solid-state nanopores, and observe a saturation phenomenon in the capture rate at low concentrations, which limits the sensitivity of the detection. To elucidate this phenomenon, we propose a competition theoretical model based on Langmuir adsorption dynamics, which identifies spatial hindrance effects caused by the competitive adsorption of DNA molecules near the nanopore entrance as the primary factor limiting detection sensitivity. This hindrance effect is influenced by the physicochemical properties of DNA, the applied voltage, and the nanopore dimensions. Through numerical simulations, we further analyze the interplay of electrophoretic forces, electroosmotic flow, and electrostatic repulsion in the translocation dynamics of DNA molecules, providing a comprehensive understanding of the underlying mechanisms. To overcome these limitations, we introduce a conformational conversion strategy that transforms ssDNA into double-stranded DNA (dsDNA) through the addition of complementary strands. This strategy significantly reduces the spatial hindrance at the nanopore inlet and lowers the energy barrier for molecular translocation. Experimental results demonstrate that the conversion to dsDNA not only enhances the capture efficiency but also transforms the concentration-response relationship from nonlinear to linear, enabling more accurate quantification of low-concentration analytes. Furthermore, the conformational conversion reverses the translocation direction of the target biomarkers, improving the specificity and sensitivity of the detection process. Our findings reveal that the dsDNA-based approach achieves dual improvements in low-concentration sensitivity and voltage-dependent capture rates, providing a robust framework for the ultrasensitive detection of low-abundance biomarkers in complex biological samples. In conclusion, the conformational conversion strategy-driven solid-state nanopore platform represents a significant step forward in the rapid, low-cost, and concentration-sensitive detection of lung cancer biomarkers, addressing critical challenges in the field and offering new opportunities for early disease diagnosis and personalized medicine.

Key words: biomarkers, solid-state nanopore, microRNA, concentration quantification, conformational conversion strategy, ssDNA, dsDNA

引用此文

尚建宇, 王超超, 高欣冉, 章寅, 沙菁㛃. 构象转换策略驱动固态纳米孔实现高灵敏生物标志物的定量检测[J]. 化学学报, 2025, 83(8): 878-886.

Jianyu Shang, Chaochao Wang, Xinran Gao, Yin Zhang, Jingjie Sha. Conformational Conversion Strategy-Driven Solid-State Nanopore Enables Concentration-Sensitive Quantification of Biomarkers[J]. Acta Chimica Sinica, 2025, 83(8): 878-886.

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图/表 6

图1. (a)使用固态纳米孔检测DNA分子的原理图. (b)实验中典型的电流阻塞事件. (c)不同条件下的离子电流信号. (d)待检测的ssDNA捕获率与浓度的非线性依赖性. 其中插图为50、100、500 pmol•L−1浓度的ssDNA溶液通过纳米孔的离子电流信号

Figure 1. (a) Principle of nanopore-based device for DNA molecules detection. (b) A typical current blockade event in experiments. (c) Ion current traces under different conditions. (d) Saturating nonlinearity relationship between concentration and the capture rate of the target ssDNA. The Inset shows current traces for 50, 100, and 500 pmol•L−1 ssDNA

图2. (a) DNA分子通过纳米孔完整动力学过程的模型示意图. 其中状态i为非捕获区的自由扩散的DNA分子, 状态ii为半捕获区内的DNA分子, 状态iii为被纳米孔捕获的DNA分子, 状态iiii到状态iiiii为DNA分子逐渐脱离纳米孔束缚的过程. (b)实际捕获率与相对吸附比的关系曲线. (c) DNA分子在易位过程中受到的合力变化. (d)单个ssDNA在纳米孔孔口受到的电泳力、电渗力与合力大小与DNA表面电荷密度的关系. 模型中的纳米孔直径为5nm. (e, f) ssDNA与dsDNA易位过程中受到的电泳力、电渗力与合力变化

Figure 2. (a) Schematic illustration of the complete kinetic process of DNA molecules translocating through a nanopore. State i represents DNA molecules freely diffusing in the non-capture region. State ii represents DNA molecules in the semi-capture region. State iii represents DNA molecules captured by the nanopore. States iiii to iiiii represent the process of DNA molecules gradually escaping from the nanopore. (b) Relationship curve between the actual capture rate and the relative adsorption ratio. (c) Variation of the total force acting on DNA molecules during the translocation process. (d) Electrophoretic force, electroosmotic force, and total force acting on a single ssDNA at the nanopore entrance as a function of DNA surface charge density. The nanopore diameter in the model is 5 nm. (e, f) Electrophoretic force, electroosmotic force, and total force acting on ssDNA and dsDNA during the translocation process

图3. (a) ssDNA具有特异性结合的性质. (b)在±500 mV外加电压下, 混合的DNA溶液通过纳米孔的装置示意图与离子电流信号. 其中上图为dsDNA溶液, 下图为n(dsDNA):n(ssDNA)=1:1的分子溶液. (c, d) ssDNA(橙)和dsDNA(蓝)通过5.3 nm纳米孔的幅值占位比和过孔时间直方图. (e) ssDNA(橙)和dsDNA(蓝)通过5.3 nm纳米孔的过孔时间与占位比分布散点图

Figure 3. (a) The specific binding property of ssDNA. (b) Schematic illustration of mixed DNA translocation through a nanopore and the corresponding current traces under ±500 mV applied voltage. The upper panel shows the dsDNA solution, while the lower panel shows the mixed solution of dsDNA:ssDNA at a ratio of 1:1. (c, d) Histograms of amplitude occupancy ratio (c) and dwell time (d) for ssDNA (orange) and dsDNA (blue) through a 5.3 nm nanopore under ±500 mV voltage. (e) Scatter plot of translocation time versus amplitude occupancy ratio for ssDNA (orange) and dsDNA (blue) through a 5.3 nm nanopore

图4. (a)不同浓度dsDNA溶液通过直径为7 nm的纳米孔的幅值占位比直方图. 溶液浓度分别为50、100、200、300、400、500 pmol•L−1. (b)不同浓度dsDNA溶液通过直径为7 nm的纳米孔的幅值占位比的频率点线图.

Figure 4. (a) Histograms of amplitude ratio of different concentrations of dsDNA solutions through a 7 nm nanopore. The solution concentrations are 50, 100, 200, 300, 400, and 500 pmol•L−1, respectively. (b) Dotted line plot of amplitude ratio of different concentrations of dsDNA solutions through a 7 nm nanopore

图5. (a)不同浓度dsDNA溶液通过直径为7 nm的纳米孔的过孔时间直方图. 溶液浓度分别为50、100、200、300、400、500 pmol•L−1. (b)不同浓度dsDNA溶液通过直径为7 nm的纳米孔的过孔时间的频率点线图

Figure 5. (a) Histograms of dwell time of different concentrations of dsDNA solutions through a 7 nm nanopore. The solution concentrations are 50, 100, 200, 300, 400, and 500 pmol•L−1, respectively. (b) Dotted line plot of dwell time of different concentrations of dsDNA solutions through a 7 nm nanopore

图6. (a) ssDNA与dsDNA的捕获率与浓度的关系. (b) ssDNA与dsDNA的捕获率与外加电压的关系

Figure 6. (a) The relationship between capture rates and diverse DNA concentrations. (b) The relationship between capture rates and diverse voltages

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