Conformational Conversion Strategy-Driven Solid-State Nanopore Enables Concentration-Sensitive Quantification of Biomarkers

doi:10.6023/A25040134

Abstract

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

Cite this article

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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Fig. & Tab. 6

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

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

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

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

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

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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