Solid-state nanopore detection of metal ions via a DNAzyme–Catalytic Hairpin Assembly reaction†

Yesheng Wang‡ ^abe, Lan Shao‡ ^ae, Chunmiao Yu ^abe, Yanru Li ^bc, Ruiping Wu ^d, Jin Yu ^bc, Lu Zhou ^f, Tianlu Wang ^f, Yan Zhao ^ag, Jinbo Zhu *^ae and Bingling Li *^bc
aDepartment of Gastric Surgery, Cancer Hospital of Dalian University of Technology, Shenyang, Liaoning 110042, P. R. China. E-mail: jinbozhu@dlut.edu.cn
^bState Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China. E-mail: binglingli@ciac.ac.cn
^cSchool of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, P. R. China
^dDepartment of Laboratory Medicine, the First Affiliated Hospital of Xi’an Medical University, Xi’an, Shaanxi 710077, P. R. China
^eSchool of Biomedical Engineering, Faculty of Medicine, Dalian University of Technology, Dalian, Liaoning 116024, P. R. China
^fDepartment of Radiotherapy, Cancer Hospital of Dalian University of Technology, Shenyang, Liaoning 110042, P. R. China
^gProvincial Key Laboratory of Interdisciplinary Medical Engineering for Gastrointestinal Carcinoma, Liaoning Cancer Hospital & Institute, Shenyang, Liaoning 110042, P. R. China

First published on 1st July 2025

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Nanopore technology is a label-free, high-sensitivity, and high-throughput single-molecule analysis method for proteins, nucleic acids, and polymers.^1–6 Under an electric field, molecules passing through a nanopore will induce current blockades,⁷ enabling characterization of dwell time, amplitude, frequency, and signal shape to derive size, concentration, and structural features.^8–10 While biological nanopores^11–13 have traditionally faced limitations in environmental stability, recent advances through Hofmeister effect modulation¹⁴ have significantly improved their operational stability and stoichiometric control, enabling reliable single-molecule detection even under challenging conditions. In comparison, solid-state nanopores^15–18 maintain inherent advantages in chemical stability, dimensional controllability, and robustness under extreme pH, temperature, and ionic strength conditions. However, limitations in resolution and preparation reproducibility hinder their applications, particularly for detecting targets with sizes significantly smaller than their pore size.

To address this challenge, we proposed the indirect transduction strategy (ITS) to amplify sub-detection-limit targets via nucleic acid circuits, converting them into larger, nanopore-detectable assemblies. ITS avoids nanopore modification, relying instead on nucleic acid reaction design for signal amplification.^19–21 Despite challenges (e.g., reaction complexity and assembly efficiency), ITS offers a novel approach to advance solid-state nanopore applications.

Heavy metal ions exhibit significant toxicity, with even trace concentrations posing severe risks to both human health and ecosystems. The detection of these ions with high sensitivity and specificity is therefore critical for environmental monitoring and biomedical applications. Among established detection tools, metal-specific DNAzymes—a type of catalytic functional nucleic acids—have emerged as particularly promising candidates due to their unique advantages. These DNAzymes demonstrate remarkable ion selectivity, targeting specific metal ions including Pb²⁺,^22,23 Cu²⁺,²⁴ Zn²⁺,²⁵ UO₂²⁺,²⁶ Hg²⁺,²⁷ Ag⁺,²⁸ Mg²⁺,²⁹etc. The detection mechanism relies on ion-induced conformational changes that activate the DNAzyme's catalytic function, resulting in precise cleavage of RNA sites within the substrate strand. This cleavage event functions as a highly specific molecular switch that enables efficient signal transduction. Owing to their rapid kinetics, programmable nature, and compatibility with diverse detection platforms, metal-specific DNAzymes have found widespread application in biosensing, environmental monitoring, and metal ion-activated biomolecular systems.²⁹ Nanopores also provide an ideal detection platform for cleavage fragments or their biosensing products^29–32 as the ionic current signatures of translocating DNA structures through the nanopores can be measured with single-molecule sensitivity.

Metal ions are undetectable by conventional solid-state nanopores due to their size falling below the resolution threshold. Based on the ITS and DNAzymes, here we developed a tandem system combining a DNAzyme with LK-3W-CHA²¹ to achieve high-resolution Pb²⁺ detection using bare glass nanopores. Through a cleavage reaction and CHA, we transformed the target to be tested from Pb²⁺ into DNA assemblies, which can offer a “signal-on” mode in solid-state nanopore detection. In this work, a GR-5 DNAzyme was employed as the Pb²⁺ recognition module, with the C1 sequence inserted into its non-catalytic region to enable subsequent CHA activation post-cleavage. As illustrated in Fig. 1A, domain 1* of the C1 sequence is embedded in the hairpin structure of GR-5, preventing its hybridization with domain 1 of H1 and thereby suppressing CHA initiation. Upon Pb²⁺ binding, GR-5 undergoes catalytic cleavage, releasing the C1 sequence from the substrate strand. The exposed domain 1* of C1 then hybridizes with domain 1 of H1, initiating a toehold-mediated strand displacement reaction that unfolds the hairpin structure of H1 and liberates domain 3. This process propagates through sequential hybridization with H2 and H3, ultimately forming an H1:H2:H3 three-way junction resembling letter “Y” (termed Y1).

The entire cascade—from DNAzyme cleavage to CHA—is designated as the “cleavage–CHA reaction”. In order to enhance the signal contrast between Pb²⁺ positive and negative signals, we implemented the LK-3W-CHA system to amplify outputs.²¹ Considering reaction efficiency and structural complexity, the trimeric product Y3 was selected as the final LK-3W-CHA product for nanopore detection.

A hairpin library was designed (Fig. 1B), comprising three distinct LK-3W-CHA substrate hairpin sets with partially complementary domains (H2-A/H1-B and H2-B/H2-C). Following the CHA pathway, three monomers (Y1A, Y1B, and Y1C) are generated (Fig. 1C). Through base-pairing interactions (a–a* and b–b* linkages), these monomers self-assemble into the trimeric complex Y3ABC via LEGO-like modular stacking (Fig. 1D).

In Fig. 2A, the C1 sequence (highlighted in green) serves as the CHA initiator, rA denotes the RNA cleavage site, and the red arrow marks represent the scissile phosphodiester bond. The GR-5 enzyme and substrate strands were conformationally constrained into a hairpin structure via a short T-rich linker (Fig. 2A), preventing C1-triggered CHA activation in solution.³² Upon Pb²⁺ recognition by GR-5, the DNAzyme catalyzes site-specific hydrolysis at the rA position,²² cutting GR-5 into two fragments: C1 and a residual strand. Post-cleavage, the weak base-pairing interaction between C1 and the enzyme strand—due to limited complementarity—combined with structural breathing dynamics, facilitates dissociation of C1 from the complex and its release into solution for the CHA reaction.

At a GR-5 concentration of 1 μM, nearly complete substrate cleavage was observed in the presence of 5 μM, 10 μM, or 50 μM Pb²⁺ (Fig. 2B, lanes 2–4), whereas no detectable cleavage occurred in the absence of Pb²⁺ (Fig. 2B, lane 1). The migration position of the shorter cleavage product aligned precisely with the 1 μM C1 control (Fig. 2B, lane 5), which confirms the functional integrity of the sequence-modified GR-5 DNAzyme.

As shown in Fig. 2C, in the absence of C1, the hairpin substrates for Y1–Y3 remained largely unconsumed, with intense bands corresponding to unreacted substrates (Fig. 2C, lanes 1–3), confirming few background signals. In the presence of C1, the substrate consumption is relatively complete, and the dark bands from Y1 to Y3 increase step by step, indicating that the products corresponding to the degree of polymerization are accurately synthesized (Fig. 2C, lanes 4–6). These size-resolved bands confirm the controlled synthesis of LK-3W-CHA products, thereby validating the feasibility of the LK-3W-CHA system.

Finally, the cleavage–CHA reaction products obtained under different conditions were characterized via 2.5% agarose gel electrophoresis (Fig. 2D). In negative controls, including no GR-5/C1 (Fig. 2D, lane 1), GR-5 alone (Fig. 2D, lane 2), and a divalent ion control (M²⁺ control, 250 μM Fe²⁺, Cu²⁺, Hg²⁺, Zn²⁺, Ca²⁺, Fig. 2D, lane 3), all with little substrate consumption, only faint bands corresponding to monomer-to-trimer assembly artifacts were observed. These backgrounds likely arise from stem-loop breathing of hairpin substrates, 3′-terminal base loss in GR-5 synthesis, or domain 1* leakage of C1 caused by respiration. In contrast, reactions containing 5 μM or 50 μM Pb²⁺ (Fig. 2D, lanes 4 and 5) exhibited robust substrate conversion, albeit with slightly reduced efficiency compared to direct C1 addition (Fig. 2D, lane 6). This discrepancy may be due to the lower actual concentration of C1 caused by the cleavage reaction, yet the system retains sufficient resolution to differentiate between assembled products.

We preliminarily assessed the assembly of Y3 in the absence/presence of C1 conditions using our glass nanopore system (Fig. 3A) to evaluate signal differentiation. As shown by the representative raw current traces in Fig. 3B, striking differences were observed between the two conditions: short current spikes dominated in the absence of C1, whereas larger current blockades were observed in the presence of C1. Event statistics derived from 300 seconds of continuous recording (Fig. 3B, thermal scatter diagram) revealed that C1 introduction enhanced both the duration time (ms) and amplitude (pA) of translocation events. The high-density region (red) in the scatter plot exhibited a pronounced positional shift, enabling clear differentiation between the two groups.

We then conducted a statistical analysis of amplitude in the two groups and plotted the corresponding histograms (Fig. 3C). Amplitude distributions for both conditions were further quantified. A 20–30 pA negative shift in amplitude was observed upon C1 addition, providing intuitive discrimination. However, amplitude values are inherently dependent on the diameter of nanopores, and robust differentiation relies primarily on scatter plot center shifts and histogram profile disparities rather than absolute amplitude magnitudes.

Based on these findings, we characterized cleavage–CHA reaction products using the glass nanopore platform. Two experimental groups were tested: negative controls (water and M²⁺ control) and Pb²⁺-positive groups (5 μM and 50 μM Pb²⁺). All groups were analyzed using the same glass nanopore and the results are summarized in Fig. 4.

Comparison of current traces (Fig. 4A) revealed significantly shorter current blockades in negative controls compared to Pb²⁺-positive groups, indicating smaller assembly products in the absence of Pb²⁺. This distinction allows preliminary Pb²⁺ detection through raw signal inspection.

Thermal scatter diagrams (Fig. 4B) and histograms (Fig. 4C) demonstrated high-density region shifts. Negative controls exhibited tight clustering near −50 pA, consistent with the nanopore data from the C1-absent group (Fig. 3B) and the electrophoresis results (Fig. 2D), indicating dominant hairpin substrates. Pb²⁺-positive groups showed broadened amplitude distributions (about −50 to −110 pA) and a prolonged duration. The obvious difference in distribution indicates the increased product size from the cleavage–CHA reaction, which can be used to determine whether Pb²⁺ is present.

To quantify the difference in current signals induced by the absence or presence of lead ions, a threshold amplitude of −70 pA was set. In Fig. 3, 72% of the translocation events in the presence of C1 exhibited amplitudes below −70 pA, compared to only 24% in the absence of C1. Similarly, in Fig. 4, the proportion of events exceeding −70 pA (the red line in Fig. 4B) was 38% for the water control and 27% for the M²⁺ control, whereas significantly higher percentages were observed for Pb²⁺-positive groups—58% at 5 μM and 65% at 50 μM. This quantification approach provides an efficient way to determine the presence or absence of Pb²⁺.

We also thoroughly investigated the response of this design to nM-level Pb²⁺. As shown in Fig. S2–S4 and Table S2 (ESI†), our measurements demonstrate a recognition capability reaching 5 nM for Pb²⁺. Based on this platform, through similar design and transduction, we also achieved nM-level detection of UO₂²⁺ using glass nanopores (Fig. S5 and S6, ESI†).

In conclusion, we used an editable LK-3W-CHA reaction system as an amplifier after a metal-specific DNAzyme cleavage reaction and used a glass nanopore to achieve specific and highly sensitive recognition of Pb²⁺. The sub-detection-limit Pb²⁺ can initiate the cleavage-CHA Reaction to produce Y3, which is precisely synthesizable and could be stably detectable by the nanopore system. Given the current limitations of glass nanopores in achieving quantitative resolution, this study focuses on qualitative recognition (presence/absence) of trace Pb²⁺ rather than concentration-dependent analysis. This work proves that our designed system has excellent editability and application potential, and also confirms the ability of the glass nanopore to distinguish the assembly of complex systems. Based on the diverse connection modes between LK-3W-CHA products and the excellent editable properties of the designed system, it is expected that this system can be used to realize simultaneous analysis of multiple metal ions and promote the practical applications of glass nanopores by designing corresponding cutting structures for various DNAzymes and optimizing the connection modes.

B. Li, Y. Wang, and J. Zhu conceived the project. Y. Wang, C. Yu and R. Wu designed the experiments. Y. Wang, L. Shao, C. Yu, Y. Li, and J. Yu performed the experiments. Y. Wang and L. Shao analysed the data. L. Zhou, T. Wang and Y. Zhao discussed the results. All authors participated in writing the manuscript.

This work was financially supported by the Fundamental Research Funds for the Central Universities of China (Grant No. DUT24YG112, DUT24YG117, DUT24YG211, and DUT23RC(3)029), the Liaoning Provincial Doctoral Research Start-up Fund (Grant No. ZX20240550), and the Liaoning Provincial Science and Technology Joint Fund (Grant No. 2023-MSLH-174).

Conflicts of interest

There are no conflicts to declare.

Data availability

All experimental data, including characterization details and analytical results, are available in the ESI† associated with this article.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5cc03429a

‡ These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2025