Solid-State Nanopore Analysis of DNA-Templated Silver Nanoclusters: Voltage-Dependent Translocation and Electrolyte Stability

Abstract

We investigate the translocation behaviors of fluorescent silver nanoclusters templated in 20- and 37-nucleotide-long DNA strands (DNA/AgNCs) through solid-state nanopores in various electrolyte solutions (1 M KNO₃ and 1 M KCl with 10 mM Tris). Using nanopores with diameters of 2.6, 3.1, 3.6, 4.8, and 5.6 nm, we analyze the stability and translocation characteristics of the DNA/AgNCs across electrolyte conditions ranging from pH 7.6 to 8.4 and applied voltages from 200 to 400 mV. Our findings reveal that AgNCs remain stable in KNO₃, resulting in distinct translocation signatures, whereas they dissociate in KCl, resulting in translocation signatures similar to bare DNA. We reveal how nanopore size and buffer conditions influence translocation behavior, providing a more comprehensive understanding of the DNA/AgNC dynamics. Conductance measurements and the corresponding nanopore diameters confirm the presence of stable AgNCs in KNO₃, with significant current blockades indicative of near-pore clogging events. Additionally, our data highlight that nanopore technology can differentiate DNA/AgNCs from bare DNA based on their translocation patterns, emphasizing the potential for advanced biosensing applications. This fundamental understanding of AgNC behaviors, combined with insights from pore-size-dependent and pH-dependent translocation patterns, not only enhances our knowledge of metallo-DNA structures but also strengthens the potential of nanopore-based analyte differentiation and biosensing applications.

Graphical Abstract

INTRODUCTION

Nanopore technology has emerged as a powerful tool in single-molecule analysis, providing unprecedented precision and versatility in investigating the properties and behaviors of various biomolecules, organic–inorganic composites, and nanoparticles.^1–6 Nanopore sensing offers several advantages over traditional methods,^7–10 including real-time sensing, label-free analysis, single-molecule detection, versatility, portability, and low sample consumption.^11–13 Solid-state nanopores (SSNs), in particular, provide increased stability, tunability of pore diameter, and potential for integration with other technologies.^14–18 These features make SSNs highly suitable for a wide range of applications, from DNA sequencing to protein analysis and biosensing.^9,10,19–21 However, despite its broad application, nanopore analysis has not yet been successfully employed to study noble metal nanoclusters templated in macromolecules such as DNA-templated silver nanoclusters (DNA/AgNCs). This limitation arises from challenges such as electrolyte compatibility issues, the heterogeneous nature of DNA/AgNC species, and the low synthetic yield of these complexes, which complicate analysis using nanopore platforms.^22–24 In this study, we sought to address these challenges by leveraging the capabilities of solid-state nanopores (SSNs) to investigate DNA/AgNCs. These AgNCs are of particular interest due to their unique optical and electronic properties, such as strong fluorescence and size-dependent characteristics, that make them highly valuable in bioanalytical applications.^25–29

AgNCs are small, atomically precise clusters of silver atoms that, when hosted on DNA strands, form enduring complexes with significant potential for bioanalytical applications such as fluorescence-based sensing and imaging.^30–36 The optical properties of DNA/AgNCs make them particularly attractive for enhancing the capabilities of nanopore sensors, as they can provide additional readouts based on fluorescence signals.^37,38 In this study, we focused on the two brightest DNA/AgNCs with simple monomeric structures identified in previous research: 20-nt and 37-nt DNA strands.³⁹ These two sequences were chosen to reduce the heterogeneity of the DNA/AgNCs species analyzed during SSN experiments, as previous studies show that DNA templates shorter than 19-nucleotide (nt) tend to form homodimeric structures (2DNA +AgNCs), while templates longer than 19-nt typically form monomeric structures (1DNA+AgNCs).^40,41,56 We aim to investigate these two AgNCs using resistive pulse nanopore sensing to enhance their potential applications.

The stability of AgNCs is highly dependent on the surrounding chemical environment.⁴² Previous studies have shown that AgNCs exhibit different stability profiles in various electrolyte solutions, which can significantly influence their behavior and detectability in nanopore experiments.⁴³ In particular, KNO₃ has been reported to stabilize AgNCs better than KCl, leading to more reliable fluorescence and translocation signatures. Understanding the influence of electrolyte composition on the stability and translocation behavior of DNA/AgNCs is essential for developing robust nanopore-based sensing platforms.

In this study, we analyzed the translocation behavior of 20-nt and 37-nt DNA and DNA/AgNCs in both KCl and KNO₃ solutions using SSNs to understand how electrolyte composition influences the stability and translocation characteristics of DNA/AgNCs. By measuring changes in ionic current as molecules pass through the nanopores, we gained insights into the size, shape, and charge of the translocating molecules, allowing us to differentiate between them. Our findings demonstrate that selecting appropriate electrolyte environments enhances the stability and fluorescence of DNA/AgNCs, thereby improving the performance of nanoporec-based biosensors. Previous studies have employed DNA nanostructures or position-encoded molecular probes with nanopipette-based nanopore systems for multiplexed detection of weakly charged analytes.^54,55 In contrast, our work leverages solid-state nanopores with DNA/AgNCs, which provide dual electrical and optical signatures, enabling novel analyte differentiation and stability characterization under varying electrolyte conditions. The current study features the first systematic investigation of DNA/AgNCs in different electrolyte environments using SSNs and lays the foundation for integrating AgNCs with nanopore technology for advanced biosensing applications such as molecular diagnostics and bioanalytical research.

RESULTS AND DISCUSSION

Characterization of Fluorescent Silver Nanoclusters.

Hairpin DNA strands effectively host fluorescent AgNCs, providing a uniform environment that promotes their nucleation and growth (Figure 1a). The hairpin structure is crucial, as it significantly influences the optical and physical properties of the AgNCs. As shown previously,³⁹ we synthesized fluorescent AgNCs using hairpin DNA templates with different lengths, 20-nt and 37-nt, that can be universally excited by UV light. The 20-nt and 37-nt DNA/AgNCs were analyzed by ESI-MS to determine the number of Ag atoms bound to each DNA template. The deconvoluted mass spectra showed the presence of various silver species bound to each respective DNA template, with gel-purified samples exhibiting more defined nanoclusters, such as Ag₁₀ for the 20-nt DNA (Figure S1, Supporting Information) and Ag₁₀-Ag₁₅ for the 37-nt DNA (Figure S2, Supporting Information). Compared to the as-synthesized DNA/AgNCs, the reduced heterogeneity achieved by gel purification is crucial for producing consistent translocation signals and fluorescence characteristics during nanopore experiments. Analysis of the isotope distributions offers additional insight into the potential composition of the number of uncharged (Ag⁰) and charged (Ag⁺) silver atoms.^50–53 Theoretical isotopic distributions of the 20-nt and 37-nt DNAs containing Ag₁₀ and Ag₁₃ were constructed and compared with the experimental ones. Alignment of the experimental and theoretical isotopic distributions with different compositions of uncharged and charged Ag atoms is shown in Figures S3 and S4. The composition with the best fit for the 20-nt DNA and 37-nt DNA incorporates 3 uncharged Ag atoms and 7 charged Ag atoms in the Ag₁₀ cluster and 4 uncharged Ag atoms and 9 charged Ag atoms in the Ag₁₃ cluster, respectively. However, it is difficult to confirm the exact number of uncharged and charged Ag atoms for each cluster since the theoretical isotopic distributions for each Ag⁰/Ag⁺ composition share similar Gaussian profiles, and several compositions could coexist and sum together to fit the experimental composition.

Figure 1.

Characterization and detection of DNA/AgNCs. (a) Schematic representation of fluorescent AgNCs hosted in hairpin DNA strands. (b) Photograph images of freshly prepared 20 μM DNA/AgNCs in SPB (pH 7.4) after 30 min of incubation with 1 M KNO₃ and 1 M KCl, under 365 nm UV excitation. 3D-EEM (excitation–emission matrix) fluorescence spectra were also obtained from samples diluted to 5 μM. (c) Detection of DNA and AgNCs using solid-state nanopores (not to scale). (d) Current–voltage (I–V) measurements of two different nanopores, with diameters of 2.6 and 3.1 nm, in 1 M KCl, 10 mM Tris, pH 8.0, used for the experiments. (e) 10 s raw current traces of 20-nt DNA and AgNCs in 1 M KCl and 1 M KNO₃ solutions, and 37-nt DNA and AgNCs in 1 M KNO₃ solution at 300 mV applied transmembrane voltage through 2.6 nm diameter pore. (f) Capture rates of 20-nt and 37-nt DNA and AgNCs in KCl and KNO₃ solutions buffered with 10 mM Tris, pH 8, with respect to applied transmembrane voltage using 2.6 nm (top panel) and 3.1 nm (bottom panel) nanopores. All data were collected with a 10 kHz low-pass Bessel filter and a sampling frequency of 250 kHz.

True color photos of freshly prepared 20 μM DNA/AgNCs in sodium phosphate buffer (SPB), pH 7.4, taken after 30 min of incubation under UV excitation at 365 nm, visually demonstrate the fluorescence properties of the DNA/AgNCs complexes (Figure 1b and Figure S5, Supporting Information). The samples were then diluted to 5 μM for 3D-EEM (excitation–emission matrix) fluorescence analysis. When incubated with 1 M KNO₃, the samples kept their bright fluorescence colors, whereas incubation with 1 M KCl significantly diminished the fluorescence, indicating the importance of the buffer in stabilizing DNA/AgNCs (Figures S6 and S7, Supporting Information). Fluorescence measurements complement the nanopore data by directly confirming the stability of DNA/AgNCs under specific conditions, such as in KNO₃, where the persistence of fluorescence signals indicates intact nanoclusters. This dual detection capability, combining ionic current analysis with fluorescence, highlights the potential of DNA/AgNCs for advanced biosensing applications.

The use of SSNs allows for the detection and differentiation of DNA and AgNCs based on their translocation signatures through a nanoscale aperture (Figure 1c). This technique’s sensitivity and specificity are highlighted by the unique current blockades generated during translocation events, providing valuable information about the size, shape, and conformation of the translocating molecules. Such differentiation is crucial for biosensing and molecular diagnostics, where the accurate identification of the molecular species is essential.

Current–voltage (I–V) measurements for nanopores with diameters of 2.6 and 3.1 nm on a 12 nm thick silicon nitride membrane are critical for understanding the electrical characteristics and ensuring the functionality of the nanopores used in our experiments (Figure 1d). The linear I–V curves confirm the reliability and stability of the nanopores, demonstrating their suitability for detecting translocation events without significant noise or artifacts. Consistent conductance values validate the fabrication quality and readiness of the nanopores for subsequent experiments involving DNA and DNA/AgNCs translocations.

Raw current traces for 20-nt DNA and AgNCs in KCl and KNO₃ solutions as well as 37-nt DNA and AgNCs in KNO₃ solution at 300 mV provide essential insights into the clear translocation signatures of DNA and DNA/AgNCs complexes through the nanopores (Figure 1e). The 20-nt DNA templates have a comparatively higher number of events due to the difference in their hydration dynamics.^44,45 However, the most impactful observation is the translocation of the 20-nt DNA/AgNCs in KCl showing much shallower events compared to KNO₃, indicating that the silver atoms dissociated from the template in KCl solution. Compared to the 37-nt DNA, the 37-nt DNA/AgNCs complexes in KNO₃ exhibited different current signatures, with larger and more frequent current blockades due to the presence of AgNCs. To determine the optimal voltage range for DNA/AgNC detection, we systematically examined the translocation behavior at 200, 300, and 400 mV. Voltages below 200 mV were excluded due to extremely low event rates, which limited statistical analysis. Figure S8 (Supporting Information) shows ionic current traces for the 20-nt and 37-nt DNAs and their AgNC complexes at 200, 300, and 400 mV in 1 M KCl and 1 M KNO₃. The 37-nt AgNCs in KNO₃ showed no translocation but clogs at 200 mV due to their larger size (Figure S8, Supporting Information), while the 20-nt DNA translocated at all voltages. Additionally, five different nanopore diameters (2.6, 3.1, 3.6, 4.8, and 5.6 nm) were tested, revealing that smaller pores (2.6 and 3.1 nm) provided the most distinct translocation signatures, whereas larger pores (4.8 and 5.6 nm) exhibited reduced steric hindrance, leading to lower current blockades and faster translocations. Given these trends, further refinement of the voltage-pore-size combinations could enhance the sensitivity of DNA/AgNC detection.

For 37-nt DNA/AgNCs in KNO₃, significant blockades distinct from those of 20-nt DNA/AgNCs reflect differences in the size and structure between the two complexes. The presence of AgNCs templated to the DNA strands alters their translocation behavior through the nanopore. Larger and more frequent current blockades for DNA/AgNCs complexes occur in KNO₃, suggesting that the AgNCs remain intact and stable in this electrolyte. In contrast, the similarity of the traces for DNA alone and DNA/AgNCs in KCl suggests that the AgNCs dissociate in KCl, resulting in current signatures of DNA/AgNCs similar to those of DNA alone.

The capture rates of 20-nt and 37-nt DNA and DNA/AgNCs complexes in KCl and KNO₃ solutions, using nanopores with diameters of 2.6 and 3.1 nm, provide insight into the efficiency and dynamics of molecule–nanopore interactions (Figure 1f). The capture rate reflects molecule–nanopore interactions influenced by electrophoretic forces, electrostatic interactions, and hydrodynamic drag. Smaller bare DNA molecules are captured more efficiently, while steric hindrance reduces capture rates for larger DNA/AgNCs in smaller pores. In KCl, AgNC dissociation leads to capture rates resembling bare DNA, whereas intact AgNCs in KNO₃ show lower rates due to their larger size. Higher capture rates for DNA/AgNCs complexes in KNO₃ suggest that the AgNCs remain stable and intact in this electrolyte, enhancing their detectability. In contrast, lower capture rates in KCl imply that the AgNCs dissociate, leading to fewer detectable translocation events. The difference in capture rates between the 2.6 and 3.1 nm nanopores highlights the impact of the nanopore size on the efficiency of molecule detection, with larger nanopores favoring higher capture rates. The capture rates of all the samples are tabulated in Table S1 (Supporting Information). To further aid visualization of these findings, a conceptual summary of the translocation behaviors of DNA and DNA/AgNCs under different electrolyte conditions is provided in Figure S17 (Supporting Information), highlighting the distinct signal profiles and molecular interactions observed in each case.

Comparative Analysis of Translocation Properties of 20-nt DNA/AgNCs in KCl and KNO₃.

The translocation behavior of 20-nt DNA and silver nanoclusters (AgNCs) through a 2.6 nm SSN in 1 M KNO₃ and 1 M KCl, both buffered with 10 mM Tris at pH 8, under varying applied voltages (200, 300, and 400 mV), is visualized through scatter plots (Figure 2a–i). These visualizations compare the dwell time versus the current blockade for different analytes at each applied voltage. Comparing the dwell time versus the current blockade for the 20-nt DNA and DNA/AgNCs in KCl revealed similar distribution patterns for both, with narrow distributions of current blockades and dwell times (Figure 2a–c). This similarity indicates that AgNCs in KCl may dissociate, resulting in translocation patterns that closely resemble those of bare DNA. The consistent and relatively uninterrupted translocations for both DNA and DNA/AgNCs in KCl support the hypothesis that AgNCs are unstable in this electrolyte and behave similarly to DNA alone during translocation.

Figure 2.

Quantitative analysis of 20-nt DNA/AgNCs in KCl and KNO₃, 10 mM Tris pH 8 at 200, 300, and 400 mV through a 2.6 nm diameter SSN. (a–c) Dwell time versus current blockade scatter plots of 20-nt DNA and 20-nt AgNCs at 200 (a), 300 (b), and 400 mV (c). Scatter plot for 20-nt DNA and DNA/AgNCs in KCl solution, showing similar distributions, indicating the dissociation of AgNCs. (d–f) Dwell time versus current blockade scatter plots of 37-nt DNA and 37-nt AgNCs at 200 (d), 300 (e), and 400 mV (f). The scatter plots comparing 20-nt DNA in KCl versus KNO₃ solutions, showing no significant difference, indicating that DNA translocation is not affected by the electrolyte type. (g-i) Dwell time versus current blockade scatter plots comparing 20-nt AgNCs and 37-nt AgNCs at 200 mV (g), 300 mV (h), and 400 mV (i). Scatter plot for 20-nt DNA/AgNCs in KNO₃, displaying a wider distribution compared to DNA/AgNCs in KCl, indicating stable AgNCs in KNO₃. All data were collected with a 10 kHz low-pass Bessel filter with a sampling frequency of 250 kHz.

Figure 2d–f provides a comparison of the dwell time versus current blockade for the 20-nt DNA in KCl and KNO₃. At 200 mV (Figure 2d), the 20-nt DNA in KNO₃ shows a slightly wider dwell time distribution, indicating a minor difference in translocation behavior. However, at 300 and 400 mV (Figure 2e,f), the scatter plots show nearly identical patterns for KCl and KNO₃, suggesting that the DNA’s translocation behavior is not significantly influenced by the electrolyte composition at higher voltages. These results imply that the structural integrity and translocation behavior of DNA alone are not substantially influenced by the change in the electrolyte from KCl to KNO₃.

The scatter plots of the 20-nt AgNCs at 200, 300, and 400 mV in KCl compared with those in KNO₃ are also presented in Figure 2g–i. For the 20-nt AgNCs, a striking difference emerges between the KCl and KNO₃ environments. In KNO₃, the 20-nt AgNCs show a much wider dwell time distribution, particularly at higher voltages (300 and 400 mV). This variability indicates more complex and varied translocation events, likely due to the increased stability and intact nature of AgNCs in KNO₃. The well-defined patterns support the hypothesis that AgNCs remain stable in KNO₃, leading to more substantial disruptions of the ionic current during translocation. The presence of intact AgNCs causes more significant disruptions, reflected by the variability in current blockade and dwell time. This indicates that in KNO₃, the AgNCs retain their structure and exhibit clear translocation behavior. Compared to KCl, the DNA/AgNCs disband and behave similarly to DNA alone.

These results confirm that the stability of AgNCs is heavily dependent on the electrolyte environment. The similarity in translocation patterns between DNA and DNA/AgNCs in KCl indicates that AgNCs are unstable and dissociate in this electrolyte (Figure 2a–c). Conversely, the unique and wider distribution patterns observed for DNA/AgNCs in KNO₃ suggest that the AgNCs remain stable and intact, causing more significant disruptions in the ionic current (Figure 2g–i). The dissociation of AgNCs in KCl is attributed to the strong interaction between the chloride ions and silver atoms, leading to the formation of silver chloride (AgCl) precipitates. This process disrupts the stability of the DNA-templated AgNCs, resulting in translocation signals that closely resemble bare DNA. This study demonstrates the potential of nanopore technology for analyzing and characterizing nanoscale complexes in various biochemical applications, particularly in environments where stability and structural integrity are critical. The comparison between DNA/AgNC behaviors in KNO₃ and those in KCl primarily reflects differences in AgNC invariability rather than direct translocation characteristics. In KNO₃, intact DNA/AgNC complexes exhibit clear translocation patterns. Contrarily, the AgNCs dissociate when analyzed from KCl, producing signatures similar to bare DNA. This outcome highlights the stabilizing effect of KNO₃ on DNA/AgNCs during nanopore translocation.

The comparative analysis of 20-nt and 37-nt purified and unpurified AgNCs at varying voltages (200, 300, and 400 mV) through SSNs, as presented in Figures S9 and S10 (Supporting Information), reveals critical insights into the translocation dynamics of these nanoclusters. Both purified and unpurified AgNCs exhibit distinct translocation behaviors, with purified AgNCs consistently showing deeper current blockades and longer dwell times, particularly at higher voltages. This suggests the presence of more structured and uniform AgNC formations, likely due to the removal of impurities. Conversely, unpurified AgNCs display scattered event distributions, with notable differences in their event density and blockade depth, implying the cotranslocation of impurities. The observed differences between 20-nt and 37-nt AgNCs, especially at 400 mV, further highlight the impact of AgNC size and purification on their interaction with the nanopore. These findings are critical for optimizing nanopore sensing protocols and understanding the molecular dynamics of AgNCs in different environments.

Comparative Analysis of Translocation Properties of 20-nt and 37-nt DNA/AgNCs.

The comparative analysis of the translocation behavior of 20-nt and 37-nt DNA and DNA/AgNCs in KNO₃ solution through a 2.6 nm SSN reveals unique characteristics under a voltage range of 200 to 400 mV in 1 M KNO₃ electrolyte solution containing 10 mM Tris at pH 8. These observations highlight the impact of the stabilizing electrolyte environment on different DNA lengths and their respective DNA/AgNCs complexes.

The 20-nt DNA exhibits a relatively narrow distribution of current blockades and dwell times, indicating consistent translocation events with minimal structural variation (Figure 3a–c). In contrast, the 20-nt DNA/AgNCs display a wider distribution with increased current blockades and longer dwell times. The stable AgNCs in KNO₃ disrupt the ionic current more significantly and take longer to pass through the nanopore, confirming the immutability and detectability of AgNCs in this electrolyte. As the voltage increases from 200 to 400 mV, 20-nt AgNCs exhibit broader distributions in dwell time compared to DNA, indicating a more complex interaction with the nanopore.

Figure 3.

Quantitative comparison of 20-nt and 37-nt DNA and AgNCs in 1 M KNO₃ and 10 mM Tris at pH 8 at 200, 300, and 400 mV through a 2.6 nm diameter SSN. (a–c) Dwell time versus current blockade scatter plots of 20-nt DNA and 20-nt AgNCs at 200 mV (a), 300 mV (b), and 400 mV (c), showing clear patterns with a wider distribution for DNA/AgNCs. (d–f) Dwell time versus current blockade scatter plots of 37-nt DNA and 37-nt AgNCs at 200 mV (d), 300 mV (e), and 400 mV (f), also showing a wider distribution for DNA/AgNCs. (g–i) Dwell time versus current blockade scatter plots comparing 20-nt AgNCs and 37-nt AgNCs at 200 mV (g), 300 mV (h), and 400 mV (i), indicating a wider range of current blockades and dwell times for 37-nt DNA/AgNCs due to their larger size and complexity. At 200 mV, 37-nt AgNCs were unable to translocate through the 2.6 nm diameter pore. These plots collectively demonstrate the impact of DNA length and AgNC size on translocation behavior, confirming the immutability and unique signatures of AgNCs in a KNO₃ solution. All data were collected with a 10 kHz low-pass Bessel filter with a sampling frequency of 250 kHz.

Similarly, dwell time versus current blockade scatter plots comparing the translocation of 37-nt DNA and DNA/AgNCs in KNO₃ solution unveil unique patterns (Figure 3d–f). The 37-nt DNA shows a narrow distribution, while the 37-nt DNA/AgNCs exhibit a broader range of current blockades and dwell times. The increased current blockades and longer dwell times for 37-nt DNA/AgNCs indicate that the larger and more complex AgNCs within 37-nt DNA cause more substantial disruptions and take longer to translocate, further supporting the stability of AgNCs in KNO₃. The 37-nt DNA translocates efficiently at all voltages, showing a narrow range of dwell times and shallower current blockades. However, at 200 mV, the 37-nt DNA/AgNCs fail to translocate through the 2.6 nm pore but showed transient clogging events (Figure S8, Supporting Information). At higher voltages (300 and 400 mV), they successfully translocate, displaying significantly longer dwell times and deeper current blockades compared to 37-nt DNA. A higher voltage is required for larger AgNCs to overcome pore restrictions and pass through, likely due to their size and interaction with the pore.

A comparison of the scatter plots of 20-nt DNA/AgNCs and 37-nt DNA/AgNCs in KNO₃ solution reveals that the 37-nt DNA/AgNCs have a wider range of current blockades and dwell times compared to 20-nt DNA/AgNCs (Figure 3g–i). This difference can be attributed to the larger size and more complex structure of the 37-nt DNA/AgNCs, resulting in greater variability in the translocation events. The scatter patterns highlight the impact of DNA length and AgNC size on translocation behavior, with longer DNA strands and larger AgNCs causing more significant current disruptions and longer translocation times. At 300 mV (h), both 20-nt and 37-nt AgNCs show overlapping translocation signatures with similar distributions, suggesting that at this voltage their interactions with the pore are comparable. At 400 mV (i), the 37-nt AgNCs show significantly deeper current blockades and longer dwell times than the 20-nt AgNCs, highlighting the impact of voltage on translocation characteristics and size-dependent behavior.

The findings from Figure 3 provide robust evidence supporting the hypothesis regarding the stability and translocation behavior of DNA/AgNCs in KNO₃ solution. The pattern differences observed in the scatter plots for 20-nt and 37-nt DNA versus their respective DNA/AgNCs complexes highlight the impact of AgNCs on the translocation characteristics. The increased current blockades and longer dwell times for DNA/AgNCs indicate the presence of sturdy nanoclusters that disrupt the ionic current more significantly than DNA alone. The comparison between 20-nt and 37-nt DNA/AgNCs further elucidates the role of DNA length and AgNCs size in translocation behavior. The wider distribution of current blockades and dwell times for 37-nt DNA/AgNCs reflects the greater complexity and variability introduced by larger and more structurally intricate AgNCs. This outcome underscores the sensitivity of nanopore technology in detecting and distinguishing between different molecular structures.

Correlation Analysis of DNA/AgNCs Translocation Dynamics.

The translocation behavior of 20-nt and 37-nt DNA and silver nanoclusters (AgNCs) through a 3.1 nm SSN in 1 M KNO₃ and 10 mM Tris at pH 8, under varying applied voltages (200, 300, and 400 mV), is visualized through scatter plots (Figure 4a–i). These visualizations compare the dwell time versus current blockade for different analytes at each applied voltage.

Figure 4.

Quantitative comparison of 20-nt and 37-nt DNA and AgNCs in 1 M KNO₃, 10 mM Tris pH 8 at 200 mV, 300 mV, and 400 mV through a 3.1 nm diameter SSN. (a–c) Dwell time versus current blockade scatter plots of 20-nt DNA and 20-nt AgNCs at 200 mV (a), 300 mV (b), and 400 mV (c). (d–f) Dwell time versus current blockade scatter plots of 37-nt DNA and 37-nt AgNCs at 200 mV (d), 300 mV (e), and 400 mV (f). (g–i) Dwell time versus current blockade scatter plots comparing 20-nt AgNCs and 37-nt AgNCs at 200 mV (g), 300 mV (h), and 400 mV (i). All data were collected with a 10 kHz low-pass Bessel filter with a sampling frequency of 250 kHz.

The scatter plots for 20-nt DNA and 20-nt AgNCs at 200, 300, and 400 mV illustrate clear translocation events. At 200 mV, 20-nt AgNCs exhibit a broader distribution in both dwell time and current blockade compared to 20-nt DNA, indicating a more heterogeneous translocation process due to the complex structure of AgNCs interacting variably with the nanopore (Figure 4a). At 300 mV, this distinction becomes even more pronounced, with 20-nt AgNCs showing a wide range of dwell times and current blockades, suggesting multiple conformations or binding states during translocation, while 20-nt DNA maintains a confined distribution (Figure 4b). At 400 mV, 20-nt AgNCs still display a well-defined pattern, though with some overlap with the 20-nt DNA. The 20-nt DNA events at this voltage exhibit longer dwell times, possibly due to voltage-induced stretching or uncoiling of the DNA strands during translocation (Figure 4c).

The comparison of 37-nt DNA and 37-nt AgNCs at 200, 300, and 400 mV similarly reveals broader distributions for 37-nt AgNCs, indicating a more complex translocation process. At 200 mV, 37-nt AgNCs show a more spread-out distribution compared to 37-nt DNA, with some instances of higher dwell times for the 37-nt DNA, potentially due to occasional entanglement or folding of the longer DNA strands (Figure 4d). At 300 mV, the 37-nt AgNCs display a wide range of dwell times, suggesting various conformations during translocation. Contrarily, the 37-nt DNA exhibits a more restricted distribution (Figure 4e). At 400 mV, 37-nt AgNCs show two distinct regions with longer dwell times, indicating the presence of at least two dominant translocation conformations or interactions, contrasting with the relatively uniform translocation of 37-nt DNA (Figure 4f).

The comparison of 20-nt and 37-nt AgNCs at 200, 300, and 400 mV reveals that both display broad dwell time distributions with noticeable patterns. At 200 mV, 37-nt AgNCs exhibit deeper current blockades than 20-nt AgNCs, reflecting their larger size and greater interaction with the nanopore (Figure 4g). This trend continues at 300 mV, with 37-nt AgNCs inducing more pronounced current blockades compared to 20-nt AgNCs, consistent with their larger volume (Figure 4h). At 400 mV, 37-nt AgNCs show two specific regions of higher dwell times, while 20-nt AgNCs maintain a single region of distribution, suggesting multiple stable conformations or interactions for the larger AgNCs during translocation (Figure 4i). While Figure 4g–i may suggest that 20-nt AgNCs exhibit longer translocation events, a detailed statistical analysis presented in Figures S15 and S16 confirms that the dwell time of 37-nt AgNCs is consistently longer than that of 20-nt AgNCs.

The results from Figure 4 provide a comprehensive analysis of the translocation behavior of DNA and DNA-templated AgNCs through SSNs under various applied voltages. The broader and more variable distributions of AgNCs compared to DNA suggest that the nanoclusters introduce significant complexity into the translocation process due to their heterogeneous structures and interactions with the nanopore. The clear translocation patterns and current blockade characteristics of AgNCs at different voltages indicate multiple conformations or binding states during translocation, unlike the more uniform behavior of DNA. The pronounced current blockades and longer dwell times of the larger 37-nt AgNCs further highlight the impact of size and structure on translocation dynamics. All these behaviors were also observed with different pore sizes (2.6 nm), as explained in Figure S9 in the Supporting Information. The proportionality between resistive pulse widths and heights observed in Figure 4a–f can be largely attributed to the dynamic behavior and heterogeneity of DNA/AgNC samples. Variations in AgNC size and conformation result in diverse translocation profiles. While temporal resolution limitations due to the 10 kHz low-pass filter may contribute to smoothing effects, the primary driver of the observed proportionality is sample-specific variability.

Analysis of 20-nt and 37-nt DNA/AgNCs in KCl and KNO₃ through Different Pores.

Figure 5 presents a comprehensive analysis of the translocation behavior of the 20-nt and 37-nt DNA and AgNCs using 2.6 and 3.1 nm SSNs in various electrolyte solutions and voltages. The histograms are organized into three columns for 200, 300, and 400 mV and three rows for different analytes and pore sizes.

Figure 5.

Histograms of relative current blockade (ΔI/I) for 20-nt and 37-nt DNA and AgNCs translocated through 2.6 and 3.1 nm SSNs at various applied voltages. Histograms of 20-nt DNA and AgNCs in both KCl and KNO₃ through a 2.6 nm pore at (a) 200 mV, (b) 300, and (c) 400 mV. Histograms of 20-nt and 37-nt DNA and AgNCs in KNO₃ through a 2.6 nm pore at (d) 200, (e) 300, and (f) 400 mV. Histograms of 20-nt and 37-nt DNA and AgNCs in KNO₃ through a 3.1 nm pore at (g) 200, (h) 300, and (i) 400 mV. Eqs S1–S3 were utilized to fit the curves to the histograms. All experiments were conducted in a 1 M electrolytic solution of either KCl or KNO₃ with 10 mM Tris at pH 8. The clear patterns observed for AgNCs in KNO₃, particularly at higher voltages and with larger pore sizes, indicate invariable and unique translocation characteristics compared to DNA, confirming the hypothesis of AgNC stability in KNO₃ and their dissociation in KCl. All data were collected with a 10 kHz low-pass Bessel filter with a sampling frequency of 250 kHz.

Histograms depicting the translocation events of 20-nt DNA and 20-nt AgNCs in KCl and KNO₃ through a 2.6 nm nanopore at 200, 300, and 400 mV are shown in Figure 5a–5c, respectively. At 200 mV, the histogram for 20-nt AgNCs in KNO₃ displays a distinct peak, indicating intact AgNCs, while the other analytes show similar distributions, suggesting the dissociation of AgNCs in KCl (Figure 5a). This trend remains consistent at 300 mV (Figure 5b) and 400 mV (Figure 5c), with 20-nt AgNCs in KNO₃ continuing to display clear peaks that reaffirm their stability in KNO₃, whereas the distributions for the other analytes remain similar, confirming that AgNCs dissociate in KCl and behave like DNA alone.

Figure 5d–5f provides histograms for 20-nt and 37-nt DNA and AgNCs in KNO₃ through a 2.6 nm pore at varying voltages. At 200 mV, no translocation events are observed for 37-nt AgNCs, presumably due to the pore size being close to or smaller than the size of the AgNCs (Figure 5d). However, 20-nt AgNCs exhibit a noticeable peak, indicating successful translocation. At 300 mV, both 20-nt and 37-nt AgNCs show a second peak around a 0.9 relative current blockade, suggesting near-clogging events due to the physical dimensions of the AgNCs relative to the nanopore size (Figure 5e). DNA histograms do not show this second peak, highlighting the difference in translocation behavior between DNA and AgNCs. At 400 mV, similar trends were observed, with 37-nt AgNCs continuing to exhibit definite translocation characteristics compared to those of DNA, confirming the immutability and unique behavior of AgNCs in KNO₃ (Figure 5f).

Histograms for 20-nt and 37-nt DNA and AgNCs in KNO₃ through a 3.1 nm pore at 200, 300, and 400 mV are shown in Figure 5g–5i, respectively. At 200 mV, both 20-nt and 37-nt AgNCs exhibit distinct patterns compared to DNA, producing broader distributions and more pronounced peaks, suggesting that the larger pore size accommodates AgNCs more and allows for clearer differentiation between AgNCs and DNA (Figure 5g). As the voltage increases to 300 mV (Figure 5h) and 400 mV (Figure 5i), the unique patterns for AgNCs become even more apparent. The 37-nt AgNCs show two specific regions of distribution, likely corresponding to different conformational states or sizes of the clusters, while the 20-nt AgNCs maintain a single broad distribution. DNA histograms remain relatively narrow and lack the pronounced peaks observed for the AgNCs. In addition to the findings presented in Figure 5, further analysis of the mean relative current blockade and mean dwell time is presented in Figure S16 (Supporting Information) as a summary of Figure 5, which reinforces the distinct translocation behavior of DNA/AgNCs in KCl and KNO₃. The results support the hypothesis that AgNCs remain stable in KNO₃, leading to higher current blockades and prolonged dwell times compared to free DNA, whereas in KCl, AgNCs disband, producing translocation signatures identical to those of DNA alone.

The results from Figure 5 provide several key insights into the behavior of DNA and AgNCs in different electrolyte solutions under varying conditions. The histograms observed for AgNCs in KNO₃, compared to DNA and AgNCs in KCl, confirm the stability of AgNCs in KNO₃ and their dissociation in KCl. This aligns with our hypothesis that AgNCs are more stable in KNO₃, leading to unique translocation characteristics. The observed peaks around a 0.9 relative current blockade for AgNCs in KNO₃ at higher voltages suggest that the size of the AgNCs is comparable to the nanopore diameter, leading to near-clogging events. This provides further evidence of the structural integrity of AgNCs in KNO₃ and their translocation behavior compared with DNA. At higher voltages, the increased electrophoretic force may lead to structural changes in DNA/AgNCs, such as stretching or partial unfolding of the DNA backbone. These changes could alter the interaction dynamics between the analyte and the nanopore, contributing to variations in ionic current blockades and dwell times that highlight the complex interplay between electrophoretic forces and molecular structure during translocation.

Further comparative analyses demonstrate that both the pore size and analyte composition significantly influence nanopore translocation dynamics. Figures S11 to S13 (Supporting Information) compare translocation signatures through 3.6, 4.8, and 5.6 nm nanopores in pH 7.6, 8.4, and 8.4, respectively. The histograms of current blockade (Figure S14, Supporting Information) and dwell time distributions (Figure S15, Supporting Information) through five different pore sizes confirm that DNA/AgNCs exhibit distinct translocation signatures compared with bare DNA, with steric hindrance and molecular interactions playing a crucial role in event characteristics. This study primarily investigates the effects of the nanopore size and applied voltage on DNA/AgNC translocation in fixed electrolyte environments (1 M KNO₃ or 1 M KCl). While pH variations were inherent to the experimental design due to different nanopore batches (ranging from pH 7.6 to pH 8.4), a systematic pH-dependent study was not within the scope of this work. Additionally, temperature and ionic strength were maintained constant to isolate nanopore and voltage effects. Future work will explore a broader range of pH conditions, ionic strengths, and temperatures to further elucidate their influence on AgNC stability and translocation behavior.

CONCLUSIONS

In this study, we investigated the translocation behavior of 20-nt and 37-nt DNA and their corresponding AgNCs through SSNs under two different electrolytes (KCl and KNO₃) and two different nanopore sizes (2.6 and 3.1 nm). Our findings revealed significant insights into the stability and translocation characteristics of AgNCs compared with bare DNA, shedding light on the complex interactions between these nanomaterials and the nanopore environment. Experiments with larger nanopores (3.6, 4.8, and 5.6 nm) further demonstrated how pore size and buffer conditions impact DNA/AgNC translocation, revealing a balance between steric effects and molecular flexibility across different pH levels (7.6–8.4).

Our experiments demonstrated that AgNCs exhibit translocation patterns in KNO₃, which were markedly different from those observed for DNA alone. The scatter plots and histograms consistently showed that AgNCs have wider distributions of dwell time and current blockade, especially in KNO₃, indicating their immutable and unique interaction with the nanopore. In contrast, in KCl solutions, AgNCs showed translocation behavior similar to that of DNA, suggesting that the silver atoms dissociate from the DNA in KCl, leaving only DNA-like distributions. The translocation patterns of DNA/AgNCs and bare DNA demonstrate the potential for future multiplexed detection strategies, enabling the differentiation of various analytes in complex samples and expanding the applicability of nanopore-based biosensing.

The compatible buffer conditions provided by KNO₃ allowed us to find the optimal nanopore for 20-nt and 37-nt DNA/AgNCs, respectively. The 2.6 nm nanopore was more suitable for 20-nt DNA/AgNCs. A 3.1 nm nanopore provides more definite translocation signatures for 37-nt DNA/AgNCs. However, more defined signatures for the 37-nt AgNCs could be observed under high voltage (400 mV) with a 2.6 nm nanopore. Although further structural analysis is required, the size difference between these nanostructures (20-nt Ag₆ vs 37-nt Ag₁₃) explains these observations. Furthermore, this suggests that a single nanopore can differentiate between AgNCs species templated by different DNAs (e.g., AgNCs templated by 20-nt and 37-nt DNA) or heterogeneous species templated by the same DNA (e.g., 20-nt Ag₆ and 20-nt Ag₁₀), as evidenced by the mass spectrometry profiles of these AgNCs shown in Figures S1 and S2 (Supporting Information).

The application of different voltages provided further insights into the behavior of these nanomaterials. At higher voltages, the patterns of AgNCs in KNO₃ became more pronounced, and we observed specific peaks in the histograms that correlated with the near-pore clogging of the AgNCs. Additionally, translocation data from larger pores provided a broader understanding of how steric interactions and electrostatic effects vary with nanopore size, demonstrating that larger pores facilitate smoother translocation with reduced current blockade depths and dwell times. These findings confirmed the hypothesis that AgNCs remain stable and maintain their unique properties in KNO₃, while they dissociate in KCl.

In conclusion, this research provides a comprehensive understanding of the translocation dynamics of DNA and AgNCs through SSNs. The observed differences in translocation behavior in KCl and KNO₃ solutions highlight the importance of the electrolyte environment in the stability and detection of AgNCs. These findings not only validate our hypothesis regarding the stability of AgNCs in KNO₃ but also pave the way for the further exploration of nanopore-based detection and analysis of complex nanomaterials. Further investigations into a wider range of electrolyte conditions, nanopore geometries, and analyte conformations will provide deeper insights into optimizing solid-state nanopores for precise molecular differentiation. Future studies can build on this work to explore other nanoparticle systems and their interactions with nanopores, potentially expanding the applications of SSN technology in biosensing and nanomaterials research.

EXPERIMENTAL SECTION

Materials.

Ultrapure type I water was produced by using a Millipore-Sigma Direct Q3 filtration system (Burlington, MA). All other chemical reagents and solvents were sourced from Sigma-Aldrich (St. Louis, MO) and were used as received without further purification unless otherwise noted.

DNA/AgNCs Preparation.

The desalted oligonucleotides (Integrated DNA Technologies, Inc. IDT) were suspended at a concentration of 500 μM in DNase-free water. To make 1 mL of 37-nt DNA/AgNCs solution, 80 μL of 500 μM DNA was added to 100 μL of 200 mM sodium phosphate buffer (SPB, pH 7.4) with 748 μL of nuclease-free water. The solution was then mixed with 48 μL of 10 mM silver nitrate (AgNO₃, Cat. No. 204390, Sigma-Aldrich) solution, vortexed, and centrifuged for 1 min at 14,000 RCF. After equilibration for 20 min, the mixture was reduced with 24 μL of 10 mM freshly prepared sodium borohydride (NaBH₄, Cat. No. 480886, Sigma-Aldrich) to form the DNA/AgNCs. For 20-nt DNA/AgNCs, the synthesis protocol was similar, with the following modifications in reagent volumes: 80 μL of 500 μM 20-nt DNA, 100 μL of 200 mM SPB (pH 7.4), and 760 μL of nuclease-free water. The solution was mixed with 40 μL of 10 mM AgNO₃ and reduced with 20 μL of 10 mM freshly prepared NaBH₄. The solution was vortexed and centrifuged again for 1 min at 14,000 RCF. The final concentrations were 40 μM DNA, 20 mM pH SPB (pH 7.4), 480 μM/400 μM AgNO₃, and 240 μM/200 μM NaBH₄. The product was stored at 4 °C for at least 1 week before further analysis. The 200 mM SPB (pH 7.4) was prepared by mixing sodium phosphate dibasic anhydrous (Na₂HPO₄, Cat. No. S375–500, Fisher Scientific) with sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O, Cat. No. S468–500, Fisher Scientific).

Excessive unbound silver atoms were initially removed using a 3 kDa molecular weight cutoff (MWCO) centrifugal filter. The DNA/AgNCs were then further purified through gel electrophoresis. The gel was run at 140 V for 300 min, visualized under a UV transilluminator, and the fluorescent gel band was excised using a gel cutter. This process can filter out nonfluorescent DNA/AgNCs. The excised gel was finely crushed and soaked in 20 mM SPB (pH 7.4) overnight. The resulting solution was transferred to a 0.45 μm polyvinylidene fluoride (PVDF) column filter and centrifuged at 7000g for 30 min to remove any remaining gel fragments.

Optical Characterization.

3D excitation–emission matrix (EEM) spectra were collected on a fluorometer (FluoroMax-4, Horiba) using a quartz cuvette (Cat. No. 16.100F-Q-10/Z15, Sterna Cells). The scan ranges for both excitation and emission were set to be 400 to 800 nm in 5 nm increments. The slit size and integration time were 5 nm and 0.1 s, respectively. Unless otherwise stated, 120 μL of 1 μM sample was used per measurement. The acquired data were postprocessed and visualized using a Python script.

Electrospray Ionization Mass Spectrometry (ESI-MS).

The DNA/AgNCs were analyzed by electrospray ionization mass spectrometry (ESI-MS) as previously described.^24,39 Solutions containing 10 μM samples (DNA or DNA/AgNCs) in 10 mM ammonium acetate were purified using a Micro Bio-Spin $P\overline{6}$ Gel Column (Bio-Rad). Octylamine was added to some solutions at approximately 0.1% (v/v) to reduce the adduction of alkali metals during the ESI process. The samples were then directly infused into an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) using Au/Pd-coated borosilicate emitters fabricated inhouse for nanoelectrospray ionization (n-ESI). Mass spectra were collected in the negative ionization mode between m/z 400 and 3000 using a resolving power of 1,20,000 (defined at m/z 200), an automatic gain control target of 1 × 10⁶ charges, and an ionization voltage of 600 V. Low in-source fragmentation (15 V) was utilized to aid in desolvation of the ions where applicable.

All mass spectra were deconvoluted by using the Xtract algorithm in FreeStyle version 1.8 (Thermo Fisher Scientific). The following deconvolution parameters were applied: signal-to-noise threshold of 3, fit factor of 80%, remainder threshold of 25%, and the nucleotide isotope table was selected. All mass spectra were interpreted and annotated manually with the aid of Mongo Oligo Mass Calculator v2.06. The mass spectrometer was calibrated using a mixture of components designed for analyses conducted in negative ionization mode to ensure high mass accuracy (for example, ≤10 ppm error).

Nanopore Fabrication.

In this work, a nanopore was fabricated using a silicon nitride membrane (NXDB-50B105 V122, Norcada) with a thickness of 12 ± 2 nm, employing the chemically tuned controlled dielectric breakdown (CT-CDB) approach.^46,47 The traditional controlled dielectric breakdown (CDB) process was modified by incorporating sodium hypochlorite (425044, Sigma-Aldrich), resulting in CT-CDB. This modification has been demonstrated to yield a more stable baseline, enhancing its utility for event analysis.

Two silicon nitride chips were positioned side by side with poly(dimethylsiloxane) (PDMS) gaskets to create a seal between two polytetrafluoroethylene flow cells. The PDMS gaskets ensured a tight seal. The flow cell reservoirs were filled with a solution containing 1 M KCl (P9333, Sigma-Aldrich) buffered with 10 mM Tris (J61036, Alfa Aesar) at pH ~ 8. Following prior research protocols, sodium hypochlorite (425044, Sigma-Aldrich) was added to this solution at a predetermined ratio of 2:9.^46,48 Ag/AgCl electrodes were inserted into each half-cell, and voltage was applied to the solution by using a specially designed circuit.

The creation of a nanopore was detected by a dramatic spike in current when the voltage was applied across the silicon nitride membrane. The conductance of the solution in the flow cell containing the nanopore was measured, and the nanopore diameter was calculated using the following equation:

$$G=\sigma {\left[\frac{4L}{\pi D^2}+\frac{1}{D}\right]}^{-1}$$

where σ represents the electrolyte solution’s conductivity, L is the nanopore’s nominal thickness, and D is the diameter of the nanopore. Short voltage pulses (1–3 s) were used to enlarge the nanopores to the desired diameter. After the CT-CDB procedure, the solution’s conductance was measured, and it was replaced with the specific electrolyte (either 1 M KCl or 1 M KNO₃) buffered with 10 mM Tris at pH ~ 8. Baseline current values were monitored throughout the experiment at each applied voltage to ensure that no impurities were present in the flow cell reservoirs. The technique produced nanopores that exhibited linear current–voltage curves and an ohmic behavior. The diameters of the resultant nanopores, calculated using eq 1, were 2.6 ± 0.2 and 3.1 ± 0.2 nm. We used 2.6 and 3.1 nm nanopores for AgNCs characterization to effectively differentiate between various sizes and conformations of AgNCs, ensuring accurate analysis of their translocation behavior.

Data Collection and Analysis.

Signal acquisition was performed using an Axopatch 200B amplifier (Molecular Devices LLC) and digitized via a Digidata 1550B device (Molecular Devices). The experiments employed a 250 kHz sampling rate, with the Axopatch 200B’s built-in 10 kHz Bessel filter applied to the raw data. Data collection was facilitated using pCLAMP 11.2 software (Molecular Devices). During the experiments, EventPro 3.0 software was used to extract data from each event, specifically focusing on resistive pulse depth (ΔI) and duration (Δt).⁴⁹ Further data analysis was carried out using OriginPro 2023.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.5c02405.

ESI-MS results for 20-nt and 37-nt DNA/AgNCs, transilluminator results, typical current traces of DNA/AgNCs through different pores, and further quantitative analyses (PDF)

ABBREVIATIONS

DNA

DNA

DNA/AgNCs

DNA-templated silver nanoclusters

SSN

solid-state nanopore

CT-CDB

chemically tuned controlled dielectric breakdown

ESI-MS

electrospray ionization mass spectrometry