Angular‐Inertia Regulated Stable and Nanoscale Sensing of Single Molecules Using Nanopore‐In‐A‐Tube

Abstract

Nanopore is commonly used for high‐resolution, label‐free sensing, and analysis of single molecules. However, controlling the speed and trajectory of molecular translocation in nanopores remains challenging, hampering sensing accuracy. Here, the study proposes a nanopore‐in‐a‐tube (NIAT) device that enables decoupling of the current signal detection from molecular translocation and provides precise angular inertia‐kinetic translocation of single molecules through a nanopore, thus ensuring stable signal readout with high signal‐to‐noise ratio (SNR). Specifically, the funnel‐shaped silicon nanopore, fabricated at a 10‐nm resolution, is placed into a centrifugal tube. A light‐induced photovoltaic effect is utilized to achieve a counter‐balanced state of electrokinetic effects in the nanopore. By controlling the inertial angle and centrifugation speed, the angular inertial force is harnessed effectively for regulating the translocation process with high precision. Consequently, the speed and trajectory of the molecules are able to be adjusted in and around the nanopore, enabling controllable and high SNR current signals. Numerical simulation reveals the decisive role of inertial angle in achieving uniform translocation trajectories and enhancing analyte‐nanopore interactions. The performance of the device is validated by discriminating rigid Au nanoparticles with a 1.6‐nm size difference and differentiating a 1.3‐nm size difference and subtle stiffness variations in flexible polyethylene glycol molecules.

The study develops a nanopore‐in‐a‐tube (NIAT) device that precisely regulates molecule translocation in a funnel‐shaped nanopore by controlling the inertial angle and centrifugation speed in a centrifuge. This ensures stable signal readout with a high signal‐to‐noise ratio, enabling nanoscale sensing of single molecules.

1. Introduction

Nanopore sensing typically involves measuring the ionic current by applying a bias voltage across a nanopore.^{[ 1} , ² , ^{3 ]} When a molecule passes through the pore, it causes a temporary change in the ionic current.^{[ 4} , ^{5 ]} This change in current can be linked to properties of the molecule, such as size, shape, and surface charge,^{[ 6} , ^{7 ]} as well as the size and geometry of the nanopore,^{[ 8} , ^{9 ]} the way the molecule passes through,^{[ 10 ]} and its transient interactions with the pore.^{[ 11} , ^{12 ]} To accurately detect subtle variations in single molecules at the nanoscale, it is important to have a consistent and stable current signal with a high signal‐to‐noise ratio (SNR).^{[ 13 ]}

To enhance the signal strength, biological nanopores have utilized protein motors to slow down the speed of translocation, enabling successful sequencing of single‐stranded DNA and peptides.^{[ 14} , ^{15 ]} However, effectively reducing the residual fluctuation in the feed rate of the protein motor remains a challenge.^{[ 16 ]} In contrast, solid‐state nanopores have been engineered to achieve similar molecular detection capabilities as biological nanopores, allowing for the detection of various biomolecules like double‐stranded DNA and proteins.^{[ 17} , ¹⁸ , ^{19 ]} Solid‐state nanopores offer clear advantages over their biological counterparts, including high chemical stability, regenerability, controllable geometry, and compatibility with existing semiconductor fabrication techniques.^{[ 20} , ^{21 ]} Moreover, solid‐state nanopores can afford a larger current drop compared to biological nanopores.^{[ 16 ]}

However, in conventional solid‐state nanopore devices, the bias voltage is used to provide the electrophoretic force to push the molecule through the nanopore and simultaneously measure current blockade dips in the detected signal.^{[ 22} , ^{23 ]} While a large ionic current increases signal swing, it leads to faster translocation speed, hence shorter dwell time and reduced SNR.^{[ 13 ]} Various approaches have been explored to slow down the translocation speed, including the incorporation of new potential gradients,^{[ 24} , ²⁵ , ²⁶ , ²⁷ , ²⁸ , ^{29 ]} optimization of test environments,^{[ 30 ]} and fine‐tuning molecular surface charges.^{[ 31} , ³² , ^{33 ]} Although these methods help effectively extend the dwell time, which trajectory molecules are translocated through the nanopore is still largely governed by stochastic factors such as thermal fluctuations.^{[ 34} , ^{35 ]} This manifests in non‐uniform dwell‐times and current drops between the detection of single molecules.^{[ 35} , ^{36 ]}

To achieve precise control over molecular translocation speed, previous works have proposed to use a nano‐positioner or an atomic force microscope cantilever.^{[ 36} , ³⁷ , ³⁸ , ^{39 ]} However, these methods require tethered molecules, which limits complete translocation. Another effective approach is to decouple current signal readout from the electrokinetic translocation mechanism.^{[ 25} , ^{27 ]} Previously, we proposed using inertial force generated through centrifugation to precisely control translocation speed and dwell time by adjusting centrifugal acceleration (or rotation speed).^{[ 40 ]} This inertial‐kinetic translocation method was found to produce more uniform signals compared to electro‐kinetic translocation.

Here, we present a novel approach utilizing angular inertia‐kinetic translocation to precisely regulate the translocation process, including both the speed and passage trajectory of molecules within the nanopore, resulting in a stable current signal with a high SNR. Our study differs from previous research by fabricating square‐base funnel‐shaped nanopores (FSN) in a micrometer‐thick single‐crystal silicon wafer. We achieve this through a home‐developed photovoltaic etch‐stop assisted anisotropic chemical etch technique, enabling better controllability of nanopore size and improved mechanical strength to withstand high levels of inertial force. The incorporation of a funnel shape in the nanopore design facilitates efficient guidance of target molecules toward the pore.

We then integrated the angle‐adjustable 10‐nm FSN into a centrifugal tube, creating a nanopore‐in‐a‐tube (NIAT) device. This allows for angular inertia‐kinetic translocation by separating molecular translocation from electrophoretic force and enables wireless measurements of current blockade signals. By maintaining a light‐regulated counter‐balanced state of electrophoretic and electroosmotic effects in FSN, we observed an appreciable controllability of inertial angle and centrifugation speed, resulting in adjustable frequency of translocation events, dwell time, and current amplitude. Here, two representative synthetic materials, Au nanoparticles (AuNPs) and polyethylene glycol (PEG) molecules, were chosen to characterize the performance of the translocation method in molecular analysis due to their controllable size, well‐defined chemical properties, and stable interaction with the pore wall.^{[ 41} , ⁴² , ^{43 ]} The results show that this controlled translocation speed and trajectory improved SNR and signal stability in FSN, effectively distinguishing rigid AuNPs and deformable PEG molecules at the nanoscale, considering their size and stiffness. These experimental findings were supported by numerical simulations, which demonstrated how the angular inertial force regulates molecular translocation and how controlled speed and trajectory produce stable signals with high SNR.

2. Results and Discussion

2.1. Angular Inertia‐Kinetic Translocation of Single Molecules in Light‐Regulated FSN

FSN was fabricated using the photovoltaic electrochemical etch‐stop technique^{[ 44} , ^{45 ]} (see Experimental Section and Note S1, Supporting Information), and it consists of an inverted funnel‐shaped structure in silicon membrane with a 10‐nm pore at the funnel tip (see Figure  1a–c and Experimental Section). When a collimated light beam with adjustable power density is directed onto the nanopore, it generates a photovoltaic surface charge surrounding the pore. This charge is reflected by a positive correlation between the slope of the I–V curve and the light power density (see Figure 1d; Note S2, Supporting Information).^{[ 31 ]} The regulated surface charge further modulates the electroosmotic force to offset the electrophoretic force exerted on molecules in nanopore until a counter‐balanced state is achieved (see Figure 1e(i,ii)). Compared to the pre‐balanced state dominated by the frequent and uncontrolled electrokinetic translocations (see Figure 1f), the balanced electroosmotic and electrophoretic forces in nanopore result in a close‐random molecular translocation of greatly reduced frequency governed by the Brownian motion (see Figure 1g, Experimental Section, and Note S3, Supporting Information).

Figure 1.

Translocation dynamics of 4.5‐nm Au nanoparticles through a light‐regulated FSN using angular inertial forces. a) SEM image and b) TEM image of the nanopore sample with a 15‐µm sided square opening of the funnel structure and a 10‐nm pore. c) Schematic representation of the NIAT device with an in‐tube flow chamber and wedge, emphasizing the adjustment of the inertial angle θ. α represents the tip angle of the wedge, and θ is defined as the angle between the center axis of the flow chamber (Line 3) and the direction of centrifugal force f_c . d) The measured I–V curves of the FSN in 1 m KCl solution when the nanopore is illuminated by a collimated light beam of different power density (central wavelength: 425 nm, beam size: 1.5 mm); I is the measured ion current and V is the applied transmembrane bias. e) Schematic diagrams showing three representative molecular translocation kinetics of AuNPs in the FSN, including (i) the traditional electrokinetic translocations when both the electrophoretic force f_EP and electroosmosis force f_EO are in actions without using the light illumination (ρ₀ = 0); (ii) the f_EP and f_EO on nanoparticles are counter‐balanced when the light power density ρ₃ of ≈12 mW mm⁻² is illuminated on nanopore and the Brownian motion dominates the molecular behaviors; (iii) the angular inertia kinetic translocations under the counter‐balanced state and when angular inertial forces f_c (decomposed into two components parallel to sidewall f_∥ = f_c sin(54.7°‐θ) and vertical to sidewall f_⊥ = f_c cos(54.7°‐θ)) are exerted on nanoparticles to compete with the Brownian motion by adjusting the centrifugal angel θ and the centrifugal acceleration rω ²; ω is the rotation speed; m is the molecular weight; and r is the nominal rotation radius of molecules, i.e., the distance between the nanopore and the spinning center of centrifuge. f,g) Representative current blockade signals of AuNPs under the electrokinetic translocation (f) and the counter‐balanced state with Brownian motion (g), respectively. h–k) Representative signals of angular inertia‐kinetic translocation of AuNPs under different centrifugal angles, i.e., 0° and 30°, and variant centrifugal accelerations, such as 1120 and 4480 g. The inserts (left of insets (f–k)) show the dwell time t_d and the current amplitude ΔI of representative blockades. l–n) Translocation frequency of nanoparticles (l), and the dwell time (m), and the current amplitude (n) of corresponding current blockades under different light power density ρ without inertial force (top); and as a function of inerital angel θ and the centrifugal acceleration rω ² when the light‐regulated balance of the electroosmotic and electrophoretic effects was achieved in FSN (bottom). Note that when the inertial angle in FSN is over the slant angle of the funnel sidewall, i.e., 54.7° (f_∥ = 0, f_⊥ = f_c ), the Brownian motion (blue dashed line) dictates the molecular translocations (bottom of inset (l)). All translocation signals were measured at room temperature with a voltage bias U of 0.3 V and a 1 m KCl solution of pH 7.0.

By integrating a light‐regulated FSN and an inertial angle adjustment function into a centrifuge tube (see Experimental Section and Note S4, Supporting Information), the proposed angular inertia‐kinetic translocation platform is achieved at the counter‐balanced state and controllable angular inertial forces are exerted on molecules to overcome the stochastic Brownian motion by adjusting the centrifugal angel and acceleration (see Figure 1e(iii)). Compared with the stochastic process observed in the electrokinetic translocation (see Figure 1f), the angular inertial‐kinetic translocation method helps achieve more controllable molecular translocations with current readouts of better SNR (see Figure 1h–k). The molecular translocation dynamics and the resultant current blockades in FSN can be tailored by adjusting the inertial angle and the centrifugal acceleration, and a higher inertial angle, such as θ = 30°, leads to a longer dwell time and a higher current amplitude in blockade signals than these acquired with the inertial angle of 0° (see Figure 1h–k).

When the light‐regulated FSN operates in the electrokinetic domain, it is found that the light power density significantly influences the molecular translocation frequency, but not the blockade signals, including their dwell times (see Note S5, Supporting Information) and current amplitudes (see top of Figure 1l–n). In comparison, when the FSN operates in a counter‐balanced state and incorporates angular inertial forces, the translocation frequency, and current blockade signals exhibit a notable dependence on the inertial angle and centrifugal acceleration (see bottom of Figure 1l–n). Specifically, the molecular translocation frequency is negatively correlated with the inertial angle and positively correlated with the centrifugal acceleration. The dwell times and current amplitudes are positively correlated with the inertial angle, while the dwell times are negatively correlated with the centrifugal acceleration. The current amplitudes, on the other hand, demonstrate a weak reliance on the centrifugal acceleration. These observations confirm that angular inertia forces govern molecular dynamics, including movement directions and translocation speeds, in FSN. This offers a controllable approach to optimize the SNR of current measurements for better sensing of single molecules.

2.2. Angular Inertial Force Stabilizing Molecular Translocations in FSN

The controlled translocation direction and speed in FSN not only improves the SNR of the signal readings (see Note S6, Supporting Information), but also greatly mitigates the stochastic motion of molecules in FSN, thus enhancing the signal stability (evaluated by the degree of scattering of data points in Figure  2a–d) and offers better temporal and spatial resolutions for single molecule sensing. In angular inertia‐kinetic translocation, the signal stability (or signal uniformity, evaluated using the coefficient of variation or CV of datapoints in Figure 2e,f) is governed by the inertial angle. When the inertial angle is beyond 30°, the CVs of dwell times and current amplitudes are ≈0.05. It is noteworthy that the TEM diameter deviation of tested AuNPs, i.e., 4.5 ± 0.4 nm, may partially contribute to the calculated CVs in addition to other stochastic factors such as thermal fluctuations (see Experimental Section and Note S7, Supporting Information).

Figure 2.

Stability study of current blockades of angular inertia‐kinetic translocation of 4.5‐nm AuNPs in FSN. a–d) Characterization of the dwell time t_d versus the current amplitude ΔI of current blockade signals measured by tuning the centrifugal angle θ and the centrifugal acceleration rω ². e,f) The stability of translocation signals is denoted by the CV of dwell time (e) and current (f) amplitude under different inertial angles and variant centrifugal accelerations. The CV values are calculated from the data shown in insets (a–d) while all translocation signals were measured at room temperature with a voltage bias U of 0.3 V, a 1 m KCl solution of pH 7.0, and an illumination light power density of 12 mW mm⁻².

Simulations of translocation trajectories and transient analyte interactions with pore were performed in FSN to understand the controlled translocations and the improved SNR and stability of current signals in our technique (see Experimental Section and Note S8, Supporting Information). Through calculating the electrical field and net ionic concentration and their spatial distributions in FSN, the sensing zone length and the thickness of the electric double layer (EDL), i.e., the Debye length, can be characterized to facilitate the study of interactions between the analyte, i.e., 4.5‐nm AuNPs, and pore for calculating the current drops (see Figure  3a–f). By randomizing the starting location entering the sensing zone and motion direction of AuNP and assuming a centrifugal acceleration of 4480 g, the motion trajectory of the nanoparticle in FSN can be calculated under different inertial angles. The simulations indicate converged trajectories and enhanced nanoparticle‐pore interactions, thus the enlarged and stabilized current drop and dwell time, with the increased inertial angle (see Figure 3g–i).

Figure 3.

Inertial angle regulating translocation trajectory in FSN for stable signals. a) Spatial distribution of relative electric field intensity (E/E_max ) in FSN of a 10‐nm pore (D) with a bias U of 0.3 V and a KCl concentration of 1 m. b) The distribution of E/E_max along z direction at x = 0 for defining ≈50‐nm sensing length of FSN as the distance between two points where the E/E_max is equal to e⁻¹. c) Spatial distribution of net ionic concentration (n₁c₁+n₂c₂) in the sensing zone of FSN. n_{1, 2,} and c_{1, 2} are the valence and concentration of two ionic species, respectively (1 for potassium ion K⁺ and 2 for chloride ion Cl⁻). d) Zoomed‐in view of area A in (c) illustrating the EDL of thickness ranging from 0.5 to 2 nm (i.e., the Debye length) formed by two ions on funnel sidewalls. e,f) Translocation of a 4.5‐nm AuNP through different locations within nanopore resulting in different spatial distributions of the net ionic concentration in the sensing zone to mimic the analyte interactions with pore. g–i) Representative trajectories simulating the translocations of AuNP in FSN (g), the calculated mean and standard deviation of current drops ΔI (h), and the calculated dwell times and their distributions (i) under different inertial angels, simulations were repeated 300 times by assuming a centrifugal acceleration of 4480 g. Notably, when the inertial angle is beyond the slant angle of the nanopore sidewall, i.e., 54.7°, the nanoparticles tend to leave the sensing zone, resulting in a decreased translocation frequency (or counts).

2.3. Nanoscale Discrimination of Rigid Molecules

AuNPs are widely used rigid sphere‐like molecules of uniform and controllable size in nanometers (see Note S9, Supporting Information and Experimental Section). To evaluate the capability of our technique for sensing and discriminating nano‐sized single molecules, AuNPs of three different TEM diameters, i.e., 4.5 ± 0.4, 6.1 ± 0.7, and 7.9 ± 0.8 nm, were tested using angular inertia kinetics in FSN (see Figure  4a; Note S5, Supporting Information). The inertial angle of 45° was used to achieve stable signals and different centrifugal accelerations were utilized to control the dwell time and current amplitude in blockade readings. The current blockade signals of different AuNP sizes display similar double‐valley trace features of different size‐dependent dwell times and current amplitudes (see Figure 4b). Dwell time exhibits a negative size dependence, while current amplitude demonstrates a positive size dependence (see Figure 4c). It is noted from the distributions of measured current amplitudes and dwell times that the tested AuNPs of ≈1.6‐nm size difference can be successfully differentiated by tuning the centrifugal acceleration and the size sensitive of current amplitude is higher than that of dwell time.

Figure 4.

AuNPs size discrimination through angular inertia‐kinetics. a) Schematic diagrams of three types of tested AuNPs with mean TEM diameters of 4.5, 6.1, and 7.9 nm, respectively, and b) their corresponding current blockade signals measured using a light‐regulated FSN operating under centrifugal accelerations of 1120 and 4480 g. c,d) The distributions of current amplitudes (ΔI) versus dwell times (t_d ) of three AuNP types after measuring 966 translocations in FSN at different centrifugal accelerations. e,f) The representative current traces and g,h) the distributions of current amplitudes (ΔI) versus dwell times (t_d ) of 478 translocation signals acquired at different centrifugal accelerations when sensing a 3‐nm mixture sample of equally mixed three AuNP types, i.e., molar ratio of 1:1:1, which are indicated with different colored circles. i) Proportions of each AuNP size among all AuNPs translocated in one minute under two different centrifugal accelerations. All translocation signals were measured at room temperature in an FSN under light‐regulated balanced state and using an inertial angle of 45°, a voltage bias of 0.3 V, and a KCl solution of 1 m concentration and pH 7.0.

Mixtures of three types of AuNPs in different molar ratios were further tested to verify the size discrimination capability of FSN (see Note S10, Supporting Information). As a prime example, the representative current traces of the mixture with a molar ratio of 1:1:1 clearly exhibit the characteristic size‐dependent signal features of three AuNPs (see Figure 4e,f). The values and the variations of each signal component measured from the AuNPs mixture test are comparable to those acquired in individual tests (see Figure 4i), demonstrating the consistent capability to resolve molecular size at nanometer level. Remarkably, within the initial minute of translocation, a correlation is observed between the portion ratio of each AuNP and its size. Specifically, larger particles in the mixture exhibit a higher frequency of translocations.

2.4. Nanosized Sensing of the Stiffness of Flexible Molecules

PEG represents a type of flexible molecule of customizable chain length and size.^{[ 46} , ^{47 ]} In addition, the stiffness of PEG can be precisely controlled by adjusting the salt concentration in the analyte solution, which induces a partial dehydration effect.^{[ 48} , ^{49 ]} The size and stiffness of PEG can be denoted with the hydrodynamic diameter D_h and the persistence length ξ_PEG , respectively.^{[ 50 ]} In this study, 10‐ and 20‐kDa PEGs of a size different less than 1.4 nm and a stiffness tailored from 0.37 to 0.34 nm (lower value means higher stiffness in high salt solution) were tested in FSN to demonstrate its capability for differentiating flexible molecules of nanometer size and their subtle variation in stiffness (see Figure  5a–c).

Figure 5.

Sensing PEGs of different sizes and stiffness. a–c) Schematics illustrating the size and stiffness of 10‐ and 20‐kDa PEGs modulated by KCl concentration from a) 0.01 m to b) 0.1 m and c) 1 m. Hydrodynamic diameter D_h indicates the size of the polymer chains. Persistence length ξ_PEG represents the stiffness of PEGs. d–f) Representative current blockade signals of respective PEGs were measured under centrifugal accelerations of 1120 and 4480 g. g–i) The distributions of current amplitudes (ΔI) and dwell times (t_d ) of two PEGs compiled from 516 translocation measurements in FSN under different centrifugal accelerations. j–m) The mean and CV of dwell time and current amplitude versus the stiffness of 10 k and 20 kDa PEGs_. The mean and CV values are calculated based on datapoints plotted in (g–i). All translocation signals were measured at room temperature in a FSN under light‐regulated balanced state and using an inertial angle of 45°, a voltage bias of 0.3 V, and different KCl concentrations with pH 7.2. The balanced states in FSN for 10‐ and 20‐ kDa PEGs were achieved by adjusting the light power density to 4.7 and 8.2 mW mm⁻² in the 0.01 m KCl solution, to 2.2 and 3.8 mW mm⁻² in the 0.1 m KCl solution, to 1.0 and 1.7 mW mm⁻² in the 1 m KCl solution, respectively.

A stable signal was achieved by using an inertial angle of 45°, while different centrifugal accelerations and solvents were employed to control the dwell time and current amplitude (see Notes S11 and S12, Supporting Information). A characteristic double‐stair feature can be identified in the current blockade signals of two PEGs, which clearly displays the size‐ and stiffness‐dependent dwell times and current amplitudes (see Figure 5d–f; Note S13, Supporting Information). Dwell time exhibits a negative size and stiffness dependence, while current amplitude demonstrates a positive size and stiffness dependence (see Figure 5g–i). By using a higher centrifugal acceleration, an ≈1.3‐nm size difference between two PEGs of variant stiffness can be differentiated and the dwell time offers a higher sensitivity to size than that of current amplitude (see Figure 5g–i). On the other hand, the increased stiffness of PEGs in high salt solution leads to a higher mean value of current amplitude and a slightly decreased mean value of dwell time, and the current amplitude is more sensitive to stiffness than that of dwell time (see Figure 5j,k).

The measured current amplitudes and dwell times also show a stiffness‐dependent degree of scattering, i.e., higher stiffness related to a lower degree of scattering or higher signal stability (evaluated using CV). The CV values of current amplitude are more sensitive to the tiny stiffness changes in PEG, i.e., a sensitivity of ≈3.5/nm, than that of dwell time of ≈0.97/nm (see Figure 5l,m). This observation may indicate that the interactions between PEG and funnel sidewall lead to potential molecule deformation and affect the measured current amplitudes. While the degree of deformation depends on the stiffness of molecule under test, stiff molecule may suffer less variation in the amplitude of current signals. However, the dwell time, which is influenced by the translocation trajectory and speed, may be less affected by the stiffness of the molecule.

3. Conclusion

In order to achieve accurate sensing and characterization of single molecules, it is essential to have control over their translocation speed and trajectory within a nanopore, which ensures a stable current blockade signal with a high SNR. To address this, we have developed an in‐tube FSN device that decouples the electrophoresis force typically used and allows for independent current blockade detection and molecular translocation control within the nanopore. By utilizing a centrifuge, the angular inertial force governs the molecular dynamics, effectively overcoming the stochastic Brownian motions within the nanopore and defining the translocation process. Both experimental and theoretical findings demonstrate the significant controllability of the inertial angle and centrifugal acceleration on the translocation speeds and trajectories of molecules in the FSN device, resulting in adjustable frequency of translocation events and current signals with a high SNR. Furthermore, the translocation frequency is found to be dependent on the mass density difference between the molecule and the solution (see Note S14, Supporting Information), suggesting a potential strategy for selectively regulating molecular translocation in FSN. The controlled translocation speed and passage trajectory within the FSN device also enhance the stability of current blockade signals, resulting in a low CV for resultant signals. For instance, when sensing AuNPs of size 4.5 ± 0.4 nm using an inertial angle larger than 30° in the FSN device, the CV of resultant dwell time and current amplitude can be ≈0.05. Furthermore, we demonstrate the capability of our proposed NIAT for the nanoscale size discrimination of different AuNPs and PEGs at resolutions of 1.6 and 1.3 nm, respectively, as well as the differentiation of subtle stiffness variations of PEGs under different salt concentrations.

Currently, the full potential of nanopore in single‐molecule sensing is limited by the challenges of achieving reliable and quantitative analysis of molecular structure and size.^{[ 51 ]} The NIAT device, with its demonstrated signal stability and high‐resolution discrimination capability, has the potential to enable precise detection of single molecules at the nanoscale. Moreover, the accurate identification of size and stiffness change in chain‐shape molecules by the new strategy may potentially enable the accurate detection of the conformational changes of biomarkers and simultaneous identification of multiple types of label‐free biomarkers, i.e., proteins, miRNA, and cfDNA^{[ 52 ]} (see Note S14, Supporting Information). Furthermore, the use of a silicon nanopore and widely available centrifuge is expected to reduce the turnaround time and associated costs of nanopore analysis. In short, the enhanced controllability offered by the inertial‐kinetic translocation technique suggests promising potential of the NIAT device for accurate single molecular fingerprinting.

4. Experimental Section

FSN Fabrication

Both sides of a double polished silicon wafer were coated with 100‐nm‐thick Si₃N₄ layers through plasma‐enhanced chemical vapor deposition (Oxford Instruments plc). Two square patterns were transferred to the Si₃N₄ layers on both sides of the silicon through photolithography (SUSS MicroTec SE) and treatment with gaseous plasma (mixture of 100‐sccm carbon tetrafluoride and 5‐sccm argon) for 20 min. The side lengths of square patterns on the front side and back side are 15 and 1000 µm, respectively. An inverted square‐base funnel‐shaped structure of ≈10.6 µm in depth was fabricated on the front side by KOH (80.0 °C) etching. Then, KOH (80.0 °C) etching was first performed to etch the back side of the silicon sample until the remaining thickness reaches ≈1300 nm by closely tracking the transmission spectral peak to 520 nm using a wideband blue light source, followed by KOH (22.0 °C) etching to achieve remaining thickness of ≈100 nm corresponding to the transmission spectral peak to 480 nm. To control the pore sizes of the nanopores, a local KOH (22.0 °C) etching step was performed on the silicon/etchant interface using a narrowband blue light source, resulting in a localized photovoltaic electrochemical etch‐stop effect (see Note S16, Supporting Information). The perforation was characterized by the rapid ramping in the current signal.^{[ 40 ]} Finally, the silicon samples underwent a cleaning process using piranha solution (a mixture of H₂SO₄ and H₂O₂ with a 3:1 volume ratio) for 30 min, followed by treatment with gaseous plasma (20‐sccm oxygen) for 10 min to generate silicon dioxide film on the surface of the nanopore sample^{[ 53 ]} (see Note S1, Supporting Information for fabrication details).

NIAT Device with Angle Adjustment Function

NIAT device consists of three key components, including flow chambers, a current signal detection module, and a light illumination module. By adding a wedge beneath the flow chamber, the inertial angle θ can be adjusted to 0°, 15°, 30°, 45°, and 60° by using wedges of different tip angles of 60°, 45°, 30°, 15°, and 0°, respectively. As for the signal detection module, the measured current signals are amplified using an ultra‐low bias Op‐AMP circuit (OPA128, Burr‐Brown Corp.) with a sensitivity of −1.1 V/nA and low root‐mean‐square circuit noise of 0.4 pA (at 50 kHz sampling rate). The amplified blockage signals were then digitized using an analog‐to‐digital converter (AD9410, Analog Devices, Inc.) and transmitted via the wireless circuits (HM‐BT4501, HOPE Microelectronics Co., Ltd.). The light illumination module utilized a 425‐nm light source, which could be adjusted for output power. Prior to being incident to FSN, the light was collimated to a beam size of 1.5 mm.

Molecule Preparation and Identification of Counter‐Balanced State in FSN

The PEGs were obtained from Thermo Fisher Scientific, Inc. The 4.5, 6.1, and 7.9 nm AuNPs were synthesised using a kinetically controlled seeded growth method, in which the 3.5‐nm‐sized Au seeds were increased to 4.8, 6.1, and 7.9 nm by repeating the growing step of diluting the seed solution and injecting aliquots of gold precursor 1, 3, and 6 times, respectively.^{[ 54 ]}

The molecules, including PEGs and AuNPs, were dispersed in 1 m KCl with a concentration of 10 nm and added to the flow chambers in NIAT for testing. The optical power directed onto the nanopore was adjusted remotely to identify the characteristic value for achieving the balanced state between the electrophoretic and electroosmotic forces in FSN. This characteristic value was identified when the measured current blockage signal indicated the lowest frequency of translocation events (see Figure 1g). Then, the molecule samples were further dispersed to 0.1 nm for minimizing the molecular interactions during the angular inertia‐kinetic translocation.

COMSOL Simulation

The electric field and ion concentration distribution in the funnel‐shaped structure were calculated by electrostatic equation and Poisson‐Nernst‐Planck equation.^{[ 55 ]} The electric field distribution indicates that the sensing zone is in the area between z = 30 nm and z = ‐20 nm (see Figure 3a,b). Both initial concentrations of K⁺ and Cl⁻ are set to 1 m. The motion trajectories of AuNPs in the nanopore were simulated using the Langevin equation.^{[ 56 ]} The current changes caused by AuNPs in the sensing zone could be evaluated by using the conductivity equation of electrolyte.^{[ 55} , ^{57 ]} Here, the maximum current change induced by AuNPs in the sensing zone is defined as the calculated current amplitude (see Figure 3g–i). Each simulation begins by assuming a single AuNP entering the nanopore (i.e., z = 30 nm) from a random direction and ends with leaving the sensing area from any cross‐section, and the duration of AuNP in the sensing zone is defined as the calculated dwell time (see Figure 3g–i).

Statistical Analysis

The molecular motions in FSN manifest in the measured current blockade signal, including the first current drop with duration t₁ associated with molecular capture and the second current drop with duration t₂ due to molecular translocation. The amplitude of current drop is defined as ΔI and the dwell time t_d is calculated as t₁ + t₂ . The molecular translocation events are identified by minimum tolerable SNR of 2.5 (see Note S13, Supporting Information). Data was expressed as mean ± SD. Each experiment in this study was repeated to ensure the reliability of the results (see Note S17, Supporting Information).

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

W.Y. and H.‐P.H. conceived the idea of the work. J.Y. and T.P. fabricated the nanopore for the initial studies. W.Y. and H.‐P.H. optimized experimental designs and fabrication protocols. J.Y. and T.P. investigated the in‐tube device assembly. W.Y., C.M., and H.‐P.H. discussed and improved the research mechanism. J.Y. and T.L. performed nanopore test, signal characterization, and other related experiments. J.Y. and T.P. prepared the manuscript with thorough editing and polishing from W.Y., C.M., and H.‐P.H.

Supporting information

Supporting Information

Yang J., Pan T., Liu T., Mao C., Ho H.‐P., Yuan W., Angular‐Inertia Regulated Stable and Nanoscale Sensing of Single Molecules Using Nanopore‐In‐A‐Tube. Adv. Mater. 2025, 37, 2400018. 10.1002/adma.202400018

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.