Sub-Femtomolar Drug Monitoring via Co-Calibration Mechanism with Nanoconfined DNA Probes

doi:10.21203/rs.3.rs-5350293/v1

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Sub-Femtomolar Drug Monitoring via Co-Calibration Mechanism with Nanoconfined DNA Probes

https://doi.org/10.21203/rs.3.rs-5350293/v1

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Synthetic drugs have fundamentally reshaped the illicit drug market due to their low cost, ease of production, and rapid manufacturing processes. However, current drug detection methods, reliant on complex instruments, face limited applicability and often neglect the influence of pH fluctuations, leading to potential bias and unreliable results. Herein, we proposed co-calibration mechanism of DNA probes in nanoconfined channel to achieve significantly enhanced target signal recognition in sweat range pH 3.0 ~ 8.0. The limit of detection (LOD) lows to 3.58 fM in artificial sweat with wide linear response range10³ ~ 10⁸ fM. Compared to the single DNA probe, the co-calibrated dual-DNA probes exhibits a broader linear response range, primarily attributed to the synergistic effects of surface wettability regulation and the hydrion capture in nanoconfined channel, which in turn reduces signal interference in proton transport. The linear response range has doubled, and the detection sensitivity has improved by 4 ~ 5 orders of magnitude compared to existing drug detection methods. This co-calibration strategy expands the application scope of aptamer-based composite probes, offering a novel approach for ultra-sensitive drug detection and showing significant potential in sweat sensing and drug monitoring fields.

Physical sciences/Chemistry/Analytical chemistry

Physical sciences/Materials science/Materials for devices/Sensors and biosensors

DNA probes

nanoconfined materials

ionic current

sweat monitoring

biosensor

The 2023 World Drug Report indicated that the global number of drug users continues to rise, with over 296 million people using drugs worldwide in 2021, a 23% increase compared to a decade ago.^[1] The ongoing expansion of the illegal drug market and the increasing flexibility of drug trafficking networks pose severe challenges to the law enforcement and healthcare systems.^[2–4] The ultra-sensitive detection and application of drugs can effectively prevent and curb crime. Traditional drug detection methods include GC-MS, LC-MS, and commercial ELISA.^[5–9] These detection methods primarily rely on large-scale instruments, which, despite their high sensitivity, are unlikely to be applicable for on-site testing. Recently, sweat samples have become ideal for real-time detection using portable devices because they are easy to collect and do not require special storage conditions.^{[10, 11]} In particular, on-site drug monitoring in sweat, compared to blood and urine testing, is both non-invasive and does not involve privacy concerns.^[12–14] However, pH variations in sweat can introduce interference, affecting detection performance. Therefore, there is an urgent need to develop sensor-based ultra-sensitive detection technologies for on-site drug testing in sweat (Fig. 1A).

In recent years, solid-state nanochannels have shown broad application potential in various technological fields due to their precise molecular-level control and tunable structural properties.^[15–20] Compared to other detection approaches, nanoconfined biosensors (N_CB_S) endow significant advantage on high sensitivity.^{[21, 22]} By integrating resistive pulse sensing technology and utilizing functional molecules (such as crown ethers,^[23–26] DNA probes,^[27–29] etc.), specific functionalities can be imparted to nanochannels, enabling regulation of effective pore size, surface wettability, and surface charge properties. When a target analyte binds to specific recognition sites on the N_CB_S, the ion transport characteristics of the electrolyte ions passing through the nanochannel under an applied voltage change, thus enabling the detection of the target analyte.^[30–33] DNA probes, single-stranded DNA or RNA oligonucleotide sequences, can be obtained through an in vitro selection process known as Systematic Evolution of Ligands by Exponential Enrichment.^[34] This endows them with high stability, strong affinity, and significant specificity for target molecules. DNA-N_CB_S hold great significance for the detection of ions and small molecules.^[35–39]

In conventional nanopore, measurements are typically conducted under a single pH condition, overlooking the impact of pH variation on detection performance. However, changes in pH directly affect the hydration state of ions and their transport behavior within the nanopore.^[40–43] When pH alters, the surface charge density of the nanopore wall and the electric potential distribution inside the channel change accordingly, affecting the arrangement of water molecules and the hydration state.^[44–46] These changes significantly influence target recognition and sensor sensitivity, especially when detecting complex biological samples like sweat, where the pH fluctuates between 3.0 ~ 8.0.^[47] This instability reduces the detection performance of conventional nanopores. The DNA-N_CB_S can maintain stable performance across different pH values and significantly enhance the recognition capability for complex samples.^[48–50] Therefore, it is essential to develop nanopore devices with co-calibration capabilities for real samples like sweat, which exhibit a wide range of pH variations.

In this work, we developed a novel co-calibration strategy with biomimetic solid-state N_CB_S with dual-DNA probes. The N_CB_S employs cathinone binding aptamer (CBA) as the gate molecule for specific sensing (AG), characterized by its transition from a long-chain structure to a hairpin structure upon binding to the target. Meanwhile, C4 DNA acts as the functional molecule (SF) regulating selective ions transport, with its structure shifting in response to pH changes, specially forming an i-Motif structure under acidic conditions upon binding with hydrion (Fig. 1B). Inspired by the functions of AG and SF in biological nanopores,^{[51, 59]} the co-calibration of dual-DNA probes for target recognition sensitivity and anti-pH interference to achieve the signal increasing in nanopores. This enhanced performance is mainly attributed to the reduced hydrophilicity of the N_CB_S due to dual-aptamer functionalization, which amplifies the current change caused by CBA binding to the target (Fig. 1C). While, C4 DNA captures hydrion from the solution, effectively reducing the signal interference from hydrion ion transport. The difference in length between the two DNA probes also increases the density of DNA-N_CB_S, resulting in a more uniformly distributed positive charge. This approaches the significant charge changes upon target binding, enhancing the target recognition signal. The investigation of drug monitoring in artificial sweat demonstrated that the CBA&C4@AAO biosensor maintained extremely ultra-sensitivity and a broad linear response range, even in complex environments. In the future, this co-calibration DNA-N_CB_S has the potential to be further developed into a wearable sensor by integrating microfluidic technology (Fig. 1A), enabling convenient, rapid, and ultra-sensitive sweat sensing and health diagnostics.^[52–54]

Design and Sensing Performance of Single DNA Probe N _C B _S (CBA@AAO).

The design strategy for the solid-state N_CB_S functionalized with single DNA probes CBA@AAO is shown in Figure S1. And the successful preparation of CBA@AAO was demonstrated in detail by SEM, EDS mapping, XPS, contact Angle and electrochemical tests (See Supporting Information for details). Subsequently, to demonstrate that the ultra-sensitivity of CBA@AAO to the target molecule is due to the specific probe CBA, we used a PolyA DNA sequence with the same number of bases as CBA but consisting entirely of adenine bases (PolyA) and grafted it onto the AAO membranes. We compared the current signal changes of CBA@AAO and PolyA@AAO after exposure to 1 fM and 1 pM Cathinone (Cat). The current increased with different concentrations of Cat for CBA@AAO, whereas PolyA@AAO showed no significant changes (Fig. 2A). Moreover, we further explored the transmembrane ionic current stability after CBA@AAO recognition of different concentrations of Cat. As shown in Fig. 2B, the ionic current increased with the increase in Cat concentration, mainly due to the increase in the effective diameter of the nanochannels resulting from the structural changes induced by probe binding to the target.

We investigated the direct detection of Cat, and the transmembrane current results demonstrated a decrease in current after CBA@AAO recognized Cat (Fig. 2C). And direct detection of Cat transmembrane transport showed a decreasing trend in I-T signal with increasing concentration. As shown in Fig. 2D, at high Cat concentrations, during the process of CBA as a specific binding probe transporting Cat, it gradually blocks the nanochannel and shields the negatively charged surface of the nanochannels, leading to varying degrees of pore blockage.^[55] The primary influencing factor for probe-modified N_CB_S is the size effect. CBA@AAO with pore sizes ranging from 40 ~ 70 nm exhibited the optimal response to Cat, with current signal change rates of 10.1% at 1 fM and 22.4% at 1 nM concentrations (Fig. 2E). This superior performance can be attributed to the appropriate pore size and porosity, which play crucial roles during the functionalization process. An optimal combination of these factors ensures not only adequate surface area for CBA modification on the N_CB_S but also sufficient probe functionalization within the pores (Figure S5, Table S9).

To emphasize the sensing performance of the CBA@AAO N_CB_S for detecting the target, we examined the relationship between transmembrane ionic current and Cat concentration. The results demonstrated a strong linear response of CBA@AAO over the Cat concentration range of 1 ~ 10⁴ fM (R² = 0.9984), with a detection limit as low as 0.40 fM (Figs. 2F, S17). This indicates the ultra-sensitivity and effectiveness of the N_CB_S for ultra-trace detection of Cat. Specificity is crucial in evaluating probe sensor performance. We tested the CBA@AAO binding to methcathinone (Met) and ethcathinone (Eth), which share similar structures with Cat. As shown in Fig. 2G, at 1 fM Cat, the current ratio before and after recognition was 1.10, whereas for Met and Eth, the ratios were only 1.03 and 1.01, respectively, at 10⁵ fM. And the ratio increased to 1.23 at 10⁵ fM Cat. We also compared responses to 11 other drugs, including norketamine (Nor), ketamine (Ket), amphetamine (Amp), MDMA, phenacetin (Phe), cocaine (Coc), heroin (Her), caffeine (Caf), procaine (Pro), paracetamol (Par), and (+)-pseudoephedrine (PSE) (Figures S19-21). CBA@AAO showed strong selectivity for Cat, with varying responses to other substances, likely due to differences in structural features (such as the number and position of amino groups, conjugated structures, etc.).

We successfully differentiated Cat, Met, and Eth using principal component analysis (PCA) based on variations in current signals before and after CBA@AAO response (Figure S24). The distribution of clusters corresponded with increasing concentrations, achieving 94% accuracy (Table S25). Building on this, we applied PCA to 14 different drugs at the same concentration, achieving 100% correct classification (Figure S23, Table S24). Additionally, hierarchical cluster analysis (HCA), an unsupervised multivariate method,^{[56, 57]} was used to distinguish analytes through dimensionality reduction (Fig. 2H). These findings indicate that, beyond specific Cat recognition, the CBA@AAO N_CB_S exhibits cross-reactivity to structurally similar drugs, offering potential for discriminative analysis in complex environments.

Sensing Properties of C4@AAO and CBA&C4@AAO at Different pH Levels

To address pH interference in Cat recognition, we introduced a pH-responsive specific probe (C4 DNA) and functionalized on N_CB_S, creating the C4@AAO sensor. As shown in Fig. 3A, the C4@AAO exhibited significant variations in I-V characteristics in response to different pH levels. At + 2 V, the transmembrane current varied notably: 85.6 ± 1.3 µA at pH 3 (open state) and 20.2 ± 0.1 µA at pH 8 (closed state). The C4-N_CB_S also showed a strong linear response between pH 3 ~ 7 (R² = 0.9881) (Fig. 3B), attributed to pH-induced structural changes in C4 DNA. Cyclic stability tests at pH 5.5 and 7.5 confirmed the C4-N_CB_S robust and reliable performance in pH analysis (Figs. 3C, S25).

To achieve co-calibration for ultra-sensitive Cat detection and pH interference resistance, we developed the CBA&C4@AAO by co-grafting CBA and C4 DNA onto N_CB_S. The biosensor is capable of simultaneously sensing the target and pH changes. We investigated the ionic current changes of CBA@AAO and CBA&C4@AAO before and after target binding under different pH conditions (Fig. 3D). The current measurements were based on I-V characteristics, reflecting the transmembrane ionic current changes before and after Cat recognition at + 2 V. The results showed that the incorporation of C4 DNA led to a significant increase in the current variation upon target recognition, compared to CBA@AAO. As shown in Fig. 3E, at pH 5.5 and 7.5, the current changes of CBA&C4@AAO after Cat binding were 20.79 µA and 15.42 µA, respectively, while for CBA@AAO, the changes were only 16.37 µA and 9.48 µA. The CBA provides target specificity, while C4 DNA responds to protons, reducing interference caused by ion transport. By compensating for the pH-induced ionic current changes, the sensitivity of CBA&C4@AAO to Cat recognition was significantly enhanced under varying pH conditions (Figs. 3F, S28). At pH 5.5, the current signal increased by 14%, 31%, and 80% for 1 fM, 1 pM, and 1 nM Cat, respectively. At pH 7.5, the signal increases were even more pronounced, reaching 159%, 201%, and 322%. These findings indicate that the CBA&C4@AAO exhibits excellent potential in resisting pH interference during Cat detection, offering a promising approach to further enhancing N_CB_S sensitivity.

We assessed the linear response ranges and minimum detection limits of two N_CB_S under pH 7.4 and 5.5 levels. As shown in Fig. 3G, the CBA&C4@AAO exhibited a broader linear response range compared to CBA@AAO at pH 7.4. Similarly, at pH 5.5, the transmembrane current change of CBA&C4@AAO upon binding to Cat was significantly higher than that of CBA@AAO, with a wider linear response range (Fig. 3H). This enhancement is primarily attributed to the introduction of C4 DNA, which slightly reduces the hydrophilicity of the nanochannels membrane, thereby lowering the initial current signal threshold. Additionally, C4 DNA provides numerous hydrion binding sites, mitigating the interference from proton transport. The mixed modification, due to the different lengths of the two DNA strands, increases the probe modification density, resulting in a more uniform charge distribution on the membrane surface. Consequently, the changes in nanochannels size, surface wettability, and surface charge before and after the binding of Cat in CBA&C4@AAO synergistically enhance the detection signal. The CBA@AAO displayed a linear response range of 1 ~ 10⁴ fM with a limit of detection (LOD) of 0.40 fM, while CBA&C4@AAO had a linear range of 0.1 ~ 10⁷ fM and an LOD of 1.25 fM at pH 7.4. At pH 5.5, CBA@AAO exhibited a linear range of 10 ~ 10⁵ fM with an LOD of 4.78 fM, whereas CBA&C4@AAO showed a linear range of 1 ~ 10⁸ fM and an LOD of 5.24 fM (Fig. 3I). Although the mixed modification slightly reduced the LOD, it significantly expanded the linear response range, optimizing the co-calibration strategy for enhancing sensitivity based on DNA probes.

Sensing Mechanism of NB Modified by DNA Probes

To investigate the reasons for the enhancement of sensitivity and specificity of the probes-N_CB_S, we used molecular docking to predict the binding mode and binding sites of the ligand interacting with the macro-molecule. Molecular docking studies can further provide information on the binding affinity between Cat and DNA. As shown in Fig. 4B, the molecular docking results indicate that CBA interacts with Cat through groove binding, primarily through non-covalent interactions (including hydrogen bonds and van der Waals forces), with a binding energy of ~ 5.6 kcal·mol^− 1. This is consistent with the results obtained from circular dichroism (CD) measurements, indicating a conformational change upon binding of CBA to the target Cat (Fig. 4C). With the continuous increase in Cat concentration, the CD signal change becomes more pronounced, accompanied by an increase in corresponding UV absorption (Fig. 4D). Therefore, the proposed mechanism primarily involves the change in the effective diameter of the nanochannel before and after recognition. During the sensing process, when voltage is applied on both sides of CBA@AAO, ions are driven to pass through the nanochannel, resulting in an open-circuit current signal. The current I of the nanochannel can be mathematically represented by formula (1),^[58] as the system can be reduced to a model of ionic conductance for a cylindrical channel:

I = V ([n₊µ₊ + n₋µ₋] e) ( \(\:\frac{4h}{\pi\:{d}^{2}}\)+ \(\:\frac{1}{d}\))⁻¹ + \(\:\frac{V\mu\:\otimes\:\pi\:d\sigma\:}{h}\) (1)

According to previous studies,^[59] when the thickness h of the nanochannel is significantly greater than its diameter d, formula (1) can be simplified to the following equation:

I = V (\(\:\frac{4h}{\pi\:{d}^{2}}\))⁻¹ (2)

Where V is the applied voltage; n₊ and n₋ represent the number densities of positive and negative ions, respectively; µ₊ and µ₋ represent the electrophoretic mobilities of positive and negative ions, respectively; e is the elementary charge; h and d are the thickness and diameter of the nanochannel. The last term in formula (1) explains the transport of ions through a highly charged inner surface, where µ represents the solution mobility of counterions adsorbed on the charged surface, and σ is the surface charge density, with its sign opposite to that of the counterions. Eq. (2) indicates that the diameter d plays a crucial role in determining the current through the nanochannel. In this work, d represents the effective diameter. Figure 4A discusses the relationship between I and the d before and after the CBA@AAO sensor recognizes the target. Before target recognition, the probe is stretched and in a dispersed state, leading to obstruction of the nanochannel current. Upon binding with Cat, the CBA probe adopts a hairpin structure, resulting in an increase in nanochannel current. The current ratio (R) and the current change (ΔI) for target recognition by the sensor can be expressed as follows:

R = I₁ / I₀ (3)

ΔI = I₁ - I₀ (4)

Where I₁ and I₀ represent the current values measured before and after the binding of CBA@AAO with Cat, respectively. By combining equations (2), (3), and (4), the current ratio and current change for the probe-N_CB_S upon target recognition can be calculated as follows:

R = \(\:\frac{{\left(d+\:\varDelta\:d\right)}^{2}}{{d}^{2}}\) > 1 (5)

ΔI > 0 (6)

The significant increase in current observed after Cat binds to the CBA@AAO can be attributed to the increase in the d of the nanochannel. To further demonstrate the specificity of the N_CB_S, we conducted CD tests using PolyA in response to different concentrations of Cat. The results indicated that PolyA did not show significant changes in CD signal due to the presence of Cat (Fig. 4E). Additionally, we examined the CD properties of Met, Eth, and Nor, which have similar structures to Cat, when interacting with CBA. The experimental results showed slight changes in the CD signals for Met and Eth.

Under higher pH conditions, C4 DNA adopts an extended linear conformation, partially blocking the nanochannels. As the pH decreases, the aptamer senses and binds to protons, causing C4 DNA to fold into an i-Motif structure, thereby increasing the effective pore size of the nanochannels (Fig. 4F). This structural transition was confirmed by circular dichroism (CD) spectroscopy. As shown in Fig. 4G, the CD characteristics remain the same at pH 3.5 and 7.5, indicating similar probe conformations. This is the most stable conformation of DNA under physiological conditions, with B-form DNA showing a negative peak around 250 nm and a positive peak around 275 nm.^{[60, 61]} When the pH drops from 5.5 to 4.5, the CD results show the negative peak shifting from 253 nm to 250 nm and the positive peak from 285 nm to 275 nm, indicating a structural transition from B-form to Z-form and back to B-form, leading to a final conformational change.^[62–64] Fig. 4H illustrates the response mechanism of the dual-aptamer calibrated N_CB_S for pH-interference-resistant Cat detection. C4 DNA binds to hydrion, reducing signal interference from mass transport, while CBA provides target specificity. By minimizing pH-induced ionic current interference, the CBA&C4@AAO significantly enhances sensitivity to Cat under different pH conditions.

Application of Co-Calibration Strategy in Complex System

Interference-free detection of target substances in complex environments is a key factor in evaluating sensor performance. For drug detection through sweat, this non-invasive method offers significant potential due to its lack of privacy concerns. However, since human sweat typically has a pH range of 3 ~ 8, pH-related signal interference remains a challenge for sensors in practical applications. To address this, we used the CBA&C4@AAO to detect various concentrations of Cat in artificial sweat. As shown in Fig. 5A, the N_CB_S exhibited a strong linear response between 1 pM ~ 100 nM, with R² = 0.9944. We selected Met, Eth and Nor, which are structurally like the Cat, as reference, while glucose (Glu) and lactate (Lac), which are common in sweat, were selected as disruptors. The results showed that the current change after incubation with Cat was 1.56 µA, while the signal change for other substances was no more than 36.32% of the target signal (Fig. 5B). This indicates that the N_CB_S produces distinct electrochemical responses for different analytes, forming the basis for discrimination. To further evaluate its discriminative capability, linear discriminant analysis (LDA) and hierarchical cluster analysis (HCA) were performed. The LDA results successfully differentiated the six substances in the complex mixture with an accuracy of 97% (Fig. 5C, Table S26). The HCA dendrogram visually conveyed the relationships between analytes, effectively illustrating the chemical similarities (Fig. 5D).

Additionally, to compare the sensitivity of the N_CB_S prepared through mixed modification, we further employed asymmetric modification by immobilizing C4 DNA and CBA on opposite sides of the nanochannels (Fig. 5F). At + 2 V, the N_CB_S current signal increased by 78.8 ± 3.4% at pH 5.5 and by 251.0 ± 3.7% at pH 7.5 after detecting 1 nM Cat, indicating that asymmetric modification enhances sensitivity under acidic conditions. We also evaluated the N_CB_S response to different concentrations of Cat in artificial sweat. As shown in Fig. 5G, the signal change rates increased with Cat concentration, reaching 29.6 ± 4.7%, 38.5 ± 1.1%, and 48.8 ± 1.5% at 1 fM, 1 pM, and 1 nM, respectively. These results demonstrate that the CBA&C4@AAO prepared via this functionalization strategy maintains excellent recognition ability for Cat in complex environments. The dual aptamer-functionalized N_CB_S, employing a co-calibration strategy for Cat recognition and pH interference resistance, exhibited outstanding sensing performance. This sensing strategy not only achieved a wider target concentration response range but also significantly improved the detection limit by 4 ~ 5 orders of magnitude compared to recent drug detection methods (Fig. 5E, Table S27).^[S1−S10] Thus, it holds great promise for drug detection in sweat, drug control, and health monitoring.

In summary, we proposed a co-calibration mechanism of DNA probes in nanoconfined channel with biomimetic N_CB_S for sub-femtomolar drug monitoring. The designed CBA DNA probe as AG, calibrates ultra-sensitive and selective Cat recognition; the C4 DNA probe as SF, calibrates selective ions transport for pH response, respectively. The molecular docking demonstrated that hydrogen bonding and van der Waals forces drive the groove binding between Cat and the CBA. The N_CB_S exhibits good selectivity and outstanding sensitivity towards Cat. An excellent linear relationship was observed between the target concentration and the output ionic current ratio in the range of 1 fM ~ 10 pM, with a LOD as low as 0.40 fM. Furthermore, the N_CB_S shows good cross-reactivity in the analysis of multiple drugs, and CBA@AAO can distinguish fourteen types of drugs with 100% accuracy. It indicates that the N_CB_S has potential applications in the discrimination and analysis of multiple analytes in complex environments. To address the interference caused by different pH levels in sweat sensing, we introduced a pH-responsive DNA probe and designed dual-probes co-calibration N_CB_S (CBA&C4@AAO). A wider range of linear responses at different pH values is achieved. The detection results in artificial sweat show that CBA&C4@AAO has good anti-interference recognition ability to Cat and has excellent linear response ability at 1 pM ~ 100 nM, and the LOD is as low as 3.58 fM. Overall, the dual-probe functionalized N_CB_S offers co-calibration for multi-target recognition, confirming better sensitivity and anti-interference for target recognition compared to traditional single probe sensors. Co-calibrated DNA probes N_CB_S provides a new approach for designing ultra-sensitive and selective drug detection biosensing devices for drug monitoring, sweat sensing, and healthcare.

Materials, and Reagents. The synthesis and purification of the DNA oligonucleotides, including the cathinone-binding probe (CBA, 5’-NH₂-(CH₂)₆-ACT GAG AAG TGT GAT TCA GTA TGT TTT CCG AAG T-3’) and the pH-responsive probe (C4 DNA, 5’-NH₂-(CH₂)₆-AAA AAA AAA ACC CTT ACC CTT ACC CTT ACC C-3’), were carried out by Sangon Biotech Co., Ltd. (Shanghai, China). Detailed sequences related to other experiments are listed in Table S1. The stock solutions of drugs, such as Cat (1.0 mg·mL^− 1), were legally sourced from the institute of evidence laboratory at China University of Political Science and Law (CUPL). For testing, these solutions were diluted to various concentrations (1 fM ~ 1 nM) using Tris-HCl buffer (0.01 M, pH 7.4). Unless otherwise specified, all chemical solutions were prepared using ultrapure water (18.2 MΩ·cm) produced by a Millipore Milli-Q system. All materials and reagents are described in detail in the Supporting Information.

Fabrication of Functionalized N _C B _S. The anodized aluminum oxide (AAO) membranes with a pore size of 40 ~ 70 nm were first sonicated in ultrapure water for 3 min. After drying, the membranes were treated with anhydrous ethanol for 2 h to ensure that the surfaces and internal channels of the AAO membranes were thoroughly cleaned. At RT, the AAO membrane surfaces were aldehyde-functionalized (GA@AAO) using a two-step chemical method. Subsequently, 1 µM CBA was grafted into the nanochannels via Schiff base reaction, resulting in CBA@AAO. Prior to -CHO modification, the AAO nanochannel membranes were first treated with a (3-aminopropyl) trimethoxysilane (APTES, 15%) isopropanol solution for 12 h to aminate the membrane surface (-NH₂). Then, the membranes underwent a Schiff base reaction with glutaraldehyde (GA, 15%) aqueous solution for 12 h to successfully modify the surface of the nanochannel membranes with -CHO groups. A similar method was employed to prepare the pH-responsive N_CB_S (C4@AAO), as well as the dual-probes nanochannels with co-calibration to Cat and pH (CBA&C4@AAO). For the preparation of CBA&C4@AAO using the asymmetric solution method, GA@AAO was placed in a custom mold, and equal amounts of 1 µM CBA and C4 DNA were added to the two separate electrolytic cells on either side of the membrane.

Current-Voltage Measurement and Real-time Current Recording. The samples were installed in a dual-chamber electrochemical cell and measured using a Keithley 6487 picoammeter (Keithley Instruments, USA). Ionic current measurements were conducted in a 0.01 M Tris-HCl (pH 7.4) electrolyte solution using a custom-made Ag/AgCl electrode. The effective area for ion conduction was approximately 5 mm². The voltage was scanned between − 2 V ~ + 2 V with a step voltage of 0.1 V to plot the current-voltage (I-V) curves. A constant voltage of + 2 V was applied, and real-time current (I-T) recordings were taken over a period of 60 s. All tests were conducted at RT, with each test performed at least three times in parallel to obtain average values.

Apparatus and Characterization. The nanochannel samples were characterized using the Zeiss GeminiSEM 360 ultra-high-resolution scanning electron microscope (SEM) at an accelerating voltage of 5.00 kV. EDS mapping of the C, N, O, and Si elements on the surface of the N_CB_S was performed using the Hitachi/SU1000 high-resolution scanning electron microscope. X-ray photoelectron spectroscopy (XPS) analysis of the AAO membrane before and after modification was conducted using the Thermo Scientific™ K-Alpha^TM+ spectrometer. The contact angles (CA) were measured at RT using the SDC-350 instrument (Shengding Precision Instrument, Dongguan, China). Circular dichroism (CD) spectra in the wavelength range of 200 to 340 nm were recorded using the Chirascan Plus spectropolarimeter (Applied Photophysics, UK). For more information, please refer to the supporting information.

Data availability

All data supporting this work and its findings are available within the article and Supplementary Information.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available online.

Correspondence and requests for materials should be addressed to Yu Huang or Fengyu Li.

Author contributions

^#Y. C., and X. L. contributed equally in this work. Y. C., Y. H., and F. L. conceived and designed the experiments. Y. C., X. Y., W. Y., Y. S., and Z. H. performed the measurements and experiments. Y. C., X. Y., Y. W., Y. H., and F. L. contributed to the data analysis. Y. C., Y. H., and F. L. wrote the manuscript. All authors reviewed and commented on the manuscript.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (22474049, 52003103, 22090050, and 22176180), the National Key R&D Program of China (2016YFC1100502 and 2021YFA1200400), the Key research and development project of Guangdong Province (Grant No.2020B010190003), and Natural Science Foundation of Zhejiang Province (Grant No. LD21B05000).

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There is NO Competing Interest.

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TOC. Utilizing multiple DNA probes leverages the combined specificity advantages of each probe. By modifying a nanoconfined biosensor (N_CB_S) with dual-DNA probes, a co-calibration strategy for both the target substance (cathinone) and pH was developed, enabling ultra-sensitive quantitative detection of cathinone in artificial sweat. This work highlights the enhanced performance of multifunctional DNA probes and nanochannels, demonstrating broader applicability in complex environments.
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Sub-Femtomolar Drug Monitoring via Co-Calibration Mechanism with Nanoconfined DNA Probes

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Sensing Properties of C4@AAO and CBA&C4@AAO at Different pH Levels

Sensing Mechanism of NB Modified by DNA Probes

Application of Co-Calibration Strategy in Complex System

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