{"paper_id":"1ce3a62c-2898-4b00-babd-acda4acb571a","body_text":"Logic-Gated Fluorescent Biosensor Integrating Aptamer Recognition and Oxidative Cleavage-Responsive DNA Circuit for Myeloperoxidase Detection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Logic-Gated Fluorescent Biosensor Integrating Aptamer Recognition and Oxidative Cleavage-Responsive DNA Circuit for Myeloperoxidase Detection Bo-Yu Shi, Li Wen, Jing Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7646512/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Feb, 2026 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Precise discrimination between protein abundance and catalytic activity of proteases remains a critical yet challenging objective in biomedical diagnostics due to overlapping biological functions, intricate regulatory mechanisms, and extensive interference from endogenous biomolecules. Herein, we report a novel dual-lock DNA biosensing platform, exemplified through myeloperoxidase (MPO), which concurrently integrates aptamer-mediated molecular recognition and hypochlorous acid (HOCl)-triggered oxidative cleavage to rigorously assess both MPO protein expression and enzymatic functionality. Specifically, MPO interaction with a conformationally structured DNA aptamer facilitates selective release of a trigger strand, while HOCl, produced enzymatically by active MPO, cleaves a strategically phosphorothioate-modified hairpin structure. Only upon simultaneous fulfillment of these two molecular conditions does the sensing mechanism activate a downstream catalytic hairpin assembly (CHA), achieving significant signal amplification. This stringent AND logic gate configuration markedly suppresses false positives and nonspecific background signals, demonstrating exceptional reliability across diverse and complex biological samples including serum, saliva, and cellular lysates. The proposed biosensing strategy thus provides a versatile, accurate, and broadly applicable analytical tool for simultaneous quantification of protease content and functional activity, holding considerable promise for advancing clinical diagnostics and pathological investigations. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The precise determination of both protease content and their catalytic activity is fundamentally important yet remains technically challenging within biomedical diagnostics 1 – 3 . Proteases play pivotal roles in numerous biological processes, including inflammation, cellular regulation, immune responses, and pathological conditions 4 . However, the overlapping substrate specificities, intricate activation pathways, and extensive interference from other biomolecules in complex biological matrices hinder the accurate quantification of protease abundance and the evaluation of their enzymatic activity. Traditional assays often focus exclusively on either protein abundance or catalytic activity, thus lacking the capacity to simultaneously interrogate both parameters and potentially generating misleading interpretations 5 . Myeloperoxidase (MPO), a lysosomal heme-containing peroxidase primarily expressed in neutrophils and monocytes, serves as an excellent model for this analytical challenge due to its significant involvement in oxidative stress-related disorders such as atherosclerosis, neurodegenerative diseases, and chronic inflammatory conditions 6 – 9 . MPO exerts its biological function by catalyzing the oxidation of chloride ions (Cl⁻) in the presence of hydrogen peroxide (H₂O₂) to produce hypochlorous acid (HOCl), a reactive oxygen species critical for antimicrobial activity and inflammatory modulation 10 , 11 . Dysregulated MPO activity is associated with extensive tissue damage, further underscoring the necessity for accurately differentiating between MPO protein levels and its active catalytic form 12 – 18 . Current MPO detection methodologies, including immunoassays and enzymatic activity-based assays, typically provide single-dimensional insights, either quantifying total protein content or monitoring enzymatic activity independently 19 – 23 . These approaches face significant limitations, including nonspecific interferences, inadequate sensitivity in complex biological environments, and an inability to discriminate effectively between active and inactive enzyme forms. However, these approaches often suffer from cross-reactivity with other peroxidases (e.g., HRP, LPO), leading to inaccurate attribution of oxidative activity to MPO alone 24 – 26 . Given the complementary nature of these two detection modalities, a unified platform capable of simultaneously assessing MPO presence and catalytic function is highly desirable. Yet, integrating both recognition and enzymatic response within a single sensing system remains technically challenging. The designs are often complex, time-consuming, and prone to inconsistent outputs due to variations in assay conditions and sample matrices. In this study, we address these analytical shortcomings by introducing a novel dual-lock DNA logic-gated biosensor design, integrating aptamer-mediated recognition of MPO with HOCl-responsive oxidative cleavage. This strategy employs two distinct biochemical events: the selective binding of MPO to release an aptamer-triggered DNA strand, and the enzymatic generation of HOCl to cleave a phosphorothioate-modified DNA hairpin 27 – 31 . Only upon simultaneous activation by both molecular triggers does the system engage in a signal amplification cascade. Here, catalytic hairpin assembly (CHA), a straightforward and widely adopted DNA amplification method, serves merely as an illustrative example to demonstrate the proof-of-concept 32 – 34 . Such a stringent AND logic gate configuration provides exceptional specificity, significantly minimizes false positives, and enhances analytical reliability in complex biological matrices. The proposed biosensor platform thus offers a robust, generalizable solution for simultaneously evaluating protease content and catalytic functionality, with significant implications for clinical diagnostics and real-time pathological monitoring. Results and Discussion (1) Design of the Dual-Lock DNA Logic-Gated Circuit In this study, we designed a molecular sensing strategy with a catalytically controlled CHA mechanism, regulated through a dual “logic-lock” configuration. This system employs two orthogonal control modules to ensure high specificity and signal fidelity (Scheme 1 ). The first lock is based on an aptamer–trigger strand (T) duplex that modulates the accessibility of the trigger. In the resting state (Scheme 1 a), the T strand is hybridized to a portion of the aptamer (Apt) through partial base pairing, forming a stable complex that sequesters the trigger and prevents downstream activation. Upon binding of MPO, which has a higher affinity for the aptamer than the T strand, the T strand is competitively displaced and released. This MPO-triggered displacement unlocks the first layer of control, exposing the toehold domain of the T strand and enabling it to initiate strand displacement reactions downstream. The second lock is encoded within a phosphorothioate (PS)-modified hairpin precursor, denoted as pH2, which functions as an inactive, protected form of the hairpin H2. In pH2, a PS modification is strategically introduced at a specific internal site within the stem region of the hairpin. This PS site acts as a chemically cleavable switch that responds selectively to HOCl, a reactive oxygen species generated by MPO in the presence of H₂O₂ and Cl⁻. Upon exposure to HOCl, the PS site undergoes oxidative cleavage, disrupting the structural integrity of the blocking segment and converting pH2 into its active form, H2 (Scheme 1 b). This structural transition exposes the toehold domain of H2, thereby unlocking the second molecular gate and allowing H2 to participate in the CHA reaction. The mechanism of HOCl-induced cleavage of phosphorothioate-modified DNA is illustrated in Figure S1 . When both locks are disengaged, the released T strand first initiates toehold-mediated strand displacement with H1, resulting in the formation of an intermediate that subsequently activates the now-accessible H2. Active H2 displaces the T strand from the H1-T duplex, forming a stable H1-H2 complex and regenerating free T to re-enter the catalytic cycle. This recursive process enables robust signal amplification (Scheme 1 c). The final H1-H2 duplex then triggers structural changes in a fluorogenic probe, releasing a measurable fluorescence signal. In essence, MPO protein serves as the first molecular key, unlocking the trigger strand through aptamer displacement, while HOCl functions as the second key, activating the previously blocked H2 hairpin through oxidative cleavage. Only in the simultaneous presence of MPO and its catalytic product (HOCl) can the full CHA cascade be initiated. This dual-lock mechanism ensures AND-gated signal output, offering precise, noise-resistant, and activity-dependent MPO detection. (2) Functional Validation of the Dual-Lock Logic-Gated DNA Circuit Modules To validate the functionality of each module within the proposed dual-lock logic-gated sensing system, we systematically examined three critical components: trigger strand (T) release, HOCl-mediated activation of the protected hairpin pH2, and signal amplification via CHA (Fig. 1 ). In Fig. 1 a and 1 b, we first verified the mechanism of the first lock, which is based on the displacement of the T strand from its duplex with the aptamer (Apt). The Apt and T strands were hybridized in a 1:1 molar ratio, with the Apt labeled at the 5′ end with a Cy3 fluorophore and the T strand labeled with a BHQ2 quencher. In the initial Apt/T duplex, fluorescence was effectively quenched. Upon addition of varying concentrations of MPO, fluorescence intensity increased proportionally, whereas the signal remained low in the absence of MPO. These results confirm that MPO can specifically bind the aptamer and displace the T strand, thereby unlocking the first gate and initiating signal transduction through toehold exposure. In Fig. 1 c and 1 d, we evaluated the second lock—the HOCl-responsive activation of the protected hairpin pH2. This structure contains an internal PS cleavage site, allowing selective response to HOCl generated in the MPO + H₂O₂ + NaCl system. Upon incubation with increasing concentrations of HOCl, polyacrylamide gel electrophoresis (PAGE, 10%) analysis demonstrated progressive cleavage of pH2 (Figure S2 ), while fluorescence recovery assays showed a dose-dependent signal enhancement. These findings indicate that HOCl efficiently cleaves the PS site, removes the structural blockade, and converts pH2 into its active H2 form by exposing its toehold, thus unlocking the second gate. In Fig. 1 e and 1 f, we tested the amplification performance and input specificity of the CHA module. A basic reaction system containing H1, H2, and a fluorogenic reporter (FQ probe) was assembled. CHA was initiated by adding varying concentrations of the released T strand, alongside a mismatched T strand group and a blank control. As expected, fluorescence increased with T strand concentration, while the mismatched and control groups showed negligible signal, confirming that the CHA circuit is selectively activated and enables efficient signal amplification only in the presence of the correct trigger. The fluorescence spectra (Fig. 1 g) further support the dose-dependent nature of the CHA response. PAGE was performed to validate the assembly and activation of the catalytic hairpin assembly (CHA) reaction under various combinations of trigger (T), hairpin H1, and hairpin H2 (Figure S3). Taken together, the results from Fig. 1 provide strong experimental validation for the modular functionality of the dual-lock biosensing system. The aptamer-mediated release of T, HOCl-induced activation of pH2, and the downstream CHA-based amplification cascade function synergistically and reliably within the proposed logic-gated architecture. (3) Construction and Response Evaluation of the Dual-Lock Logic-Gated DNA Sensing System To enable precise molecular recognition and signal transduction, we constructed a DNA nanodevice that integrates aptamer binding and oxidative cleavage within a dual-lock logic-gated architecture, capable of detecting active MPO. As shown in Fig. 2 a, the sensing system incorporates two input-dependent control elements, corresponding to two molecular “keys.” Input 1 (MPO): The trigger strand (T) is initially hybridized with the MPO-specific aptamer (Apt), forming a locked duplex that inhibits signal propagation. Only when MPO is present does it competitively bind the aptamer and release the T strand, enabling downstream activation of the CHA cascade. Input 2 (HOCl): HOCl, generated by MPO in the presence of H₂O₂ and Cl⁻, selectively cleaves the internal PS site of the protected hairpin pH2. This cleavage disrupts the blocking segment and exposes the toehold of H2, converting pH2 into its active conformation. These two inputs act cooperatively to regulate signal output. Only when both the T strand and activated H2 are present can the CHA reaction proceed, fulfilling an AND logic gate function. To experimentally validate the logic behavior, Fig. 2 b shows fluorescence spectra under different component combinations. Introducing only the T strand (FQ + H1 + pH2 + T) or T with MPO (FQ + H1 + pH2 + T + MPO) failed to induce significant fluorescence. In contrast, only when the full MPO catalytic system (MPO + H₂O₂ + Cl⁻) was introduced did the system generate a strong fluorescence signal, confirming that both molecular locks must be disengaged for activation. Figure 2 c provides the corresponding quantitative analysis of relative fluorescence intensity (R/R₀), clearly demonstrating the cooperative nature of the dual-input activation and the high specificity of the system. (4) Optimization of Reaction Conditions for the Dual-Lock MPO Sensing System To ensure the stability and sensitivity of the sensing system under practical conditions, we systematically evaluated the influence of several key environmental parameters, including pH, temperature, Cl⁻ concentration, and H₂O₂ concentration (Fig. 3 ). As shown in Fig. 3 a, the fluorescence intensity peaked at pH 6.0, which aligns with the optimal catalytic activity of MPO under mildly acidic conditions. Alkaline pH (e.g., 9.2) significantly reduced the signal, indicating impaired enzymatic efficiency. Temperature also had a notable impact on the sensing response (Fig. 3 b). The strongest fluorescence signal was observed at 37°C, while lower signals were recorded at 25°C and 30°C. The signal at 42°C was comparable to that at 37°C, indicating that the system maintains optimal reactivity and structural stability near physiological temperature. Since MPO catalysis relies on both Cl⁻ and H₂O₂ as substrates, we next examined their concentration-dependent effects. As shown in Fig. 3 c, the fluorescence signal increased with Cl⁻ concentration and plateaued at 20 mM, suggesting this concentration is sufficient to saturate MPO’s catalytic requirement. Higher concentrations (≥ 40 mM) did not yield further enhancement, implying a reaction plateau. Similarly, Fig. 3 d shows that the system exhibited the strongest fluorescence at 20 µM H₂O₂, with a stable signal range from 20–100 µM. In contrast, lower concentrations (5–10 µM) resulted in significantly weaker fluorescence, indicating that 20 µM is the threshold for effective MPO activation in this system. Taken together, the optimal conditions for the dual-lock sensing platform are: pH 6.0, temperature 37°C, Cl⁻ 20–100 mM, and H₂O₂ 20–100 µM. These parameters ensure robust performance of the sensor under physiologically relevant environments. (5) Analytical Performance Evaluation of the Dual-Lock Fluorescent MPO Sensing Platform To systematically evaluate the performance of the dual-lock logic-gated MPO sensing system, we assessed its specificity, sensitivity, quantification ability, and enzyme activity dependence, as summarized in Fig. 4 . Specificity analysis was conducted using a range of protein and redox interferents. As shown in Fig. 4 a and 4 b, only catalytically active MPO in the presence of H₂O₂ and Cl⁻, or exogenous MPO and HOCl, triggered a strong fluorescence response. Other conditions—including MPO alone, inactive MPO, or single substrates—produced negligible signals, confirming strict dual-input gating and minimal false positives. In Fig. 4 c, further selectivity was demonstrated by testing structurally and functionally related biomolecules commonly encountered in oxidative stress environments, such bovine serum albumin (BSA), horseradish peroxidase (HRP), NADPH, catalase (CAT), glucose oxidase (GOx), xanthine oxidase and glutathione reductase (GR). Only catalytically active MPO induced a significant fluorescence signal. This result confirms the high specificity of the system in discriminating MPO from structurally and functionally related oxidoreductases. To further assess the anti-interference capacity of the platform, we conducted interference assays under physiologically relevant conditions (Fig. 4 d). The system was challenged with a panel of redox-active species (e.g., cysteine, GSH, ascorbic acid), oxidants (e.g., HOCl, H₂O₂, NO₃⁻), and abundant cations found in biological fluids (e.g., Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺). None of these interferents triggered notable fluorescence increases, while MPO consistently activated the system. These findings underscore the excellent selectivity and robustness of the platform under complex biological conditions. We next assessed the sensitivity and dynamic range of the platform. As shown in Fig. 4 e, the fluorescence intensity increased proportionally with MPO concentration from 0.5 to 30 ng/mL, forming a clear kinetic response profile. Endpoint fluorescence spectra (Fig. 4 f) further validated this trend, showing dose-dependent enhancement centered at ~ 570 nm. A calibration curve was constructed based on endpoint intensity (Fig. 4 g), exhibiting excellent linearity (R² = 0.9834) with the regression equation Y = 9.023X + 43.34 . The limit of detection (LOD) was estimated to be 1.73 ng/mL. These results demonstrate the system’s high sensitivity and broad dynamic range for quantitative MPO detection. To verify that the fluorescence output originated specifically from MPO enzymatic activity rather than probe self-activation or nonspecific reactions, we performed inhibition assays using 4-aminobenzoic acid hydrazide (4-ABAH), a selective MPO inhibitor that binds to and inactivates its catalytic site. As shown in Fig. 4 h, increasing concentrations of 4-ABAH (0–16 µM) led to a dose-dependent suppression of the fluorescence signal, confirming that the readout was indeed driven by MPO catalysis. Taken together, the dual-lock fluorescent sensing platform exhibits outstanding performance in terms of specificity, sensitivity, quantitative accuracy, and interference resistance. These features highlight its potential for reliable detection of MPO content and activity in complex biological samples. (6) Recovery Analysis in Biological Samples and Long-Term Stability Evaluation of the Biosensor To assess the applicability of the developed MPO biosensing system in complex biological matrices, recovery experiments were conducted in human serum, cell lysate, and saliva samples. Known concentrations of MPO (5, 10, and 15 ng/mL) were spiked into each sample type, and the corresponding fluorescence responses were recorded. The results are summarized in Table S2 . In all tested matrices, the biosensor accurately quantified the spiked MPO, yielding recoveries ranging from 99.78% to 106.42%, with relative standard deviations (RSDs) below 4.85%, demonstrating excellent accuracy and reproducibility. These findings confirm the feasibility of direct MPO quantification in biological samples without significant matrix interference, highlighting the robustness and practicality of the sensing platform. To further investigate the long-term performance of the fluorescent biosensor, stability studies were carried out under refrigerated storage conditions (4°C, protected from light) for up to six months. The sensor was stored under two conditions: in the presence and absence of MPO, and fluorescence responses were periodically measured. As illustrated in Figure S4, the fluorescence intensity in the MPO group remained consistently high over the 6-month period, with only a slight, non-significant decrease, indicating stable sensor performance over time. In contrast, the fluorescence signals in the control group without MPO remained at a low background level throughout the entire duration, showing no evidence of spontaneous signal activation. These results confirm the excellent storage stability and anti-background triggering capacity of the system. In summary, the proposed MPO biosensor exhibits high detection accuracy across various biological matrices and retains excellent stability under standard storage conditions. These properties make it highly suitable for preclinical sample analysis and long-term storage following batch fabrication. Conclusion In summary, we have established a dual-lock, logic-gated DNA biosensing platform that enables the concurrent and highly specific detection of both the presence and enzymatic activity of myeloperoxidase (MPO), a key oxidative enzyme implicated in numerous inflammation-associated pathologies. By integrating aptamer-mediated molecular recognition with HOCl-triggered oxidative cleavage, the system achieves a stringent AND logic gating mechanism, wherein signal output is initiated only upon simultaneous satisfaction of two biochemical criteria: MPO protein binding and catalytic generation of HOCl. This molecular logic architecture offers several critical advantages, including minimal background leakage, high analytical specificity, and resistance to false-positive signals arising from inactive MPO or related peroxidases. Under optimized conditions, the sensor exhibits a broad linear detection range (0.5–30 ng/mL), a low detection limit (~ 1.73 ng/mL), and excellent selectivity against interfering species. Furthermore, its robust performance in diverse biological matrices—such as human serum, saliva, and cell lysate—combined with long-term storage stability, underscores its practical applicability in real-world biosensing scenarios. Beyond methodological innovation, this work provides a generalizable framework for constructing activity-dependent, logic-controlled DNA nanodevices, with potential implications in dynamic biomarker monitoring, precision diagnostics, and point-of-care testing. Nonetheless, some limitations persist. The oxidative cleavage reaction, while effective, is inherently sensitive to redox fluctuations in complex samples, necessitating further refinements for in vivo robustness. Moreover, real-time kinetic monitoring and integration into miniaturized platforms remain important future directions. Overall, this dual-lock strategy exemplifies the fusion of programmable nucleic acid circuitry with biochemical reactivity, offering a powerful paradigm for next-generation biosensor design and molecular logic computation in biomedical diagnostics. Declarations Data availability The authors declared that all data supporting the conclusions of this research is available. Acknowledgements Not applicable. Funding This research was supported by the National Natural Science Foundation of China (22404124), the Tianjin Education Commission research project (2023KJ074), and the Beijing Dadi Medical Charity Foundation (DDYL-A-KT-20241111-0112). Authors and Affiliations Department of Clinical Biochemistry and Molecular Diagnostics, College of Medical Technology, Tianjin Medical University, Tianjin, 300203, P. R. China Bo-Yu Shi, Li Wen & Jing Wang Contributions Bo-Yu Shi: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. Li Wen: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Jing Wang: Writing – review & editing, Writing – original draft, Validation, Supervision, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. Corresponding authors Correspondence to Jing Wang. Ethics declarations Ethics approval and consent to participate Our research was approved by the local ethics committee based on the Declaration of Helsinki. Written consent was obtained from each patient before surgery. 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Label-free electrochemical sensing platform for sensitive detection of ampicillin by combining nucleic acid isothermal enzyme-free amplification circuits with CRISPR/Cas12a. Talanta 273 , 125950, doi:https://doi.org/10.1016/j.talanta.2024.125950 (2024). Scheme 1 Scheme 1 is available in the Supplementary Files section. Supplementary Files supplementaryfile.docx floatimage1.jpeg Scheme 1. Schematic of the dual-lock logic-gated DNA circuit for MPO detection. The system integrates aptamer recognition and HOCl-responsive activation to enable concurrent evaluation of MPO content and enzymatic activity. (a) In the resting state, the trigger strand (T) is hybridized with the MPO-specific aptamer, while H2 remains in an inactive precursor form (pH2) containing an internal PS site. (b) MPO binding displaces T from the aptamer, and MPO-generated HOCl cleaves the PS site in pH2, converting it into active H2 with an exposed toehold. (c) Only when both T and active H2 are present is the CHA circuit initiated, leading to fluorescence output. Cite Share Download PDF Status: Published Journal Publication published 02 Feb, 2026 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 18 Nov, 2025 Reviewers invited by journal 27 Sep, 2025 Editor assigned by journal 19 Sep, 2025 First submitted to journal 17 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7646512\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":521572983,\"identity\":\"22dbe154-7209-4f61-875b-f081f99e76e6\",\"order_by\":0,\"name\":\"Bo-Yu Shi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tianjin Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Bo-Yu\",\"middleName\":\"\",\"lastName\":\"Shi\",\"suffix\":\"\"},{\"id\":521572984,\"identity\":\"08ba52a8-1ea7-4c84-a6f9-3258f561a7ba\",\"order_by\":1,\"name\":\"Li Wen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Tianjin Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Li\",\"middleName\":\"\",\"lastName\":\"Wen\",\"suffix\":\"\"},{\"id\":521572985,\"identity\":\"41dedf2e-b15d-4bff-ae5b-06b805d27218\",\"order_by\":2,\"name\":\"Jing Wang\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYLACCQMGBn725oMPINwEIrVI9hxLNjhAtBYQMLiRYyZBlBaD42cPv7AouGM3syHBrPpjzmGgC3MMGH7uwKPlTF6ahYTBs+R+hgNpNw5uOwx04RsDxt4zuLWYHcgxM5AwOJws2dhwDKwF6EIDZsY2PFrOv4FoMTjM2FYA0mJPUMuNHOMHQC12BseY2RjAtkgQ0GJ/440ZMJAPJ0j2sDFLnN2WziNx5lnBwV48WiT7c4w/S/w5bM8v//7jh8pt1nL87ckbH/zEowUI2KQlGBgSG6A8HhBxAK8GBgbmjx+ADiSgaBSMglEwCkYyAACUdFjT42UvVwAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0009-0009-9097-627X\",\"institution\":\"Tianjin Medical 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10:13:40\",\"extension\":\"html\",\"order_by\":18,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"acdc-reference\",\"size\":91811,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"earlyproof.html\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7646512/v1/30798f7a28fba24dc4be3e7e.html\"},{\"id\":93122996,\"identity\":\"9351fb34-d255-4721-b6dd-0563b457db3b\",\"added_by\":\"auto\",\"created_at\":\"2025-10-09 09:57:40\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":580014,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunctional validation of the dual-lock logic-gated sensing system.\\u003c/strong\\u003e (a) Schematic illustration of the first gate: MPO binding induces aptamer conformational change, releasing the quenched trigger strand (T). (b) Fluorescence recovery of Cy3-labeled Apt/T duplex with increasing MPO concentrations (5–1000 ng/mL). (c) Schematic of the second gate: HOCl cleaves the PS site in pH2, converting it to active H2. (d) Fluorescence kinetics of pH2 activation under various HOCl concentrations (0–50 μM). (e) Schematic of CHA initiated by released T and H2, leading to FQ probe activation. (f) Fluorescence spectra of CHA triggered by different T concentrations (200–50 pM), mismatched T, and control. (g) Time-dependent fluorescence curves of CHA with increasing T input.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7646512/v1/b376670c4b2d5becbe9cb21c.png\"},{\"id\":93122998,\"identity\":\"7392ea54-fd46-4667-aad9-c54d7ebec293\",\"added_by\":\"auto\",\"created_at\":\"2025-10-09 09:57:40\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":458311,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDesign and fluorescence verification of the dual-lock DNA logic gate.\\u003c/strong\\u003e (a) Schematic representation of the dual-input sensing mechanism. Input 1: MPO binds the aptamer, releasing trigger T strand. Input 2: HOCl , generated by the MPO–H₂O₂–Cl⁻ system, cleaves pH2 to expose the H2 toehold. Only when both inputs are present is the CHA reaction activated, generating fluorescence. (b) Fluorescence spectra for different component combinations. Signal appears only under dual-input conditions, consistent with AND logic behavior. (c) Normalized fluorescence intensity (R/R₀) corresponding to panel (b). Significant signal enhancement is observed only with both MPO and catalytic substrates. *p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001, ****p \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7646512/v1/4f6e7bf8a3d1d6b42c714ae2.png\"},{\"id\":93125072,\"identity\":\"97cbd4b2-8a4f-4021-b0c3-c0ea632730e1\",\"added_by\":\"auto\",\"created_at\":\"2025-10-09 10:21:40\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":269152,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eOptimization of key reaction conditions for the dual-lock logic-gated MPO sensing system.\\u003c/strong\\u003e (a) Fluorescence measurements under different pH conditions to evaluate the influence of acidity on system performance. (b) Assessment of fluorescence output at various incubation temperatures (25 °C, 30 °C, 37 °C, and 42 °C). (c) Fluorescence response as a function of Cl⁻ concentration ranging from 0 to 30 mM. (d) Evaluation of fluorescence intensity across a gradient of H₂O₂ concentrations (5–100 μM).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7646512/v1/76040b9ff97be35942a32eb5.png\"},{\"id\":93123313,\"identity\":\"915e8ae8-d2bb-4dc9-b6ab-d3897f28cfb5\",\"added_by\":\"auto\",\"created_at\":\"2025-10-09 10:05:40\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":619275,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnalytical performance evaluation of the dual-lock MPO fluorescent sensing platform. \\u003c/strong\\u003e(a) Fluorescence spectra under seven representative biochemical conditions (e.g., MPO, inactive MPO, HOCl, partial combinations). (b) Quantified R/R₀ values from (a), highlighting selective activation by catalytically active MPO. Background corresponds to the condition containing only FQ-labeled H1 and pH2. (c) Fluorescence response of the system to various redox-related proteins and small molecules, including BSA, BSA, HRP, NADPH, CAT, GOx, and GR. (d) Interference assessment under physiologically relevant conditions. Nineteen potential interfering substances—including reducing agents (Cys, GSH, ascorbic acid), oxidants (HOCl, H₂O₂, NO₃⁻), and common metal cations (Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺). (e) Fluorescence kinetic curves at different MPO concentrations (0.5–30 ng/mL), showing dose-dependent signal amplification. (f) Steady-state fluorescence spectra corresponding to (e), with increasing emission intensities at ~570 nm in response to higher MPO concentrations. (g) Calibration curve based on endpoint fluorescence intensities. Linear relationship observed within 0.5–30 ng/mL (R² = 0.9834; Y = 9.023X + 43.34). (h) Fluorescence inhibition by MPO-specific inhibitor 4-ABAH. Signal intensity decreased with increasing 4-ABAH concentration (0–16 μM).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7646512/v1/980c5c23bbb70f5b083e9a44.png\"},{\"id\":102235197,\"identity\":\"8a92d5f0-6530-4dea-9de2-6eb1889827e3\",\"added_by\":\"auto\",\"created_at\":\"2026-02-09 16:15:42\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2767036,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7646512/v1/529229c9-02be-466e-9074-795ea2451e62.pdf\"},{\"id\":93123316,\"identity\":\"1f9a3712-ccc5-49e7-a20e-110be1571083\",\"added_by\":\"auto\",\"created_at\":\"2025-10-09 10:05:40\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":13345545,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"supplementaryfile.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7646512/v1/f2ddced2f5d93260255cd0fe.docx\"},{\"id\":93124676,\"identity\":\"309c356b-070c-4932-a59c-22b91bf8a805\",\"added_by\":\"auto\",\"created_at\":\"2025-10-09 10:13:40\",\"extension\":\"jpeg\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":750031,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eScheme 1. Schematic of the dual-lock logic-gated DNA circuit for MPO detection.\\u003c/strong\\u003e The system integrates aptamer recognition and HOCl-responsive activation to enable concurrent evaluation of MPO content and enzymatic activity. (a) In the resting state, the trigger strand (T) is hybridized with the MPO-specific aptamer, while H2 remains in an inactive precursor form (pH2) containing an internal PS site. (b) MPO binding displaces T from the aptamer, and MPO-generated HOCl cleaves the PS site in pH2, converting it into active H2 with an exposed toehold. (c) Only when both T and active H2 are present is the CHA circuit initiated, leading to fluorescence output.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7646512/v1/73ce9e8fa7438daa9d5ed30e.jpeg\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Logic-Gated Fluorescent Biosensor Integrating Aptamer Recognition and Oxidative Cleavage-Responsive DNA Circuit for Myeloperoxidase Detection\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe precise determination of both protease content and their catalytic activity is fundamentally important yet remains technically challenging within biomedical diagnostics\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR2\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e\\u003c/sup\\u003e. Proteases play pivotal roles in numerous biological processes, including inflammation, cellular regulation, immune responses, and pathological conditions\\u003csup\\u003e\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u003c/sup\\u003e. However, the overlapping substrate specificities, intricate activation pathways, and extensive interference from other biomolecules in complex biological matrices hinder the accurate quantification of protease abundance and the evaluation of their enzymatic activity. Traditional assays often focus exclusively on either protein abundance or catalytic activity, thus lacking the capacity to simultaneously interrogate both parameters and potentially generating misleading interpretations\\u003csup\\u003e\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u003c/sup\\u003e. Myeloperoxidase (MPO), a lysosomal heme-containing peroxidase primarily expressed in neutrophils and monocytes, serves as an excellent model for this analytical challenge due to its significant involvement in oxidative stress-related disorders such as atherosclerosis, neurodegenerative diseases, and chronic inflammatory conditions\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR7 CR8\\\" citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e\\u003c/sup\\u003e. MPO exerts its biological function by catalyzing the oxidation of chloride ions (Cl⁻) in the presence of hydrogen peroxide (H₂O₂) to produce hypochlorous acid (HOCl), a reactive oxygen species critical for antimicrobial activity and inflammatory modulation\\u003csup\\u003e\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e,\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u003c/sup\\u003e. Dysregulated MPO activity is associated with extensive tissue damage, further underscoring the necessity for accurately differentiating between MPO protein levels and its active catalytic form\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR13 CR14 CR15 CR16 CR17\\\" citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003eCurrent MPO detection methodologies, including immunoassays and enzymatic activity-based assays, typically provide single-dimensional insights, either quantifying total protein content or monitoring enzymatic activity independently\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR20 CR21 CR22\\\" citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u003c/sup\\u003e. These approaches face significant limitations, including nonspecific interferences, inadequate sensitivity in complex biological environments, and an inability to discriminate effectively between active and inactive enzyme forms. However, these approaches often suffer from cross-reactivity with other peroxidases (e.g., HRP, LPO), leading to inaccurate attribution of oxidative activity to MPO alone\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR25\\\" citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e\\u003c/sup\\u003e. Given the complementary nature of these two detection modalities, a unified platform capable of simultaneously assessing MPO presence and catalytic function is highly desirable. Yet, integrating both recognition and enzymatic response within a single sensing system remains technically challenging. The designs are often complex, time-consuming, and prone to inconsistent outputs due to variations in assay conditions and sample matrices.\\u003c/p\\u003e\\u003cp\\u003eIn this study, we address these analytical shortcomings by introducing a novel dual-lock DNA logic-gated biosensor design, integrating aptamer-mediated recognition of MPO with HOCl-responsive oxidative cleavage. This strategy employs two distinct biochemical events: the selective binding of MPO to release an aptamer-triggered DNA strand, and the enzymatic generation of HOCl to cleave a phosphorothioate-modified DNA hairpin\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR28 CR29 CR30\\\" citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e\\u003c/sup\\u003e. Only upon simultaneous activation by both molecular triggers does the system engage in a signal amplification cascade. Here, catalytic hairpin assembly (CHA), a straightforward and widely adopted DNA amplification method, serves merely as an illustrative example to demonstrate the proof-of-concept\\u003csup\\u003e\\u003cspan additionalcitationids=\\\"CR33\\\" citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e\\u003c/sup\\u003e. Such a stringent AND logic gate configuration provides exceptional specificity, significantly minimizes false positives, and enhances analytical reliability in complex biological matrices. The proposed biosensor platform thus offers a robust, generalizable solution for simultaneously evaluating protease content and catalytic functionality, with significant implications for clinical diagnostics and real-time pathological monitoring.\\u003c/p\\u003e\"},{\"header\":\"Results and Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e(1) Design of the Dual-Lock DNA Logic-Gated Circuit\\u003c/h2\\u003e\\u003cp\\u003eIn this study, we designed a molecular sensing strategy with a catalytically controlled CHA mechanism, regulated through a dual \\u0026ldquo;logic-lock\\u0026rdquo; configuration. This system employs two orthogonal control modules to ensure high specificity and signal fidelity (Scheme \\u003cspan refid=\\\"Sch1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). The first lock is based on an aptamer\\u0026ndash;trigger strand (T) duplex that modulates the accessibility of the trigger. In the resting state (Scheme \\u003cspan refid=\\\"Sch1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea), the T strand is hybridized to a portion of the aptamer (Apt) through partial base pairing, forming a stable complex that sequesters the trigger and prevents downstream activation. Upon binding of MPO, which has a higher affinity for the aptamer than the T strand, the T strand is competitively displaced and released. This MPO-triggered displacement unlocks the first layer of control, exposing the toehold domain of the T strand and enabling it to initiate strand displacement reactions downstream. The second lock is encoded within a phosphorothioate (PS)-modified hairpin precursor, denoted as pH2, which functions as an inactive, protected form of the hairpin H2. In pH2, a PS modification is strategically introduced at a specific internal site within the stem region of the hairpin. This PS site acts as a chemically cleavable switch that responds selectively to HOCl, a reactive oxygen species generated by MPO in the presence of H₂O₂ and Cl⁻. Upon exposure to HOCl, the PS site undergoes oxidative cleavage, disrupting the structural integrity of the blocking segment and converting pH2 into its active form, H2 (Scheme \\u003cspan refid=\\\"Sch1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb). This structural transition exposes the toehold domain of H2, thereby unlocking the second molecular gate and allowing H2 to participate in the CHA reaction. The mechanism of HOCl-induced cleavage of phosphorothioate-modified DNA is illustrated in Figure \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e. When both locks are disengaged, the released T strand first initiates toehold-mediated strand displacement with H1, resulting in the formation of an intermediate that subsequently activates the now-accessible H2. Active H2 displaces the T strand from the H1-T duplex, forming a stable H1-H2 complex and regenerating free T to re-enter the catalytic cycle. This recursive process enables robust signal amplification (Scheme \\u003cspan refid=\\\"Sch1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec). The final H1-H2 duplex then triggers structural changes in a fluorogenic probe, releasing a measurable fluorescence signal. In essence, MPO protein serves as the first molecular key, unlocking the trigger strand through aptamer displacement, while HOCl functions as the second key, activating the previously blocked H2 hairpin through oxidative cleavage. Only in the simultaneous presence of MPO and its catalytic product (HOCl) can the full CHA cascade be initiated. This dual-lock mechanism ensures AND-gated signal output, offering precise, noise-resistant, and activity-dependent MPO detection.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003e(2) Functional Validation of the Dual-Lock Logic-Gated DNA Circuit Modules\\u003c/h3\\u003e\\n\\u003cp\\u003eTo validate the functionality of each module within the proposed dual-lock logic-gated sensing system, we systematically examined three critical components: trigger strand (T) release, HOCl-mediated activation of the protected hairpin pH2, and signal amplification via CHA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). In Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea and \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb, we first verified the mechanism of the first lock, which is based on the displacement of the T strand from its duplex with the aptamer (Apt). The Apt and T strands were hybridized in a 1:1 molar ratio, with the Apt labeled at the 5\\u0026prime; end with a Cy3 fluorophore and the T strand labeled with a BHQ2 quencher. In the initial Apt/T duplex, fluorescence was effectively quenched. Upon addition of varying concentrations of MPO, fluorescence intensity increased proportionally, whereas the signal remained low in the absence of MPO. These results confirm that MPO can specifically bind the aptamer and displace the T strand, thereby unlocking the first gate and initiating signal transduction through toehold exposure. In Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec and \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed, we evaluated the second lock\\u0026mdash;the HOCl-responsive activation of the protected hairpin pH2. This structure contains an internal PS cleavage site, allowing selective response to HOCl generated in the MPO\\u0026thinsp;+\\u0026thinsp;H₂O₂ + NaCl system. Upon incubation with increasing concentrations of HOCl, polyacrylamide gel electrophoresis (PAGE, 10%) analysis demonstrated progressive cleavage of pH2 (Figure \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e), while fluorescence recovery assays showed a dose-dependent signal enhancement. These findings indicate that HOCl efficiently cleaves the PS site, removes the structural blockade, and converts pH2 into its active H2 form by exposing its toehold, thus unlocking the second gate. In Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee and \\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef, we tested the amplification performance and input specificity of the CHA module. A basic reaction system containing H1, H2, and a fluorogenic reporter (FQ probe) was assembled. CHA was initiated by adding varying concentrations of the released T strand, alongside a mismatched T strand group and a blank control. As expected, fluorescence increased with T strand concentration, while the mismatched and control groups showed negligible signal, confirming that the CHA circuit is selectively activated and enables efficient signal amplification only in the presence of the correct trigger. The fluorescence spectra (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eg) further support the dose-dependent nature of the CHA response. PAGE was performed to validate the assembly and activation of the catalytic hairpin assembly (CHA) reaction under various combinations of trigger (T), hairpin H1, and hairpin H2 (Figure S3). Taken together, the results from Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e provide strong experimental validation for the modular functionality of the dual-lock biosensing system. The aptamer-mediated release of T, HOCl-induced activation of pH2, and the downstream CHA-based amplification cascade function synergistically and reliably within the proposed logic-gated architecture.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\n\\u003ch3\\u003e(3) Construction and Response Evaluation of the Dual-Lock Logic-Gated DNA Sensing System\\u003c/h3\\u003e\\n\\u003cp\\u003eTo enable precise molecular recognition and signal transduction, we constructed a DNA nanodevice that integrates aptamer binding and oxidative cleavage within a dual-lock logic-gated architecture, capable of detecting active MPO. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, the sensing system incorporates two input-dependent control elements, corresponding to two molecular \\u0026ldquo;keys.\\u0026rdquo; Input 1 (MPO): The trigger strand (T) is initially hybridized with the MPO-specific aptamer (Apt), forming a locked duplex that inhibits signal propagation. Only when MPO is present does it competitively bind the aptamer and release the T strand, enabling downstream activation of the CHA cascade. Input 2 (HOCl): HOCl, generated by MPO in the presence of H₂O₂ and Cl⁻, selectively cleaves the internal PS site of the protected hairpin pH2. This cleavage disrupts the blocking segment and exposes the toehold of H2, converting pH2 into its active conformation. These two inputs act cooperatively to regulate signal output. Only when both the T strand and activated H2 are present can the CHA reaction proceed, fulfilling an AND logic gate function. To experimentally validate the logic behavior, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb shows fluorescence spectra under different component combinations. Introducing only the T strand (FQ\\u0026thinsp;+\\u0026thinsp;H1\\u0026thinsp;+\\u0026thinsp;pH2\\u0026thinsp;+\\u0026thinsp;T) or T with MPO (FQ\\u0026thinsp;+\\u0026thinsp;H1\\u0026thinsp;+\\u0026thinsp;pH2\\u0026thinsp;+\\u0026thinsp;T\\u0026thinsp;+\\u0026thinsp;MPO) failed to induce significant fluorescence. In contrast, only when the full MPO catalytic system (MPO\\u0026thinsp;+\\u0026thinsp;H₂O₂ + Cl⁻) was introduced did the system generate a strong fluorescence signal, confirming that both molecular locks must be disengaged for activation. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec provides the corresponding quantitative analysis of relative fluorescence intensity (R/R₀), clearly demonstrating the cooperative nature of the dual-input activation and the high specificity of the system.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\n\\u003ch3\\u003e(4) Optimization of Reaction Conditions for the Dual-Lock MPO Sensing System\\u003c/h3\\u003e\\n\\u003cp\\u003eTo ensure the stability and sensitivity of the sensing system under practical conditions, we systematically evaluated the influence of several key environmental parameters, including pH, temperature, Cl⁻ concentration, and H₂O₂ concentration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, the fluorescence intensity peaked at pH 6.0, which aligns with the optimal catalytic activity of MPO under mildly acidic conditions. Alkaline pH (e.g., 9.2) significantly reduced the signal, indicating impaired enzymatic efficiency. Temperature also had a notable impact on the sensing response (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb). The strongest fluorescence signal was observed at 37\\u0026deg;C, while lower signals were recorded at 25\\u0026deg;C and 30\\u0026deg;C. The signal at 42\\u0026deg;C was comparable to that at 37\\u0026deg;C, indicating that the system maintains optimal reactivity and structural stability near physiological temperature. Since MPO catalysis relies on both Cl⁻ and H₂O₂ as substrates, we next examined their concentration-dependent effects. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec, the fluorescence signal increased with Cl⁻ concentration and plateaued at 20 mM, suggesting this concentration is sufficient to saturate MPO\\u0026rsquo;s catalytic requirement. Higher concentrations (\\u0026ge;\\u0026thinsp;40 mM) did not yield further enhancement, implying a reaction plateau. Similarly, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed shows that the system exhibited the strongest fluorescence at 20 \\u0026micro;M H₂O₂, with a stable signal range from 20\\u0026ndash;100 \\u0026micro;M. In contrast, lower concentrations (5\\u0026ndash;10 \\u0026micro;M) resulted in significantly weaker fluorescence, indicating that 20 \\u0026micro;M is the threshold for effective MPO activation in this system. Taken together, the optimal conditions for the dual-lock sensing platform are: pH 6.0, temperature 37\\u0026deg;C, Cl⁻ 20\\u0026ndash;100 mM, and H₂O₂ 20\\u0026ndash;100 \\u0026micro;M. These parameters ensure robust performance of the sensor under physiologically relevant environments.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\n\\u003ch3\\u003e(5) Analytical Performance Evaluation of the Dual-Lock Fluorescent MPO Sensing Platform\\u003c/h3\\u003e\\n\\u003cp\\u003eTo systematically evaluate the performance of the dual-lock logic-gated MPO sensing system, we assessed its specificity, sensitivity, quantification ability, and enzyme activity dependence, as summarized in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e. Specificity analysis was conducted using a range of protein and redox interferents. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea and \\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb, only catalytically active MPO in the presence of H₂O₂ and Cl⁻, or exogenous MPO and HOCl, triggered a strong fluorescence response. Other conditions\\u0026mdash;including MPO alone, inactive MPO, or single substrates\\u0026mdash;produced negligible signals, confirming strict dual-input gating and minimal false positives. In Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec, further selectivity was demonstrated by testing structurally and functionally related biomolecules commonly encountered in oxidative stress environments, such bovine serum albumin (BSA), horseradish peroxidase (HRP), NADPH, catalase (CAT), glucose oxidase (GOx), xanthine oxidase and glutathione reductase (GR). Only catalytically active MPO induced a significant fluorescence signal. This result confirms the high specificity of the system in discriminating MPO from structurally and functionally related oxidoreductases. To further assess the anti-interference capacity of the platform, we conducted interference assays under physiologically relevant conditions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed). The system was challenged with a panel of redox-active species (e.g., cysteine, GSH, ascorbic acid), oxidants (e.g., HOCl, H₂O₂, NO₃⁻), and abundant cations found in biological fluids (e.g., Na⁺, K⁺, Ca\\u0026sup2;⁺, Mg\\u0026sup2;⁺, Fe\\u0026sup3;⁺). None of these interferents triggered notable fluorescence increases, while MPO consistently activated the system. These findings underscore the excellent selectivity and robustness of the platform under complex biological conditions.\\u003c/p\\u003e\\u003cp\\u003eWe next assessed the sensitivity and dynamic range of the platform. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee, the fluorescence intensity increased proportionally with MPO concentration from 0.5 to 30 ng/mL, forming a clear kinetic response profile. Endpoint fluorescence spectra (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef) further validated this trend, showing dose-dependent enhancement centered at ~\\u0026thinsp;570 nm. A calibration curve was constructed based on endpoint intensity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eg), exhibiting excellent linearity (R\\u0026sup2; = 0.9834) with the regression equation \\u003cem\\u003eY\\u0026thinsp;=\\u0026thinsp;9.023X\\u0026thinsp;+\\u0026thinsp;43.34\\u003c/em\\u003e. The limit of detection (LOD) was estimated to be 1.73 ng/mL. These results demonstrate the system\\u0026rsquo;s high sensitivity and broad dynamic range for quantitative MPO detection. To verify that the fluorescence output originated specifically from MPO enzymatic activity rather than probe self-activation or nonspecific reactions, we performed inhibition assays using 4-aminobenzoic acid hydrazide (4-ABAH), a selective MPO inhibitor that binds to and inactivates its catalytic site. As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eh, increasing concentrations of 4-ABAH (0\\u0026ndash;16 \\u0026micro;M) led to a dose-dependent suppression of the fluorescence signal, confirming that the readout was indeed driven by MPO catalysis. Taken together, the dual-lock fluorescent sensing platform exhibits outstanding performance in terms of specificity, sensitivity, quantitative accuracy, and interference resistance. These features highlight its potential for reliable detection of MPO content and activity in complex biological samples.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e(6) Recovery Analysis in Biological Samples and Long-Term Stability Evaluation of the Biosensor\\u003c/h2\\u003e\\u003cp\\u003eTo assess the applicability of the developed MPO biosensing system in complex biological matrices, recovery experiments were conducted in human serum, cell lysate, and saliva samples. Known concentrations of MPO (5, 10, and 15 ng/mL) were spiked into each sample type, and the corresponding fluorescence responses were recorded. The results are summarized in Table \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e. In all tested matrices, the biosensor accurately quantified the spiked MPO, yielding recoveries ranging from 99.78% to 106.42%, with relative standard deviations (RSDs) below 4.85%, demonstrating excellent accuracy and reproducibility. These findings confirm the feasibility of direct MPO quantification in biological samples without significant matrix interference, highlighting the robustness and practicality of the sensing platform. To further investigate the long-term performance of the fluorescent biosensor, stability studies were carried out under refrigerated storage conditions (4\\u0026deg;C, protected from light) for up to six months. The sensor was stored under two conditions: in the presence and absence of MPO, and fluorescence responses were periodically measured. As illustrated in Figure S4, the fluorescence intensity in the MPO group remained consistently high over the 6-month period, with only a slight, non-significant decrease, indicating stable sensor performance over time. In contrast, the fluorescence signals in the control group without MPO remained at a low background level throughout the entire duration, showing no evidence of spontaneous signal activation. These results confirm the excellent storage stability and anti-background triggering capacity of the system. In summary, the proposed MPO biosensor exhibits high detection accuracy across various biological matrices and retains excellent stability under standard storage conditions. These properties make it highly suitable for preclinical sample analysis and long-term storage following batch fabrication.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eIn summary, we have established a dual-lock, logic-gated DNA biosensing platform that enables the concurrent and highly specific detection of both the presence and enzymatic activity of myeloperoxidase (MPO), a key oxidative enzyme implicated in numerous inflammation-associated pathologies. By integrating aptamer-mediated molecular recognition with HOCl-triggered oxidative cleavage, the system achieves a stringent AND logic gating mechanism, wherein signal output is initiated only upon simultaneous satisfaction of two biochemical criteria: MPO protein binding and catalytic generation of HOCl. This molecular logic architecture offers several critical advantages, including minimal background leakage, high analytical specificity, and resistance to false-positive signals arising from inactive MPO or related peroxidases. Under optimized conditions, the sensor exhibits a broad linear detection range (0.5\\u0026ndash;30 ng/mL), a low detection limit (~\\u0026thinsp;1.73 ng/mL), and excellent selectivity against interfering species. Furthermore, its robust performance in diverse biological matrices\\u0026mdash;such as human serum, saliva, and cell lysate\\u0026mdash;combined with long-term storage stability, underscores its practical applicability in real-world biosensing scenarios. Beyond methodological innovation, this work provides a generalizable framework for constructing activity-dependent, logic-controlled DNA nanodevices, with potential implications in dynamic biomarker monitoring, precision diagnostics, and point-of-care testing. Nonetheless, some limitations persist. The oxidative cleavage reaction, while effective, is inherently sensitive to redox fluctuations in complex samples, necessitating further refinements for in vivo robustness. Moreover, real-time kinetic monitoring and integration into miniaturized platforms remain important future directions. Overall, this dual-lock strategy exemplifies the fusion of programmable nucleic acid circuitry with biochemical reactivity, offering a powerful paradigm for next-generation biosensor design and molecular logic computation in biomedical diagnostics.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declared that all data supporting the conclusions of this research is available.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis research was supported by the National Natural Science Foundation of China (22404124), the Tianjin Education Commission research project (2023KJ074), and the Beijing Dadi Medical Charity Foundation (DDYL-A-KT-20241111-0112).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors and Affiliations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDepartment of Clinical Biochemistry and Molecular Diagnostics, College of Medical Technology, Tianjin Medical University, Tianjin, 300203, P. R. China\\u003c/p\\u003e\\n\\u003cp\\u003eBo-Yu Shi, Li Wen\\u0026nbsp;\\u0026amp; Jing Wang\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eContributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBo-Yu Shi: Writing \\u0026ndash; review \\u0026amp; editing, Writing \\u0026ndash; original draft, Investigation, Formal analysis, Data curation. Li Wen: Writing \\u0026ndash; review \\u0026amp; editing, Writing \\u0026ndash; original draft, Validation, Methodology, Investigation, Formal analysis, Data curation. Jing Wang: Writing \\u0026ndash; review \\u0026amp; editing, Writing \\u0026ndash; original draft, Validation, Supervision, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCorresponding authors\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCorrespondence to Jing Wang.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics declarations\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOur research was approved by the local ethics committee based on the Declaration of Helsinki. Written consent was obtained from each patient before surgery. Our research has received approval from the ethics committee of Affiliated Hospital with Jiangnan University.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors have reviewed and approved the final version of the manuscript for publication.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNo conflict of interest was declared by the authors.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eSoleimany, A. P., Martin-Alonso, C., Anahtar, M., Wang, C. S. \\u0026amp; Bhatia, S. N. Protease Activity Analysis: A Toolkit for Analyzing Enzyme Activity Data. \\u003cem\\u003eACS Omega\\u003c/em\\u003e \\u003cstrong\\u003e7\\u003c/strong\\u003e, 24292-24301, doi:10.1021/acsomega.2c01559 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eOng, I. L. H. \\u0026amp; Yang, K.-L. 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Herein, we report a novel dual-lock DNA biosensing platform, exemplified through myeloperoxidase (MPO), which concurrently integrates aptamer-mediated molecular recognition and hypochlorous acid (HOCl)-triggered oxidative cleavage to rigorously assess both MPO protein expression and enzymatic functionality. Specifically, MPO interaction with a conformationally structured DNA aptamer facilitates selective release of a trigger strand, while HOCl, produced enzymatically by active MPO, cleaves a strategically phosphorothioate-modified hairpin structure. Only upon simultaneous fulfillment of these two molecular conditions does the sensing mechanism activate a downstream catalytic hairpin assembly (CHA), achieving significant signal amplification. This stringent AND logic gate configuration markedly suppresses false positives and nonspecific background signals, demonstrating exceptional reliability across diverse and complex biological samples including serum, saliva, and cellular lysates. The proposed biosensing strategy thus provides a versatile, accurate, and broadly applicable analytical tool for simultaneous quantification of protease content and functional activity, holding considerable promise for advancing clinical diagnostics and pathological investigations.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Logic-Gated Fluorescent Biosensor Integrating Aptamer Recognition and Oxidative Cleavage-Responsive DNA Circuit for Myeloperoxidase Detection\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-10-09 09:57:35\",\"doi\":\"10.21203/rs.3.rs-7646512/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2025-11-19T04:36:36+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-09-27T17:59:19+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-09-19T15:20:57+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Translational Medicine\",\"date\":\"2025-09-18T03:33:52+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-translational-medicine\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jtrm\",\"sideBox\":\"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/jtrm/default.aspx\",\"title\":\"Journal of Translational Medicine\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"52110241-9dfe-4ef1-9f0f-fae2e360e2d3\",\"owner\":[],\"postedDate\":\"October 9th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-02-09T16:11:51+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7646512\",\"link\":\"https://doi.org/10.1186/s12967-026-07780-4\",\"journal\":{\"identity\":\"journal-of-translational-medicine\",\"isVorOnly\":false,\"title\":\"Journal of Translational Medicine\"},\"publishedOn\":\"2026-02-02 15:58:35\",\"publishedOnDateReadable\":\"February 2nd, 2026\"},\"versionCreatedAt\":\"2025-10-09 09:57:35\",\"video\":\"\",\"vorDoi\":\"10.1186/s12967-026-07780-4\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12967-026-07780-4\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7646512\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7646512\",\"identity\":\"rs-7646512\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}