Heparin’s Inhibition Capability on DNA Polymerase Enables Probe-Free Heparin Quantification

Heparin’s Inhibition Capability on DNA Polymerase Enables Probe-Free Heparin Quantification | 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 Heparin’s Inhibition Capability on DNA Polymerase Enables Probe-Free Heparin Quantification YI XIE, Yi Li, Xiong Ding, Meilin Weng, Kun Yin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7559255/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Accurate monitoring of heparin is essential for safe and effective anticoagulant therapy. Current detection methods often rely on synthetic probes and are vulnerable to interference from complex biological matrices. Here, we introduce HIPR ( H eparin- i nhibited P olymerase R eaction), a novel probe-free assay that leverages the selective inhibitory effect of heparin on a rationally engineered DNA polymerase, Zst. Instead of relying on charge interactions, HIPR translates van der Waals and hydrogen bonds between heparin and the enzyme into measurable inhibition during loop-mediated isothermal amplification (LAMP) primer extension. The assay achieves high sensitivity (limit of detection: 0.5 µM) and specificity, with recovery rates of 95–108% in plasma. HIPR offers a robust, rapid, and user-friendly platform for point-of-care heparin monitoring and extends the application of isothermal amplification methods to non-nucleic acid targets. Analytical Biochemistry Heparin detection Probe-free Loop-mediated isothermal amplification Polymerase inhibition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Coagulation system disorders, including both thrombotic and hemorrhagic conditions, represent major clinical challenges due to their association with increased morbidity and mortality worldwide 1 . Effective clinical treatments rely heavily on accurate evaluation of coagulation function, particularly in patients receiving anticoagulant therapy 2 . Heparin is a widely administered anticoagulant that exerts its effect by enhancing the activity of antithrombin, thereby inhibiting key serine proteases involved in the coagulation cascade 3 . Due to its narrow therapeutic index 4 , even small deviations in heparin levels can lead to severe complications—either thrombotic or hemorrhagic. This necessitates precise and timely monitoring during clinical administration 5 . Traditional heparin monitoring methods such as activated clotting time (ACT) 6 , 7 , activated partial thromboplastin time (aPTT) 8 , and chromogenic anti-factor Xa assays 9 , are commonly used in clinical settings. However, these assays have significant limitations, such as variability between individuals, susceptibility to physiological and procedural interferences, and difficulties with assay standardization and cost. These factors can impair the accuracy of heparin quantification and complicate dose adjustment. Therefore, there is a clear and pressing need for the development of simpler, more robust, and readily deployable methods for heparin detection, particularly those amenable to real-time and point-of-care testing. To solve these limitations, many molecular diagnostic methods for heparin detection have been developed recently. The colorimetric sensors 10 – 12 combined with gold nanoparticles 11 , 13 as well as fluorescent probes 13 , 14 offer high sensitivity. However, they need complex probe synthesis and are vulnerable to biological factors interference. Techniques such as LC-MS offer excellent analytical precision but are impractical for routine use due to their cost, technical complexity, and reliance on specialized instrumentation 15 . Other biosensing strategies, including aptamer-based sensors 16 and fluorescence resonance energy transfer (FRET) assays 17 , achieve high sensitivity and selectivity, yet are frequently compromised by sample matrix effects and the labor-intensive process of aptamer development. These challenges highlight the urgent need for a simple, reliable, and interference-resistant method for heparin detection. Here, we report a novel assay termed HIPR ( H eparin- i nhibited P olymerase R eaction) for the rapid, probe-free detection of heparin. This method capitalizes on the specific inhibitory effect of heparin on an engineered Zst DNA polymerase, translating molecular interactions—primarily van der Waals forces and hydrogen bonding—into quantifiable amplification signals. Unlike traditional charge-based detection strategies, HIPR offers high sensitivity and specificity without complex probe design and remains effective in plasma. While demonstrated using isothermal amplification (e.g., LAMP), the underlying principle is generalizable to other amplification platforms, including PCR, underscoring its broad applicability. HIPR thus represents a versatile and robust strategy for heparin monitoring, expanding the utility of nucleic acid amplification technologies to non-nucleic acid targets. Methods/Experimental Section Evaluation of Heparin-Polymerase Interaction via Real-Time ZE-LAMP To assess the interaction between heparin and various DNA polymerases, real-time loop-mediated isothermal amplification (LAMP) reactions were conducted. Three enzymes were evaluated: Bst 2.0 WarmStart and Bst 3.0 (New England Biolabs), and a recombinant Zst DNA polymerase prepared in-house as previously described 18 . Each ZE-LAMP reaction (Zst-mediated LAMP) was prepared in a 25 µL total volume containing primers, dNTPs, EvaGreen dye, and reaction buffer (see below), with or without heparin. Heparin was added to the reaction at a final concentration of 10 µM. Following a 2-minute equilibration at room temperature (25°C), the reactions were incubated at 65°C for 60 minutes. Fluorescence signals were recorded every 60 seconds using a Roche LightCycler 480 (Roche Diagnostics). Amplification kinetics were analyzed by quantifying threshold time (Cp values) to assess the inhibitory effect of heparin on each polymerase. Agarose Gel Electrophoresis for Product Analysis Agarose Gel Electrophoresis for Product Analysis LAMP amplification products were analyzed via 1.5% (w/v) agarose gel electrophoresis to further evaluate heparin-induced inhibition. A 1.5% agarose gel (prepared in 1× TAE buffer) containing GelRed nucleic acid stain was used. Electrophoresis was conducted at 150 V for 40 minutes. Gels were imaged using a Gel Doc XR + system (Bio-Rad), and band intensities were quantified with Image Lab software (v6.1). Relative band intensities were normalized to internal controls, and triplicate measurements were used to calculate mean values ± standard deviation. Protein–Carbohydrate Interaction via Molecular Docking Molecular docking of heparin to the Zst DNA polymerase structure was performed using the SwissDock online platform ( http://www.swissdock.ch/ ). The predicted tertiary structure of Zst (modeled via SWISS-MODEL) was submitted along with a heparin pentasaccharide (PubChem CID: 444410). Binding poses were visualized using UCSF ChimeraX and PyMOL v3.0, with hydrogen bonding, electrostatic, and hydrophobic interactions annotated. Potential binding pockets and interacting residues were identified and selected for further molecular dynamics simulation. Molecular Dynamics (MD) Simulations and Free Energy Calculations All-atom molecular dynamics (MD) simulations were conducted using GROMACS v2020.6 with the AMBER99SB-ILDN force field. The docked Zst–heparin complex served as the initial structure, solvated in a TIP3P water box with periodic boundary conditions and neutralized with Na⁺ counterions. The system was energy minimized and equilibrated in NVT and NPT ensembles before a 50-ns production run at 300 K. Trajectories were analyzed for root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and time-resolved hydrogen bond formation. The binding free energy was estimated using the MM/GBSA method as implemented in the g_mmpbsa tool. The decomposition of van der Waals, electrostatic, and solvation energy terms was used to evaluate binding contributions. Heparin Detection via Real-time ZE-LAMP Heparin quantification was performed using real-time ZE-LAMP assays. Each 15 µL reaction contained 1.2 mM dNTPs (with 0.2 mM dUTP), 1×custom-formulated reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% Tween-20), 6 mM MgSO₄, 0.5 µM each of FIP/BIP, 0.2 µM each of F3/B3 primers, 1× EvaGreen dye, and heparin at final concentrations ranging from 0.1 to 10 µM. After 2 minutes of pre-incubation at room temperature, the DNA template (100 copies/µL) and Zst polymerase (0.48 U) were added. The reactions were incubated at 65°C for 80 minutes in a Roche LightCycler 480 system, with fluorescence signals collected every 60 seconds. Cp values were determined based on the second derivative maximum of fluorescence curves. Clinical Sample Collection and Spiking Protocol Venous blood was collected from three healthy adult volunteers under informed consent, with ethical approval granted by [Ethics Committee Approval No.]. Plasma was isolated by centrifugation at 3,500 rpm for 10 minutes at 4°C. Aliquots (100 µL) of plasma were spiked with heparin standard solutions to achieve final concentrations of 40, 60, and 80 µg/mL (corresponding to ~ 5.3, 8.0, and 10.7 µM, respectively). These spiked samples were diluted 1:4 in reaction buffer and used directly in ZE-LAMP assays as described above. Each concentration was tested in triplicate, and recovery rates were calculated by comparing observed values to theoretical spiked concentrations. Results and Discussion Overview of Heparin-inhibited Polymerase Reaction (HIPR) By leveraging heparin's ability to inhibit DNA polymerase activity, we developed a direct, probe-free method for a quantitative heparin detection assay. In the absence of heparin, the engineered Zst polymerase efficiently drives strand-displacing amplification through a loop-mediated mechanism, generating robust fluorescence signals under isothermal conditions (Fig. 1 A). When heparin is added to the reaction tube, it binds to the specific activity region of Zst polymerase competitively, thereby delaying its combination with the DNA substrate (Fig. 1 B), which results in weakened or suppressed amplification signals. Meanwhile, this inhibition is evident as a heparin concentration-dependent increase in Cp value or reduction in endpoint fluorescence. Herein, this assay converts polymerase inhibition into a visible switch-off LAMP reaction, enabling sensitive and accurate quantification of heparin. With no need for complicated design nano-probes, this turn-off isothermal reaction assay makes it well-suited for real-time heparin monitoring in point-of-care settings, especially where laboratory infrastructure is limited. Heparin Dose-Dependently Inhibits ZE-LAMP Amplification To assess the inhibitory range of heparin in the ZE-LAMP system, we performed reactions with serial concentrations of heparin from 2.5×10 − 2 to 2.5×10 − 4 g/mL (Group 1: 2.5 × 10 − 2 g/mL; Group 2, Group 3: 2.5 × 10 − 3 g/mL; Group 4: 2.5 × 10 − 4 g/mL; Group 5: no template control). As shown in Figure S1 , high concentrations of heparin (Group 1, Group 2, and Group 3) led to complete suppression of amplification, with fluorescence signals remaining at baseline throughout the 60-minute assay. At a lower concentration (G4: 2.5×10 − 4 g/mL), a delayed but slowly rising signal was observed, indicating partial inhibition. The no-template control (G5) showed no signal, confirming the reaction's specificity. These results indicate a clear dose-dependent inhibition of ZE-LAMP by heparin. Total inhibition at ≥ 2.5×10 − 3 g/mL suggests that heparin at these levels is sufficient to fully disrupt polymerase-driven amplification. Partial inhibition at 2.5×10 − 4 g/mL suggests that polymerase activity remains functional but impaired. This concentration-dependent effect provides a potential working window for quantitative detection of heparin using the ZE-LAMP platform. Selective Inhibition of Zst Polymerase by Heparin To determine whether heparin-mediated inhibition is polymerase-specific, we compared its effects on Zst, Bst 2.0 WarmStart, and Bst 3.0 polymerases under identical LAMP conditions with or without heparin (2.5×10 − 4 g/mL). Fluorescence amplification curves and endpoint gel electrophoresis were used to evaluate polymerase activity. In the absence of heparin, Zst polymerase produced a rapid and robust amplification signal, characterized by early Cp values and strong fluorescence (Fig. 2A). However, once heparin was added, no fluorescence increase was observed throughout the reaction period, indicating complete inhibition. Gel analysis confirmed the absence of amplicons under these conditions (Fig. 2B). In contrast, reactions catalyzed by Bst 2.0 and Bst 3.0 proceeded efficiently in both the presence and absence of heparin, with consistent fluorescence signals and visible bands on the gel. Further comparison of Cp values (Fig. 2C) showed that Zst reactions with heparin yielded no detectable signal (ND), while Bst-based reactions showed no statistically significant differences between treated and untreated groups (NS). These results clearly indicate that Zst polymerase exhibits unique sensitivity to heparin-mediated inhibition at low concentrations, while Bst 2.0 and Bst 3.0 retain full enzymatic activity under the same conditions. This polymerase-specific inhibition suggests a distinct binding interface between heparin and Zst, likely arising from structural or electrostatic features not present in the Bst enzymes. Molecular Insights into Heparin-Mediated Inhibition of Zst Polymerase To clarify the molecular basis of heparin inhibition, we continue to perform molecular docking and molecular dynamics (MD) simulations using the predicted structure of Zst polymerase and the heparin oligosaccharide sequence (Fig. 3 B). Docking showed that heparin binds within a specific cavity on Zst, stabilized by multiple hydrogen bonds and salt bridges (Fig. 3 , C-D). Structural validation using the SAVES 6.0 platform supported the reliability of the model, with an ERRAT score of 95.57 and a VERIFY 3D score of 88.33%. MD simulations confirmed the stability of the complex throughout a 50-ns trajectory. RMSD analysis indicated rapid convergence, stabilizing near 0.4 nm (Fig. 3 E), while RMSF analysis identified moderate flexibility at the termini and a surface-exposed loop (Fig. 3 F). Heparin maintained dynamic yet consistent interactions within the binding pocket, forming 2–5 hydrogen bonds throughout the simulation (Fig. 3 G). MM/GBSA energy decomposition showed that van der Waals forces (-158.59 kcal/mol) were the main contributors to binding, with limited electrostatic interaction (− 5.25 kcal/mol) and a modest solvation penalty (+ 6.01 kcal/mol), leading to a net binding free energy of -157.82 kcal/mol (Fig. 3 H, Table S1 ). Key interacting residues—Leu181, Leu271, Arg281, Thr284, Ile316, and Leu502—were identified as potential sites for future engineering (Fig. 3 I). Together, these results provide the first structural explanation of how heparin inhibits DNA polymerase in isothermal amplification, offering a foundation for optimizing Zst as a heparin-responsive feature in molecular diagnostics. Optimization of Reaction Conditions for HIPR To enhance assay performance, we systematically optimized three critical parameters: DNA substrate concentration, reaction buffer composition, and Zst polymerase dosage. A template concentration of 10 6 copies/µL yielded the most consistent linear relationship between heparin concentration and Cp value ( Figure S4 ). The use of a custom-formulated reaction buffer significantly amplified the inhibitory effect of heparin on the amplification reaction (Fig. 4 A). Additionally, 0.48U of Zst polymerase was identified as the optimal enzyme concentration, balancing both amplification efficiency and assay stability (Fig. 4 B, 4 C). These optimized conditions collectively improved the sensitivity, reproducibility, and quantitative accuracy of the signal-off LAMP-based heparin assay. Development and Validation of Clinical Heparin Detection Accurate heparin monitoring remains crucial for safe anticoagulation therapy, yet traditional assays often depend on indirect measurement methods and can be affected by interference from biological matrices. Building on our discovery of Zst DNA polymerase inhibition by heparin, we established a LAMP-based detection assay that enables direct, probe-free quantification of heparin with minimal complexity. Under optimized conditions, the assay exhibited a strong linear relationship between Cp values and heparin concentrations (y = 5.960x + 28.21, R 2 = 0.9530), with a detection limit of 0.5 µM. This range is clinically relevant, covering heparin doses used in cardiovascular surgery (2–8 U/mL, approximately 17–67 µM) and for long-term management (0.2–1.2 U/mL, about 1.7–10 µM). The HIPR assay exhibited clear dose-dependent amplification suppression, as evidenced by prolonged Cp values and reduced fluorescence output. This correlation enables accurate quantitative assessment of heparin concentrations across clinically relevant ranges. Specificity tests showed that common anions (e.g., glucose, sodium citrate) and biological substances (e.g., BSA, EDTA) did not interfere with the readout or negate heparin’s inhibitory effect. In human plasma spiked with different concentrations of heparin, the assay produced recovery rates of 95–108% with low relative standard deviation (RSD < 4.51%), supporting its robustness and suitability for clinical use. These findings confirm that the method is accurate, highly specific, and ideal for rapid, point-of-care heparin monitoring. Table 1. Recovery of heparin from spiked human plasma samples measured by ZE-LAMP (n = 3). Results are expressed as mean ± RSD. SD: Standard Deviation; RSD: Relative Standard Deviation. Sample Heparin dosage(µM) Heparin detection(µM) Recovery Rate (%) Relative Standard Deviation(%) 1 1.5 1.40 ± 0.03 107.14 2.31 2.5 2.61 ± 0.09 95.79 3.52 3.5 3.44 ± 0.06 101.74 1.81 4 4.12 ± 0.17 97.09 4.01 2 1.5 1.53 ± 0.05 98.04 3.12 2.5 2.46 ± 0.05 101.63 2.12 3.5 3.49 ± 0.07 100.29 2.03 4 3.94 ± 0.07 101.52 1.90 3 1.5 1.52 ± 0.07 98.68 4.51 2.5 2.53 ± 0.06 98.81 2.20 3.5 3.46 ± 0.11 101.16 3.09 4 3.69 ± 0.11 108.40 3.01 Discussion This study presents a novel and probe-free method for heparin detection based on the inhibition of Zst DNA polymerase in LAMP assay. The assay offers substantial improvements over traditional detection methods, such as activated clotting time (ACT) and chromogenic tests, which can be indirect and prone to interference. Our results show that the assay is highly sensitive, with a detection limit of 0.5 µM, which covers the clinically relevant ranges of heparin concentrations typically encountered during cardiovascular surgery (2–8 U/mL, 17–67 µM) and post-operative therapy (0.2–1.2 U/mL, 1.7–10 µM). The assay demonstrated a strong linear correlation between heparin concentration and the Cp value, with an R² value of 0.99530, confirming its accuracy and reliability. The simplicity and efficiency of the assay make it an appealing alternative for point-of-care testing. It functions under isothermal conditions, requires no complex equipment, and provides a clear fluorescence readout, which is especially beneficial for environments with limited resources. Additionally, the method's high specificity against common anions and biological substances, like glucose and BSA, further boosts its suitability for clinical use by reducing false positives or interference often seen with other methods. One of the key strengths of the assay lies in its mechanistic foundation. Heparin interacts specifically with Zst DNA polymerase, inhibiting its activity by forming non-covalent interactions such as van der Waals forces and hydrogen bonds. This interaction causes a delay or complete inhibition of the amplification reaction, translating into measurable changes in the Cp value. This mechanism enables the detection of heparin directly, without the need for additional probes, and offers a novel approach to detecting non-nucleic acid targets with LAMP. The validation of the assay in human plasma, with recovery rates between 95% and 108%, demonstrates its strong performance in complex biological samples. Additionally, its potential for use in personalized medicine looks promising. The ability to quickly and accurately measure heparin levels could enhance patient monitoring and help optimize treatment plans in clinical settings. Looking ahead, the use of this assay could expand beyond heparin detection to other glycosaminoglycans, such as heparan sulfate or chondroitin sulfate, by utilizing similar protein-ligand interactions. Additionally, the insights gained from the heparin-Zst interaction might lead to further improvements in engineered polymerases for better detection. Future research should focus on integrating this assay with microfluidic platforms, which could offer greater portability and lower reagent use, making it suitable for widespread application in resource-limited settings. Conclusion In summary, we report a novel, probe-free assay for heparin detection based on the selective inhibition of Zst DNA polymerase during loop-mediated isothermal amplification. This method achieves a low detection limit of 0.5 µM and offers excellent linearity, specificity, and reproducibility in complex biological matrices. Its simplicity, minimal instrumentation requirements, and compatibility with clinical plasma samples make it a promising tool for real-time, point-of-care heparin monitoring. With no need for complex instruments or probes, this assay is well-suited for point-of-care testing. Its simplicity and potential for integration into microfluidic platforms make it an ideal solution for resource-limited environments. The assay could also be adapted for detecting other glycosaminoglycans, contributing to the advancement of diagnostic tools. Overall, this work introduces a novel approach for heparin monitoring that has the potential to improve clinical care, particularly in personalized medicine and point-of-care settings. Declarations Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 82372502), Shanghai 2023 Medical Innovation Research Special Funding (No. 23Y11902300), Zhongshan Hospital Clinical Research Project (No. ZSLCYJ202348), Zhongshan Hospital Science and Technology Innovation Fund-Cultivation Project (No. 2024-ZSCX07), and China International Medical Exchange Funding (No. Z-2017-24-2421). Author Contributions Y.X. contributed equally to this work. Y.X. contributed to the conceptualization, data curation, formal analysis, and writing the original draft. Y. L. contributed to the visualization. M. L. W contributed to investigation. M. L. W and X. D. contributed to validation, review, editing, and funding acquisition. K.Y. contributed to supervision, review, editing, and funding acquisition. Notes The authors declare no competing financial interest. References Triplett DA (2000) Coagulation and bleeding disorders: review and update. 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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-7559255","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511572435,"identity":"bfa730c5-3133-4824-9a1f-d23021f74976","order_by":0,"name":"YI XIE","email":"","orcid":"","institution":"1School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, People’s Republic of China 2One Health Center, Shanghai Jiao Tong University-The University of Edinburgh, Shanghai, 200025, People’s Republic of China2Department of Anesthesiology, Zhongshan Hospital, Fudan University, Shanghai 200032, China","correspondingAuthor":false,"prefix":"","firstName":"YI","middleName":"","lastName":"XIE","suffix":""},{"id":511572436,"identity":"3d5947d4-b50c-4ac6-b29f-4caafb6432a4","order_by":1,"name":"Yi Li","email":"","orcid":"","institution":"Department of Anesthesiology, Zhongshan hospital, Fudan University, Shanghai 200032, China","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Li","suffix":""},{"id":511576025,"identity":"6d6e43b5-3b00-4356-959b-405e958f0110","order_by":2,"name":"Xiong Ding","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACZhDBZsFgwN7Y+AAilECUFgkGA57DzQbEaWGAaZFIb5MgSovBceZjD7+UScibMyS2Vf6oOMzAz55jwPBzB24tks1s6cYy5yQMdzYcbLvNc+Ywg2TPGwPG3jO4tfAz85hJS7ZJMG442Nh2m7HtMIPBjRwDZsY2PL6AarHfcJixrfDnv8MM9oS0gGyR/NgmkbjhGFAZbwPQFgkCWoB+SZNmOCeRvLOHsVma51g6j8SZZwUHe/FoMTh/+JjkjzIb2+3yzx9+/FFjLcffnrzxwU88WkCAmQeJA2YfwK+BgYHxByEVo2AUjIJRMLIBANAXTNThwcfgAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0437-0589","institution":"Key Laboratory of Environmental Medicine and Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing 210009, P. 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China","correspondingAuthor":true,"prefix":"","firstName":"Xiong","middleName":"","lastName":"Ding","suffix":""},{"id":511576026,"identity":"3f82708d-7e8c-4ab2-8e09-21144e39fc17","order_by":3,"name":"Meilin Weng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYFACxsYDCVDWgwoGZjBLgoCWBpgWZoMzxGlhYDgApdkkiNKi297ccODhDobEfun2axUH26zlzRmYD97mYbDLw6XF7MzBhgOJZxgSZ845U3bjYFu64c4GtmRrHobkYpxabiQCtbQx5G64kZN2+2PbYcYNB3jMpHkYDiQ24NJy/yFEy36gloKDbYftNxzg/4Zfyw1GqC0S6ccYgFoSgbaw4ddyBuKw+hk3cpglDpxLT95wmM3Yco5BMm4tx48/fPizjcGYf0b6ww8HyqxtNxxvfnjjTYUdTi1Q8B+IeQwgbHDUGOBXDwXsD4hSNgpGwSgYBSMPAAA2JmP0jLK4hQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0004-6766-0713","institution":"Department of Anesthesiology, Zhongshan hospital, Fudan University, Shanghai 200032, China","correspondingAuthor":true,"prefix":"","firstName":"Meilin","middleName":"","lastName":"Weng","suffix":""},{"id":511576027,"identity":"376de191-5034-4d62-8405-3bae465cd2e0","order_by":4,"name":"Kun Yin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACCQbGBoaEChsefhAvoYBYLQ/OpMlJNoC0GBClhYGB8WHbYWODAyAuMVokZyS3PUhsO5y4+fzqxA8PDBjk+cUO4NciLZHYbpBwLj1x2423myWADjOcOTsBvxY5icQ2iYQya6CWsxtAWhIMbhOlhY05cfOMs5t/EKVFGqylzdnYgL93G3G2SPY8BGoBBrLEDd5tFgkGEoT9InE8/ZnkD1BU9p/dfBPIkOeXJqCFQQCmQALMkCCgHAT4D6AzRsEoGAWjYBSgAQCXQEfsqrsLZQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6300-6985","institution":"School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, P. R. China; One Health Center, Shanghai Jiao Tong University-The University of Edinburgh, Shanghai 200127, P. R. China","correspondingAuthor":true,"prefix":"","firstName":"Kun","middleName":"","lastName":"Yin","suffix":""}],"badges":[],"createdAt":"2025-09-08 02:35:38","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-7559255/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7559255/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90879395,"identity":"26247f77-3335-44fa-95fc-8caa023d1a13","added_by":"auto","created_at":"2025-09-09 09:35:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":565344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of the HIPR assay mechanism.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) In the absence of heparin, Zst polymerase catalyzes efficient loop-mediated amplification, generating strong fluorescence signals.\u003c/p\u003e\n\u003cp\u003e(B) Heparin binds competitively to Zst polymerase, blocking the formation of the primer-enzyme-substrate complex and suppressing DNA extension, resulting in delayed or inhibited amplification.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7559255/v1/b9e698308e5d4d7e1dac50a5.png"},{"id":90879394,"identity":"19f550c5-cc34-4a77-9755-65382ee1e085","added_by":"auto","created_at":"2025-09-09 09:35:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":305951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSelective inhibition of Zst polymerase by heparin compared with Bst polymerases.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Real-time fluorescence amplification curves for Zst, Bst 2.0, and Bst 3.0 in the presence or absence of 2.5×10\u003csup\u003e-4\u003c/sup\u003e g/mL heparin.\u003c/p\u003e\n\u003cp\u003e(B) Comparison of threshold time (Cp values) under each condition, showing complete inhibition in the Zst group only.\u003c/p\u003e\n\u003cp\u003e(C) Agarose gel electrophoresis (1.5%) showing amplicon bands generated by each polymerase ± heparin. M: DNA ladder (100 bp).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7559255/v1/c48a5d03f0684d3539544fbd.png"},{"id":90879402,"identity":"6d5b2c76-0183-4293-980e-587478f19edf","added_by":"auto","created_at":"2025-09-09 09:35:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":844002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking and dynamics simulation of Zst-heparin interaction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Workflow of structure prediction, docking, and MD analysis.\u003c/p\u003e\n\u003cp\u003e(B) Predicted 3D model of the Zst-heparin complex generated via SWISS-MODEL.\u003c/p\u003e\n\u003cp\u003e(C-D) Zoomed-in view of binding interface showing key hydrogen bonds and salt bridges.\u003c/p\u003e\n\u003cp\u003e(E) RMSD plot showing convergence of the complex during 50-ns MD simulation.\u003c/p\u003e\n\u003cp\u003e(F) RMSF analysis indicating flexibility at terminal loops.\u003c/p\u003e\n\u003cp\u003e(G) Time-resolved hydrogen bond count between heparin and Zst.\u003c/p\u003e\n\u003cp\u003e(H) MM/GBSA energy decomposition: van der Waals interactions dominate.\u003c/p\u003e\n\u003cp\u003e(I) Key residues involved in binding.\u003c/p\u003e\n\u003cp\u003e(J) Structural overlay of Zst–heparin complex at 0 ns and 50 ns.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7559255/v1/fbe72c94af88e36e3b71f6a3.png"},{"id":90879396,"identity":"d9a780da-c414-4464-bbec-ce47848c9fbc","added_by":"auto","created_at":"2025-09-09 09:35:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":386212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOptimization of key reaction parameters for ZE-LAMP performance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Effect of buffer formulation on amplification inhibition in the presence of 2.5 µM heparin.\u003c/p\u003e\n\u003cp\u003e(B) Effect of varying Zst polymerase concentration on fluorescence kinetics.\u003c/p\u003e\n\u003cp\u003e(C) Corresponding Cp values under different enzyme concentrations, identifying 0.48 U as optimal.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7559255/v1/5d368d355bb4ec434d931641.png"},{"id":90881413,"identity":"901c38e1-18ea-4f19-b1ac-bf8151f4e9db","added_by":"auto","created_at":"2025-09-09 09:43:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":272246,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalytical validation of the HIPR assay for heparin quantification.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Sensitivity analysis showing dose-dependent fluorescence suppression.\u003c/p\u003e\n\u003cp\u003e(B) Calibration curve correlating heparin concentration with Cp value (R² = 0.9530).\u003c/p\u003e\n\u003cp\u003e(C) Specificity test with common anions and biomolecules; no significant interference observed (amplification curves see \u003cstrong\u003eFigure S3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e(D) Anti-interference analysis confirming robustness in complex matrices (amplification curves see \u003cstrong\u003eFigure S4\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7559255/v1/978c6c3fb31bd79fef695763.png"},{"id":90885471,"identity":"d251d241-a5b2-4d46-b45f-12c840b0ca0f","added_by":"auto","created_at":"2025-09-09 10:07:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3117115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7559255/v1/961d47db-7dea-46c2-bb33-7dd2cb3e7cbe.pdf"},{"id":90881896,"identity":"eed96b79-430d-45d4-be9f-645fdbb33ab4","added_by":"auto","created_at":"2025-09-09 09:51:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":463059,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7559255/v1/77517423df4c0eb121733025.docx"},{"id":90881411,"identity":"7ab3d4f7-b7ef-4b78-a30a-26d339d588ba","added_by":"auto","created_at":"2025-09-09 09:43:04","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":418225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTOC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"TOC.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7559255/v1/a181b42bc0b5417dfec47924.jpg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHeparin’s Inhibition Capability on DNA Polymerase Enables Probe-Free Heparin Quantification\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoagulation system disorders, including both thrombotic and hemorrhagic conditions, represent major clinical challenges due to their association with increased morbidity and mortality worldwide \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Effective clinical treatments rely heavily on accurate evaluation of coagulation function, particularly in patients receiving anticoagulant therapy \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Heparin is a widely administered anticoagulant that exerts its effect by enhancing the activity of antithrombin, thereby inhibiting key serine proteases involved in the coagulation cascade \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Due to its narrow therapeutic index \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, even small deviations in heparin levels can lead to severe complications\u0026mdash;either thrombotic or hemorrhagic. This necessitates precise and timely monitoring during clinical administration \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTraditional heparin monitoring methods such as activated clotting time (ACT) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, activated partial thromboplastin time (aPTT) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and chromogenic anti-factor Xa assays \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, are commonly used in clinical settings. However, these assays have significant limitations, such as variability between individuals, susceptibility to physiological and procedural interferences, and difficulties with assay standardization and cost. These factors can impair the accuracy of heparin quantification and complicate dose adjustment. Therefore, there is a clear and pressing need for the development of simpler, more robust, and readily deployable methods for heparin detection, particularly those amenable to real-time and point-of-care testing.\u003c/p\u003e\u003cp\u003eTo solve these limitations, many molecular diagnostic methods for heparin detection have been developed recently. The colorimetric sensors \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e combined with gold nanoparticles \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e as well as fluorescent probes \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e offer high sensitivity. However, they need complex probe synthesis and are vulnerable to biological factors interference. Techniques such as LC-MS offer excellent analytical precision but are impractical for routine use due to their cost, technical complexity, and reliance on specialized instrumentation \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Other biosensing strategies, including aptamer-based sensors \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and fluorescence resonance energy transfer (FRET) assays \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, achieve high sensitivity and selectivity, yet are frequently compromised by sample matrix effects and the labor-intensive process of aptamer development. These challenges highlight the urgent need for a simple, reliable, and interference-resistant method for heparin detection.\u003c/p\u003e\u003cp\u003eHere, we report a novel assay termed \u003cb\u003eHIPR\u003c/b\u003e (\u003cb\u003eH\u003c/b\u003eeparin-\u003cb\u003ei\u003c/b\u003enhibited \u003cb\u003eP\u003c/b\u003eolymerase \u003cb\u003eR\u003c/b\u003eeaction) for the rapid, probe-free detection of heparin. This method capitalizes on the specific inhibitory effect of heparin on an engineered Zst DNA polymerase, translating molecular interactions\u0026mdash;primarily van der Waals forces and hydrogen bonding\u0026mdash;into quantifiable amplification signals. Unlike traditional charge-based detection strategies, HIPR offers high sensitivity and specificity without complex probe design and remains effective in plasma. While demonstrated using isothermal amplification (e.g., LAMP), the underlying principle is generalizable to other amplification platforms, including PCR, underscoring its broad applicability. HIPR thus represents a versatile and robust strategy for heparin monitoring, expanding the utility of nucleic acid amplification technologies to non-nucleic acid targets.\u003c/p\u003e"},{"header":"Methods/Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of Heparin-Polymerase Interaction via Real-Time ZE-LAMP\u003c/h2\u003e\u003cp\u003eTo assess the interaction between heparin and various DNA polymerases, real-time loop-mediated isothermal amplification (LAMP) reactions were conducted. Three enzymes were evaluated: Bst 2.0 WarmStart and Bst 3.0 (New England Biolabs), and a recombinant Zst DNA polymerase prepared in-house as previously described \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Each ZE-LAMP reaction (Zst-mediated LAMP) was prepared in a 25 \u0026micro;L total volume containing primers, dNTPs, EvaGreen dye, and reaction buffer (see below), with or without heparin. Heparin was added to the reaction at a final concentration of 10 \u0026micro;M. Following a 2-minute equilibration at room temperature (25\u0026deg;C), the reactions were incubated at 65\u0026deg;C for 60 minutes. Fluorescence signals were recorded every 60 seconds using a Roche LightCycler 480 (Roche Diagnostics). Amplification kinetics were analyzed by quantifying threshold time (Cp values) to assess the inhibitory effect of heparin on each polymerase.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAgarose Gel Electrophoresis for Product Analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eAgarose Gel Electrophoresis for Product Analysis\u003c/div\u003e\u003cp\u003eLAMP amplification products were analyzed via 1.5% (w/v) agarose gel electrophoresis to further evaluate heparin-induced inhibition. A 1.5% agarose gel (prepared in 1\u0026times; TAE buffer) containing GelRed nucleic acid stain was used. Electrophoresis was conducted at 150 V for 40 minutes. Gels were imaged using a Gel Doc XR\u0026thinsp;+\u0026thinsp;system (Bio-Rad), and band intensities were quantified with Image Lab software (v6.1). Relative band intensities were normalized to internal controls, and triplicate measurements were used to calculate mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\n\u003ch3\u003eProtein–Carbohydrate Interaction via Molecular Docking\u003c/h3\u003e\n\u003cp\u003eMolecular docking of heparin to the Zst DNA polymerase structure was performed using the SwissDock online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissdock.ch/\u003c/span\u003e\u003cspan address=\"http://www.swissdock.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The predicted tertiary structure of Zst (modeled via SWISS-MODEL) was submitted along with a heparin pentasaccharide (PubChem CID: 444410). Binding poses were visualized using UCSF ChimeraX and PyMOL v3.0, with hydrogen bonding, electrostatic, and hydrophobic interactions annotated. Potential binding pockets and interacting residues were identified and selected for further molecular dynamics simulation.\u003c/p\u003e\n\u003ch3\u003eMolecular Dynamics (MD) Simulations and Free Energy Calculations\u003c/h3\u003e\n\u003cp\u003eAll-atom molecular dynamics (MD) simulations were conducted using GROMACS v2020.6 with the AMBER99SB-ILDN force field. The docked Zst\u0026ndash;heparin complex served as the initial structure, solvated in a TIP3P water box with periodic boundary conditions and neutralized with Na⁺ counterions. The system was energy minimized and equilibrated in NVT and NPT ensembles before a 50-ns production run at 300 K. Trajectories were analyzed for root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and time-resolved hydrogen bond formation. The binding free energy was estimated using the MM/GBSA method as implemented in the g_mmpbsa tool. The decomposition of van der Waals, electrostatic, and solvation energy terms was used to evaluate binding contributions.\u003c/p\u003e\n\u003ch3\u003eHeparin Detection via Real-time ZE-LAMP\u003c/h3\u003e\n\u003cp\u003eHeparin quantification was performed using real-time ZE-LAMP assays. Each 15 \u0026micro;L reaction contained 1.2 mM dNTPs (with 0.2 mM dUTP), 1\u0026times;custom-formulated reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH₄)₂SO₄, 0.1% Tween-20), 6 mM MgSO₄, 0.5 \u0026micro;M each of FIP/BIP, 0.2 \u0026micro;M each of F3/B3 primers, 1\u0026times; EvaGreen dye, and heparin at final concentrations ranging from 0.1 to 10 \u0026micro;M. After 2 minutes of pre-incubation at room temperature, the DNA template (100 copies/\u0026micro;L) and Zst polymerase (0.48 U) were added. The reactions were incubated at 65\u0026deg;C for 80 minutes in a Roche LightCycler 480 system, with fluorescence signals collected every 60 seconds. Cp values were determined based on the second derivative maximum of fluorescence curves.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eClinical Sample Collection and Spiking Protocol\u003c/h2\u003e\u003cp\u003eVenous blood was collected from three healthy adult volunteers under informed consent, with ethical approval granted by [Ethics Committee Approval No.]. Plasma was isolated by centrifugation at 3,500 rpm for 10 minutes at 4\u0026deg;C. Aliquots (100 \u0026micro;L) of plasma were spiked with heparin standard solutions to achieve final concentrations of 40, 60, and 80 \u0026micro;g/mL (corresponding to ~\u0026thinsp;5.3, 8.0, and 10.7 \u0026micro;M, respectively). These spiked samples were diluted 1:4 in reaction buffer and used directly in ZE-LAMP assays as described above. Each concentration was tested in triplicate, and recovery rates were calculated by comparing observed values to theoretical spiked concentrations.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eOverview of Heparin-inhibited Polymerase Reaction (HIPR)\u003c/h2\u003e\n \u003cp\u003eBy leveraging heparin\u0026apos;s ability to inhibit DNA polymerase activity, we developed a direct, probe-free method for a quantitative heparin detection assay. In the absence of heparin, the engineered Zst polymerase efficiently drives strand-displacing amplification through a loop-mediated mechanism, generating robust fluorescence signals under isothermal conditions (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). When heparin is added to the reaction tube, it binds to the specific activity region of Zst polymerase competitively, thereby delaying its combination with the DNA substrate (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB), which results in weakened or suppressed amplification signals. Meanwhile, this inhibition is evident as a heparin concentration-dependent increase in Cp value or reduction in endpoint fluorescence. Herein, this assay converts polymerase inhibition into a visible switch-off LAMP reaction, enabling sensitive and accurate quantification of heparin. With no need for complicated design nano-probes, this turn-off isothermal reaction assay makes it well-suited for real-time heparin monitoring in point-of-care settings, especially where laboratory infrastructure is limited.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eHeparin Dose-Dependently Inhibits ZE-LAMP Amplification\u003c/h2\u003e\n \u003cp\u003eTo assess the inhibitory range of heparin in the ZE-LAMP system, we performed reactions with serial concentrations of heparin from 2.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 2.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/mL (Group 1: 2.5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e g/mL; Group 2, Group 3: 2.5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e g/mL; Group 4: 2.5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/mL; Group 5: no template control). As shown in \u003cstrong\u003eFigure S1\u003c/strong\u003e, high concentrations of heparin (Group 1, Group 2, and Group 3) led to complete suppression of amplification, with fluorescence signals remaining at baseline throughout the 60-minute assay. At a lower concentration (G4: 2.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/mL), a delayed but slowly rising signal was observed, indicating partial inhibition. The no-template control (G5) showed no signal, confirming the reaction\u0026apos;s specificity.\u003c/p\u003e\n \u003cp\u003eThese results indicate a clear dose-dependent inhibition of ZE-LAMP by heparin. Total inhibition at \u0026ge;\u0026thinsp;2.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e g/mL suggests that heparin at these levels is sufficient to fully disrupt polymerase-driven amplification. Partial inhibition at 2.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/mL suggests that polymerase activity remains functional but impaired. This concentration-dependent effect provides a potential working window for quantitative detection of heparin using the ZE-LAMP platform.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eSelective Inhibition of Zst Polymerase by Heparin\u003c/h2\u003e\n \u003cp\u003eTo determine whether heparin-mediated inhibition is polymerase-specific, we compared its effects on Zst, Bst 2.0 WarmStart, and Bst 3.0 polymerases under identical LAMP conditions with or without heparin (2.5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/mL). Fluorescence amplification curves and endpoint gel electrophoresis were used to evaluate polymerase activity.\u003c/p\u003e\n \u003cp\u003eIn the absence of heparin, Zst polymerase produced a rapid and robust amplification signal, characterized by early Cp values and strong fluorescence (Fig.\u0026nbsp;2A). However, once heparin was added, no fluorescence increase was observed throughout the reaction period, indicating complete inhibition. Gel analysis confirmed the absence of amplicons under these conditions (Fig.\u0026nbsp;2B). In contrast, reactions catalyzed by Bst 2.0 and Bst 3.0 proceeded efficiently in both the presence and absence of heparin, with consistent fluorescence signals and visible bands on the gel.\u003c/p\u003e\n \u003cp\u003eFurther comparison of Cp values (Fig.\u0026nbsp;2C) showed that Zst reactions with heparin yielded no detectable signal (ND), while Bst-based reactions showed no statistically significant differences between treated and untreated groups (NS).\u003c/p\u003e\n \u003cp\u003eThese results clearly indicate that Zst polymerase exhibits unique sensitivity to heparin-mediated inhibition at low concentrations, while Bst 2.0 and Bst 3.0 retain full enzymatic activity under the same conditions. This polymerase-specific inhibition suggests a distinct binding interface between heparin and Zst, likely arising from structural or electrostatic features not present in the Bst enzymes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMolecular Insights into Heparin-Mediated Inhibition of Zst Polymerase\u003c/h2\u003e\n \u003cp\u003eTo clarify the molecular basis of heparin inhibition, we continue to perform molecular docking and molecular dynamics (MD) simulations using the predicted structure of Zst polymerase and the heparin oligosaccharide sequence (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Docking showed that heparin binds within a specific cavity on Zst, stabilized by multiple hydrogen bonds and salt bridges (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, C-D). Structural validation using the SAVES 6.0 platform supported the reliability of the model, with an ERRAT score of 95.57 and a VERIFY 3D score of 88.33%. MD simulations confirmed the stability of the complex throughout a 50-ns trajectory. RMSD analysis indicated rapid convergence, stabilizing near 0.4 nm (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE), while RMSF analysis identified moderate flexibility at the termini and a surface-exposed loop (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). Heparin maintained dynamic yet consistent interactions within the binding pocket, forming 2\u0026ndash;5 hydrogen bonds throughout the simulation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG). MM/GBSA energy decomposition showed that van der Waals forces (-158.59 kcal/mol) were the main contributors to binding, with limited electrostatic interaction (\u0026minus;\u0026thinsp;5.25 kcal/mol) and a modest solvation penalty (+\u0026thinsp;6.01 kcal/mol), leading to a net binding free energy of -157.82 kcal/mol (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH, \u003cstrong\u003eTable S1\u003c/strong\u003e). Key interacting residues\u0026mdash;Leu181, Leu271, Arg281, Thr284, Ile316, and Leu502\u0026mdash;were identified as potential sites for future engineering (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI). Together, these results provide the first structural explanation of how heparin inhibits DNA polymerase in isothermal amplification, offering a foundation for optimizing Zst as a heparin-responsive feature in molecular diagnostics.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eOptimization of Reaction Conditions for HIPR\u003c/h2\u003e\n \u003cp\u003eTo enhance assay performance, we systematically optimized three critical parameters: DNA substrate concentration, reaction buffer composition, and Zst polymerase dosage. A template concentration of 10\u003csup\u003e6\u003c/sup\u003e copies/\u0026micro;L yielded the most consistent linear relationship between heparin concentration and Cp value (\u003cstrong\u003eFigure S4\u003c/strong\u003e). The use of a custom-formulated reaction buffer significantly amplified the inhibitory effect of heparin on the amplification reaction (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, 0.48U of Zst polymerase was identified as the optimal enzyme concentration, balancing both amplification efficiency and assay stability (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). These optimized conditions collectively improved the sensitivity, reproducibility, and quantitative accuracy of the signal-off LAMP-based heparin assay.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eDevelopment and Validation of Clinical Heparin Detection\u003c/h2\u003e\n \u003cp\u003eAccurate heparin monitoring remains crucial for safe anticoagulation therapy, yet traditional assays often depend on indirect measurement methods and can be affected by interference from biological matrices. Building on our discovery of Zst DNA polymerase inhibition by heparin, we established a LAMP-based detection assay that enables direct, probe-free quantification of heparin with minimal complexity. Under optimized conditions, the assay exhibited a strong linear relationship between Cp values and heparin concentrations (y\u0026thinsp;=\u0026thinsp;5.960x\u0026thinsp;+\u0026thinsp;28.21, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9530), with a detection limit of 0.5 \u0026micro;M. This range is clinically relevant, covering heparin doses used in cardiovascular surgery (2\u0026ndash;8 U/mL, approximately 17\u0026ndash;67 \u0026micro;M) and for long-term management (0.2\u0026ndash;1.2 U/mL, about 1.7\u0026ndash;10 \u0026micro;M).\u003c/p\u003e\n \u003cp\u003eThe HIPR assay exhibited clear dose-dependent amplification suppression, as evidenced by prolonged Cp values and reduced fluorescence output. This correlation enables accurate quantitative assessment of heparin concentrations across clinically relevant ranges. Specificity tests showed that common anions (e.g., glucose, sodium citrate) and biological substances (e.g., BSA, EDTA) did not interfere with the readout or negate heparin\u0026rsquo;s inhibitory effect. In human plasma spiked with different concentrations of heparin, the assay produced recovery rates of 95\u0026ndash;108% with low relative standard deviation (RSD\u0026thinsp;\u0026lt;\u0026thinsp;4.51%), supporting its robustness and suitability for clinical use. These findings confirm that the method is accurate, highly specific, and ideal for rapid, point-of-care heparin monitoring.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;1.\u003c/strong\u003e Recovery of heparin from spiked human plasma samples measured by ZE-LAMP (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n \u003cp\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;RSD.\u003c/p\u003e\n \u003cp\u003eSD: Standard Deviation; RSD: Relative Standard Deviation.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHeparin dosage(\u0026micro;M)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHeparin detection(\u0026micro;M)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRecovery Rate (%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRelative Standard Deviation(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e107.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e101.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e101.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e100.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e101.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e98.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e101.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e108.40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study presents a novel and probe-free method for heparin detection based on the inhibition of Zst DNA polymerase in LAMP assay. The assay offers substantial improvements over traditional detection methods, such as activated clotting time (ACT) and chromogenic tests, which can be indirect and prone to interference. Our results show that the assay is highly sensitive, with a detection limit of 0.5 \u0026micro;M, which covers the clinically relevant ranges of heparin concentrations typically encountered during cardiovascular surgery (2\u0026ndash;8 U/mL, 17\u0026ndash;67 \u0026micro;M) and post-operative therapy (0.2\u0026ndash;1.2 U/mL, 1.7\u0026ndash;10 \u0026micro;M). The assay demonstrated a strong linear correlation between heparin concentration and the Cp value, with an R\u0026sup2; value of 0.99530, confirming its accuracy and reliability.\u003c/p\u003e\u003cp\u003eThe simplicity and efficiency of the assay make it an appealing alternative for point-of-care testing. It functions under isothermal conditions, requires no complex equipment, and provides a clear fluorescence readout, which is especially beneficial for environments with limited resources. Additionally, the method's high specificity against common anions and biological substances, like glucose and BSA, further boosts its suitability for clinical use by reducing false positives or interference often seen with other methods.\u003c/p\u003e\u003cp\u003eOne of the key strengths of the assay lies in its mechanistic foundation. Heparin interacts specifically with Zst DNA polymerase, inhibiting its activity by forming non-covalent interactions such as van der Waals forces and hydrogen bonds. This interaction causes a delay or complete inhibition of the amplification reaction, translating into measurable changes in the Cp value. This mechanism enables the detection of heparin directly, without the need for additional probes, and offers a novel approach to detecting non-nucleic acid targets with LAMP.\u003c/p\u003e\u003cp\u003eThe validation of the assay in human plasma, with recovery rates between 95% and 108%, demonstrates its strong performance in complex biological samples. Additionally, its potential for use in personalized medicine looks promising. The ability to quickly and accurately measure heparin levels could enhance patient monitoring and help optimize treatment plans in clinical settings.\u003c/p\u003e\u003cp\u003eLooking ahead, the use of this assay could expand beyond heparin detection to other glycosaminoglycans, such as heparan sulfate or chondroitin sulfate, by utilizing similar protein-ligand interactions. Additionally, the insights gained from the heparin-Zst interaction might lead to further improvements in engineered polymerases for better detection. Future research should focus on integrating this assay with microfluidic platforms, which could offer greater portability and lower reagent use, making it suitable for widespread application in resource-limited settings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we report a novel, probe-free assay for heparin detection based on the selective inhibition of Zst DNA polymerase during loop-mediated isothermal amplification. This method achieves a low detection limit of 0.5 \u0026micro;M and offers excellent linearity, specificity, and reproducibility in complex biological matrices. Its simplicity, minimal instrumentation requirements, and compatibility with clinical plasma samples make it a promising tool for real-time, point-of-care heparin monitoring.\u003c/p\u003e\u003cp\u003eWith no need for complex instruments or probes, this assay is well-suited for point-of-care testing. Its simplicity and potential for integration into microfluidic platforms make it an ideal solution for resource-limited environments. The assay could also be adapted for detecting other glycosaminoglycans, contributing to the advancement of diagnostic tools.\u003c/p\u003e\u003cp\u003eOverall, this work introduces a novel approach for heparin monitoring that has the potential to improve clinical care, particularly in personalized medicine and point-of-care settings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 82372502), Shanghai 2023 Medical Innovation Research Special Funding (No. 23Y11902300), Zhongshan Hospital Clinical Research Project (No. ZSLCYJ202348), Zhongshan Hospital Science and Technology Innovation Fund-Cultivation Project (No. 2024-ZSCX07), and China International Medical Exchange Funding (No. Z-2017-24-2421).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.X. contributed equally to this work. Y.X. contributed to the conceptualization, data curation, formal analysis, and writing the original draft. Y. L. contributed to the visualization. M. L. W contributed to investigation. M. L. W and X. D. contributed to validation, review, editing, and funding acquisition. K.Y. contributed to supervision, review, editing, and funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTriplett DA (2000) Coagulation and bleeding disorders: review and update. Clin Chem 46(8):1260\u0026ndash;1269\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAgarwal B, Wright G, Gatt A, Riddell A, Vemala V, Mallett S, Chowdary P, Davenport A, Jalan R, Burroughs A (2012) Evaluation of coagulation abnormalities in acute liver failure. J Hepatol 57(4):780\u0026ndash;786\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDamus PS, Hicks M, Rosenberg RD (1973) Anticoagulant action of heparin. Nature 246(5432):355\u0026ndash;357\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHirsh J, Warkentin TE, Raschke R, Granger C (1998) Heparin and low-molecular-weight heparin: mechanisms of action, pharmacokinetics, dosing considerations, monitoring, efficacy, and safety. Chest 114(5):S489\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHirsh J, Anand SS, Halperin JL, Fuster V (2001) Guide to anticoagulant therapy: Heparin: a statement for healthcare professionals from the American Heart Association. Circulation 103(24):2994\u0026ndash;3018\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEsposito RA, Culliford AT, Colvin SB, Thomas SJ, Lackner H, Spencer FC (1983) The role of the activated clotting time in heparin administration and neutralization for cardiopulmonary bypass. 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Anal Chem 96(38):15493\u0026ndash;15502\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"e5685e17-8c84-44f4-8819-8c810a609466","identifier":"10.13039/501100003347","name":"Fudan University","awardNumber":"National Natural Science Foundation of China (No. 82372502)","order_by":0},{"identity":"700f70b8-1665-401f-8541-22ea36817e68","identifier":"10.13039/501100003347","name":"Fudan University","awardNumber":"Shanghai 2023 Medical Innovation Research Special Funding (No. 23Y11902300)","order_by":1},{"identity":"4e61494e-b0bd-48c1-9b43-6625e84fecdc","identifier":"10.13039/501100003347","name":"Fudan University","awardNumber":"Zhongshan Hospital Clinical Research Project (No. ZSLCYJ202348)","order_by":2},{"identity":"523384af-5933-46c1-af0c-a2c89b355111","identifier":"10.13039/501100003347","name":"Fudan University","awardNumber":"Zhongshan Hospital Science and Technology Innovation Fund - Cultivation Project (No. 2024-ZSCX07)","order_by":3},{"identity":"6c6cfd49-7af9-4c27-b8fb-1ccf809d641b","identifier":"10.13039/501100003347","name":"Fudan University","awardNumber":"China International Medical Exchange Funding (No. Z-2017-24-2421)","order_by":4}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Fudan University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heparin detection, Probe-free, Loop-mediated isothermal amplification, Polymerase inhibition","lastPublishedDoi":"10.21203/rs.3.rs-7559255/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7559255/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAccurate monitoring of heparin is essential for safe and effective anticoagulant therapy. Current detection methods often rely on synthetic probes and are vulnerable to interference from complex biological matrices. Here, we introduce \u003cb\u003eHIPR\u003c/b\u003e (\u003cb\u003eH\u003c/b\u003eeparin-\u003cb\u003ei\u003c/b\u003enhibited \u003cb\u003eP\u003c/b\u003eolymerase \u003cb\u003eR\u003c/b\u003eeaction), a novel probe-free assay that leverages the selective inhibitory effect of heparin on a rationally engineered DNA polymerase, Zst. Instead of relying on charge interactions, HIPR translates van der Waals and hydrogen bonds between heparin and the enzyme into measurable inhibition during loop-mediated isothermal amplification (LAMP) primer extension. The assay achieves high sensitivity (limit of detection: 0.5 \u0026micro;M) and specificity, with recovery rates of 95\u0026ndash;108% in plasma. HIPR offers a robust, rapid, and user-friendly platform for point-of-care heparin monitoring and extends the application of isothermal amplification methods to non-nucleic acid targets.\u003c/p\u003e","manuscriptTitle":"Heparin’s Inhibition Capability on DNA Polymerase Enables Probe-Free Heparin Quantification","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 09:34:59","doi":"10.21203/rs.3.rs-7559255/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"393f3af2-25dd-4ed5-b952-3db6f12ea0e0","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54332683,"name":"Analytical Biochemistry"}],"tags":[],"updatedAt":"2025-09-09T09:35:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 09:34:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7559255","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7559255","identity":"rs-7559255","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}