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

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 (Heparin-inhibited Polymerase Reaction), 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.