Hydrogel bead-based isothermal detection (BEAD-ID) for assessing the activity of DNA-modifying enzymes

DNA-modifying enzymes are crucial in biological processes and have significant clinical implications. Traditional quantification methods often overlook enzymatic activity, the true determinants of enzymes' functions. We present hydrogel Bead-based Isothermal Detection (BEAD-ID), utilizing uniform hydrogel bead-based microreactors to evaluate DNA-modifying enzyme activity on-bead. We fabricated homogeneous oligo-conjugated polyacrylamide (oligo-PAA) beads <i>via</i> droplet microfluidics, optimized for capturing and amplifying enzyme-modified nanosensors. By incorporating DNA oligos within the hydrogel network, BEAD-ID retains isothermally amplified products, facilitating <i>in situ</i> detection of enzyme activities on-bead. We validate BEAD-ID by quantifying human topoisomerase I (TOP1) and restriction endonuclease EcoRI, showing a direct correlation between enzyme concentration and fluorescence intensity, demonstrating the platform's sensitivity (6.25 nM TOP1, 6.25 U/μL EcoRI) and reliability in food matrix (25 U/μL EcoRI). Additionally, a customized flow cytometry-mimicking setup allows high-throughput detection at 352 Hz with objective assessment. BEAD-ID, offering flexibility and scalability, is a promising tool for studying DNA-modifying enzymes.