Hydrogel bead-based isothermal detection (BEAD-ID) for assessing the activity of DNA-modifying enzymes
Kathrine Nygaard Borg,
Ayush Shetty,
Guangyao Cheng,
Shaodi Zhu,
Tianle Wang,
Wu Yuan,
Ho Pui Ho,
Birgitta Ruth Knudsen,
Cinzia Tesauro,
Yi-Ping Ho
Affiliations
Kathrine Nygaard Borg
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China
Ayush Shetty
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China
Guangyao Cheng
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China
Shaodi Zhu
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China
Tianle Wang
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China
Wu Yuan
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China
Ho Pui Ho
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China
Birgitta Ruth Knudsen
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark
Cinzia Tesauro
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark
Yi-Ping Ho
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China; Centre for Biomaterials, The Chinese University of Hong Kong, Hong Kong SAR, China; Hong Kong Branch of CAS Center for Excellence in Animal Evolution and Genetics, Hong Kong SAR, China; State Key Laboratory of Marine Pollution, City University of Hong Kong, Hong Kong SAR, China; Corresponding author
Summary: 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 via 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 in situ 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.
