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

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

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 u...

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Bibliographic Details
Main Authors: Kathrine Nygaard Borg, Ayush Shetty, Guangyao Cheng, Shaodi Zhu, Tianle Wang, Wu Yuan, Ho Pui Ho, Birgitta Ruth Knudsen, Cinzia Tesauro, Yi-Ping Ho
Format: Article
Language:English
Published: Elsevier 2024-12-01
Series:iScience
Subjects:
Sensor
Fluidics
Nanotechnology
Methodology in biological sciences
Online Access:http://www.sciencedirect.com/science/article/pii/S2589004224025574
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Summary: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.
ISSN:2589-0042

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