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

doi:10.1016/j.isci.2024.111332

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^*

^*Corresponding author for this work

Research output: Journal Publications and Reviews › RGC 21 - Publication in refereed journal › peer-review

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Abstract

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. © 2024 The Author(s).

Original language	English
Article number	111332
Journal	iScience
Volume	27
Issue number	12
Online published	6 Nov 2024
DOIs	https://doi.org/10.1016/j.isci.2024.111332
Publication status	Published - 20 Dec 2024
Externally published	Yes

Funding

The authors would like to express their gratitude the support received by the Research Grants Council of Hong Kong Special Administrative Region, China (project #: CUHK 14207121, 14219922 and C5005-23WF), the VC Discretionary Fund provided by the Chinese University of Hong Kong, Hong Kong Special Administrative Region, China (project #: 8601014) and the State Key Laboratory in Marine Pollution (SKLMP), the City University of Hong Kong, Hong Kong Special Administrative Region, China, under the scheme of Seed Collaborative Research Fund (project #: SKLMP/SCRF/0042).

Research Keywords

Fluidics
Methodology in biological sciences
Nanotechnology
Sensor

Publisher's Copyright Statement

This full text is made available under CC-BY-NC-ND 4.0. https://creativecommons.org/licenses/by-nc-nd/4.0/

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

abstract = "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{\textquoteright} 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. {\textcopyright} 2024 The Author(s).",

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T1 - Hydrogel bead-based isothermal detection (BEAD-ID) for assessing the activity of DNA-modifying enzymes

AU - Borg, Kathrine Nygaard

AU - Shetty, Ayush

AU - Cheng, Guangyao

AU - Zhu, Shaodi

AU - Wang, Tianle

AU - Yuan, Wu

AU - Ho, Ho Pui

AU - Knudsen, Birgitta Ruth

AU - Tesauro, Cinzia

AU - Ho, Yi-Ping

PY - 2024/12/20

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N2 - 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. © 2024 The Author(s).

AB - 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. © 2024 The Author(s).

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

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