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; Ho Pui Ho; Birgitta R. 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, Ho Pui Ho, Birgitta R. Knudsen, Cinzia Tesauro, Yi-Ping Ho

Department of Molecular Biology and Genetics - Cellular Health, Intervention, and Nutrition

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

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.

Original language	English
Article number	111332
Journal	iScience
Volume	27
Issue	12
Number of pages	13
ISSN	2589-0042
DOIs	https://doi.org/10.1016/j.isci.2024.111332
Publication status	Published - 20 Dec 2024

Keywords

Fluidics
Methodology in biological sciences
Nanotechnology
Sensor

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10.1016/j.isci.2024.111332Licence: CC BY-NC-ND

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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.",

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author = "Borg, {Kathrine Nygaard} and Ayush Shetty and Guangyao Cheng and Shaodi Zhu and Tianle Wang and Ho, {Ho Pui} and Knudsen, {Birgitta R.} and Cinzia Tesauro and Yi-Ping Ho",

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AU - Borg, Kathrine Nygaard

AU - Shetty, Ayush

AU - Cheng, Guangyao

AU - Zhu, Shaodi

AU - Wang, Tianle

AU - Ho, Ho Pui

AU - Knudsen, Birgitta R.

AU - Tesauro, Cinzia

AU - Ho, Yi-Ping

PY - 2024/12/20

Y1 - 2024/12/20

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.

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.

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

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