Borg Kathrine Nygaard, Shetty Ayush, Cheng Guangyao, Zhu Shaodi, Wang Tianle, Yuan Wu, Ho Ho Pui, Knudsen Birgitta Ruth, Tesauro Cinzia, Ho Yi-Ping
Department of Biomedical Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong SAR 00000, China.
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
iScience. 2024 Nov 6;27(12):111332. doi: 10.1016/j.isci.2024.111332. eCollection 2024 Dec 20.
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 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 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.
DNA修饰酶在生物过程中至关重要，具有重要的临床意义。传统的定量方法往往忽略了酶活性，而酶活性才是酶功能的真正决定因素。我们提出了基于水凝胶珠的等温检测法（BEAD-ID），利用基于均匀水凝胶珠的微反应器在珠上评估DNA修饰酶的活性。我们通过液滴微流控技术制备了均匀的寡核苷酸共轭聚丙烯酰胺（oligo-PAA）珠，该技术针对捕获和扩增酶修饰的纳米传感器进行了优化。通过将DNA寡核苷酸纳入水凝胶网络，BEAD-ID保留了等温扩增产物，便于检测珠上的酶活性。我们通过定量人类拓扑异构酶I（TOP1）和限制性内切酶EcoRI来验证BEAD-ID，结果表明酶浓度与荧光强度之间存在直接相关性，证明了该平台在食品基质中的灵敏度（6.25 nM TOP1，6.25 U/μL EcoRI）和可靠性（25 U/μL EcoRI）。此外，定制的类似流式细胞术的设置允许以352 Hz的频率进行高通量检测，并进行客观评估。BEAD-ID具有灵活性和可扩展性，是研究DNA修饰酶的一种很有前景的工具。
