具有液滴生成、孵育和连续荧光检测功能的集成液滴式数字环路介导等温扩增微流控芯片

Biosensors Pub Date : 2024-07-08 DOI:10.3390/bios14070334
Yen-Heng Lin, Yuan-Ting Hung, Wei Chang, Chiuan-Chian Chiou
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引用次数: 0

摘要

这项研究在单个微流控芯片上集成了样品分装、孵育和连续荧光检测功能,用于基于液滴的核酸数字环介导等温扩增(LAMP)。这种集成消除了在不同平台之间转移反应的需要,避免了样品污染和损失。反应前,在通道中注入油相,并在芯片顶部添加玻璃盖玻片,解决了 LAMP 反应期间因加热而在通道中产生气泡的问题。此外,使用两个荧光强度阈值可以在单个荧光检测通道内同时检测和计数阳性和阴性液滴。该芯片可在 10 分钟内从 5 µL 样品中分离出约 6000 个液滴,液滴直径约为 110 µm,变异系数 (CV) 值为 0.82%。金黄色葡萄球菌通过拟议的平台进行了定量检测。结果表明,相关系数(R = 0.9998)非常准确,检测限达到 1.7 × 102 拷贝/微升。液滴数字 LAMP 反应的整个过程,从液滴产生到培养再到定量结果,最长只需 70 分钟。
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Integrated Droplet-Based Digital Loop-Mediated Isothermal Amplification Microfluidic Chip with Droplet Generation, Incubation, and Continuous Fluorescence Detection
This study integrated sample partition, incubation, and continuous fluorescence detection on a single microfluidic chip for droplet-based digital Loop-Mediated Isothermal Amplification (LAMP) of nucleic acids. This integration eliminated the need to transfer reactions between different platforms, avoiding sample contamination and loss. Prior to the reaction, filling the channels with an oil phase and adding a glass cover slip on top of the chip overcame the problem of bubble generation in the channels during the LAMP reaction due to heating. Additionally, using two fluorescence intensity thresholds enabled simultaneous detection and counting of positive and negative droplets within a single fluorescence detection channel. The chip can partition approximately 6000 droplets from a 5 µL sample within 10 min, with a droplet diameter of around 110 µm and a coefficient of variation (CV) value of 0.82%. Staphylococcus aureus was quantified via the proposed platform. The results demonstrated a highly accurate correlation coefficient (R = 0.9998), and the detection limit reached a concentration of 1.7 × 102 copies/µL. The entire process of the droplet digital LAMP reaction, from droplet generation to incubation to quantitative results, took a maximum of 70 min.
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