Zhang, Z., Zhang, X., Chen, K., Wang, J., Wu, Z., Shao, M., Tang, B. and Cheng, Y., 2025. Magnetic nanoconfined metabolic biosensing for rapid and high-sensitivity detecting viable E. coli O157: H7 in aquatic matrices. Sensors and Actuators B: Chemical, p.139132.

山东大学使用我司链霉亲和素磁珠偶联适配体捕获细菌发表在Sensors and Actuators B: Chemical

Magnetic nanoconfined metabolic biosensing for rapid and high-sensitivity detecting viable E. coli O157:H7 in aquatic matrices

Abstract
The accurate and viability-specific detection of E. coli O157:H7 in water environments is of vital importance for public health safety. In this work, we developed a Magnetic-capture Cellular Metabolism Droplet (MCCMD) biosensor combining magnetic capture, bacterial metabolism, and droplet microfluidics for rapid and high-sensitivity detection of E. coli O157:H7 in aquatic environments. To our knowledge, the magnetic nanoconfined metabolic biosensing represents the first report on analyzing the metabolic activity of E. coli O157:H7 in droplets. Specifically, bacterial metabolized β-galactosidase, induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), produced fluorescence signals by degrading fluorescein di-β-D-galactopyranoside (FDG) in the droplets. The miniaturized droplet volume (nL) significantly enhanced the local enzyme concentration and shortened detection time. More importantly, the biosensor allowed to distinguish between live and dead cells, eliminating the interference of dead E. coli O157:H7. With the aid of magnetic capture, E. coli O157:H7 could be enriched from the real sample. Compared to qPCR and plate counting method, the biosensor yields more accurate results and a wider detection range (∼5 orders of magnitude) within 3 h. It overcomes the limitations of traditional group-level detection methods, achieving single-cell detection by integrating the metabolic activities of living cells with droplet microfluidic technology. We applied the biosensor to detect E. coli O157:H7 in drinking water, rivers and a wastewater treatment plant, and it demonstrated robust performance in complex environmental matrices. It is expected to be a powerful alternative for rapid epidemiological detection, with significant potential for enhancing public health safety alerts and facilitating timely responses to waterborne pathogen outbreaks.

摘要
水环境中大肠杆菌 O157:H7 的精准、活菌特异性检测对公共卫生安全至关重要。本研究开发了一种结合磁捕获、细菌代谢与微流控液滴技术的磁捕获细胞代谢液滴（MCCMD）生物传感器，用于水环境中大肠杆菌 O157:H7 的快速高灵敏度检测。据我们所知，磁纳米限制代谢生物传感技术是首次报道用于分析液滴内大肠杆菌 O157:H7 的代谢活性。具体而言，异丙基 -β-D - 硫代半乳糖苷（IPTG）诱导细菌产生代谢酶 β- 半乳糖苷酶，该酶在液滴内降解荧光素二 -β-D - 半乳糖苷（FDG）产生荧光信号。纳升级（nL）的微型液滴体积显著提高了局部酶浓度，缩短了检测时间。更重要的是，该生物传感器可区分活菌与死菌，排除死体大肠杆菌 O157:H7 的干扰。借助磁捕获技术，可从实际样品中富集大肠杆菌 O157:H7。与实时荧光定量 PCR（qPCR）和平板计数法相比，该生物传感器在 3 小时内可获得更准确的结果，且检测范围更广（约 5 个数量级）。它克服了传统群体水平检测方法的局限性，通过整合活细胞代谢活性与微流控液滴技术实现了单细胞检测。将该生物传感器应用于饮用水、河水及污水处理厂样品中大肠杆菌 O157:H7 的检测，其在复杂环境基质中表现出稳定性能。该传感器有望成为快速流行病学检测的有力替代工具，在提升公共卫生安全预警、促进食源性致病菌暴发的及时响应方面具有重要潜力。

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2. Experimental section
2.1. Preparation of aptamer-functionalized magnetic nanoparticles
Biotinylated aptamers were conjugated to streptavidin-coated magnetic nanoparticles. The aptamer sequence is 5 ´ -BCC GGA CGC TTA TGC CTT GCC ATC TAC AGA GCA GGT GTG ACG G-3 ´ [29]. The process consists of the following six steps. (1) Vortexed the G-Streptavidin magnetic nanoparticles (PuriMag Biotechnology Ltd.) for 20 s before use. (2) Pipetted 20 μL of magnetic nanoparticles into a nuclease-free microcentrifuge tube. (3) Removed the supernatant and resuspended nanoparticles in 20 μL binding wash buffer (20 mM Tris Cl, 1.0 M NaCl, 1 mM EDTA, 0.02 % Triton ® X-100; pH 7.8). Repeated this washing step three times to remove any residual stabilizers. (4) Added 20 μL of biotinylated aptamers (Sangon Biotech (Shanghai) Co., Ltd.) to the magnetic nanoparticles suspension at a final concentration of 75 pM. Incubated for 15 min at room temperature with gentle mixing. (5) Separated the nanoparticles and washed the nanoparticles three times with an appropriate amount of binding wash buffer. (6) Resuspend the aptamer-functionalized nanoparticles in PBS (pH 7.2) containing 1 % bovine serum albumin (BSA) to block any remaining non-specific binding sites. Stored the nanoparticles at 4 ℃ until further use.