He Hong, Wang Xiaoli, Tan Haolan, Xiang Songtao, Xu Yi
Key Disciplines Lab of Novel Micro-Nano Devices and System Technology, Key Laboratory of Optoelectronic Technology and Systems, Ministry of Education, Chongqing University, Shapingba, Chongqing, 400044, China; School of Optoelectronics Engineering, Chongqing University, Shapingba, Chongqing, 400044, China.
School of Chemistry and Chemical Engineering, Chongqing University, Shapingba, Chongqing, 401331, China.
Talanta. 2025 Apr 1;285:127395. doi: 10.1016/j.talanta.2024.127395. Epub 2024 Dec 16.
A multifunctional microfluidic chip integrated with perfusion cell culture and in situ SERS detection of cell secretion was designed and developed for the detection of IL-6 secretion from LPS-stimulation of A549 cells in this paper. Researching works were focused on A549 cell activity and secretion in the constructed LPS-stimulated A549 cells model. On the designed microchip, a bubble trap chamber was designed to remove the bubbles in the culture medium which could also be simultaneously preheated by a split hot plate. Then, a long-time perfusion culture process of 549 cells could be realized. Under the optimized conditions the A549 cells could be cultured and kept in good activity for more than 36 h. Subsequently, the model of interaction between LPS and A549 cells was established on the designed microchip. When LPS-stimulated A549 cells, the IL-6 which was one of the secretions formed in this process was detected quantitatively by SERS spectral technique. The silver-coated gold nano-stars were prepared and taken as a sensitive enhancing probe for the SERS detection of IL-6 secreted from LPS-stimulated A549 cells. The immunomagnetic beads, IL-6 antigen, and SERS probes were mixed and incubated in the microchip and form a sandwich structure which was captured by the permanent magnet in the detection zone for SERS detection. The reference material of IL-6 was used to establish the calibration curve, and the linear range and detection limit were 1-10000 pg/mL and 0.75 pg/mL, respectively. Then, the IL-6 secretion from LPS-stimulated A549 cells was detected hourly for 7 h by this established method. The process of LPS stimulation of A594 cells did not lead to a sustained increase in the SERS spectral signature of IL-6. Instead, IL6 secretion initially increased sharply, then decreased and eventually stabilized. It could be due to a potential mechanism that the cells self-regulated to mitigate the inflammatory effects in response to sustained stimulation. The proposed multifunctional microfluidic chip, characterized by high sensitivity and the ability to perform continuous hourly detection, exhibited significant application prospects in the study of external stimulation on cells.
本文设计并开发了一种集成灌注细胞培养和细胞分泌原位表面增强拉曼光谱（SERS）检测功能的多功能微流控芯片，用于检测脂多糖（LPS）刺激A549细胞后白细胞介素-6（IL-6）的分泌情况。研究工作聚焦于构建的LPS刺激A549细胞模型中A549细胞的活性和分泌情况。在设计的微芯片上，设计了一个气泡捕获腔室，用于去除培养基中的气泡，同时该腔室还可通过分体式热板进行预热。然后，可实现A549细胞的长时间灌注培养过程。在优化条件下，A549细胞可培养并保持良好活性超过36小时。随后，在设计的微芯片上建立了LPS与A549细胞之间的相互作用模型。当LPS刺激A549细胞时，通过SERS光谱技术对该过程中形成的分泌产物之一IL-6进行定量检测。制备了镀银金纳米星，并将其作为灵敏的增强探针用于SERS检测LPS刺激A549细胞分泌的IL-6。将免疫磁珠、IL-抗原和SERS探针在微芯片中混合孵育，形成夹心结构，在检测区被永久磁铁捕获以进行SERS检测。使用IL-6的参考物质建立校准曲线，线性范围和检测限分别为1 - 10000 pg/mL和0.75 pg/mL。然后，用此建立的方法每小时检测LPS刺激A549细胞7小时的IL-分泌情况。LPS刺激A594细胞的过程并未导致IL-6的SERS光谱特征持续增加。相反，IL-6分泌最初急剧增加，然后下降并最终稳定。这可能是由于一种潜在机制，即细胞通过自我调节来减轻持续刺激引起的炎症效应。所提出的多功能微流控芯片具有高灵敏度和每小时连续检测的能力，在细胞外部刺激研究中展现出显著的应用前景。
