Klyusko Illya, Scalise Stefania, Guzzi Francesco, Randazzini Luigi, Zaccone Simona, Parrotta Elvira Immacolata, Lucchino Valeria, Merola Alessio, Cosentino Carlo, Krühne Ulrich, Aquila Isabella, Cuda Giovanni, Di Fabrizio Enzo, Candeloro Patrizio, Perozziello Gerardo
Department of Experimental and Clinical Medicine, University of Catanzaro, Germaneto, 88100 Catanzaro, Italy.
Department of Chemistry and Biochemical Engineering, Technical University of Denmark, DK-2800 Kgs. Lyhgby, Denmark.
Biosensors (Basel). 2025 Jul 16;15(7):459. doi: 10.3390/bios15070459.
We present a miniaturized, inexpensive, and user-friendly microfluidic platform to support biological applications. The system integrates a mini-incubator providing controlled environmental conditions and housing a microfluidic device for long-term cell culture experiments. The incubator is designed to be compatible with standard inverted optical microscopes and Raman spectrometers, allowing for the non-invasive imaging and spectroscopic analysis of cell cultures in vitro. The microfluidic device, which reproduces a dynamic environment, was optimized to sustain a passive, gravity-driven flow of medium, eliminating the need for an external pumping system and reducing mechanical stress on the cells. The platform was tested using Raman analysis and adherent tumoral cells to assess proliferation prior and subsequent to hydrogen peroxide treatment for oxidative stress induction. The results demonstrated a successful adhesion of cells onto the substrate and their proliferation. Furthermore, the platform is suitable for carrying out optical monitoring of cultures and Raman analysis. In fact, it was possible to discriminate spectra deriving from control and hydrogen peroxide-treated cells in terms of DNA backbone and cellular membrane modification effects provoked by reactive oxygen species (ROS) activity. The 800-1100 cm band highlights the destructive effects of ROS on the DNA backbone's structure, as its rupture modifies its vibration; moreover, unpaired nucleotides are increased in treated sample, as shown in the 1154-1185 cm band. Protein synthesis deterioration, led by DNA structure damage, is highlighted in the 1257-1341 cm, 1440-1450 cm, and 1640-1670 cm bands. Furthermore, membrane damage is emphasized in changes in the 1270, 1301, and 1738 cm frequencies, as phospholipid synthesis is accelerated in an attempt to compensate for the membrane damage brought about by the ROS attack. This study highlights the potential use of this platform as an alternative to conventional culturing and analysis procedures, considering that cell culturing, optical imaging, and Raman spectroscopy can be performed simultaneously on living cells with minimal cellular stress and without the need for labeling or fixation.
我们展示了一个小型化、低成本且用户友好的微流控平台,以支持生物应用。该系统集成了一个小型培养箱,可提供可控的环境条件,并容纳用于长期细胞培养实验的微流控装置。该培养箱设计为与标准倒置光学显微镜和拉曼光谱仪兼容,允许对体外细胞培养进行非侵入性成像和光谱分析。微流控装置可再现动态环境,经过优化以维持培养基的被动重力驱动流动,无需外部泵送系统,并减少对细胞的机械应力。该平台使用拉曼分析和贴壁肿瘤细胞进行测试,以评估过氧化氢处理诱导氧化应激之前和之后的细胞增殖情况。结果表明细胞成功粘附在基质上并增殖。此外,该平台适用于对培养物进行光学监测和拉曼分析。事实上,根据活性氧(ROS)活性引起的DNA主链和细胞膜修饰效应,可以区分来自对照细胞和过氧化氢处理细胞的光谱。800 - 1100 cm波段突出了ROS对DNA主链结构的破坏作用,因为其断裂会改变其振动;此外,处理后的样品中未配对核苷酸增加,如1154 - 1185 cm波段所示。由DNA结构损伤导致的蛋白质合成恶化在1257 - 1341 cm、1440 - 1450 cm和1640 - 1670 cm波段中得到突出显示。此外,在1270、1301和1738 cm频率的变化中强调了膜损伤,因为磷脂合成加速以试图补偿ROS攻击造成的膜损伤。这项研究突出了该平台作为传统培养和分析程序替代方案的潜在用途,因为细胞培养、光学成像和拉曼光谱可以在活细胞上同时进行,对细胞的压力最小,且无需标记或固定。
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