Free proline, termed proline, is a biomarker used for diagnosing drought stress in plants. A previously developed proline-ninhydrin reaction-based paper sensor could quickly and easily detect proline, but it was limited by low sensitivity. In this study, we developed an enclosed multilayer paper-based microfluidic sensor with high sensitivity for the quantitative detection of proline in plants. The multilayer paper-based sensor was manufactured using simple wax printing and origami methods, and contained an internal mixing channel to allow good mixing of the proline with ninhydrin, increasing the proline-ninhydrin reactivity and providing accurate and sensitive proline detection. By preloading ninhydrin onto the sample loading area, uniform coloration of the sensing window was achieved, allowing quantitative analysis of various proline concentrations using a constant reaction time. Only the sensing window and sample loading area were exposed to limit sample evaporation and contamination from the external environment. The LOD of the fabricated sensor was 23 μM, which is approximately 29-fold lower than that of the previously proposed paper sensor (657 μM). Samples were extracted from A. thaliana plants subjected to drought stress for proline detection. The proline concentrations measured using the developed paper sensor and a spectrophotometric method were not statistically significant at a confidence level of 95%. Therefore, the developed sensor can be applied to measure proline concentrations precisely in the field with a low detection limit. The developed paper-based sensor can be used to detect the early stages of drought in plants and thus improve crop productivity.

中文翻译：

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用于定量植物中脯氨酸的高灵敏度封闭式多层纸基微流体传感器

游离脯氨酸，称为脯氨酸，是用于诊断植物干旱胁迫的生物标志物。先前开发的基于脯氨酸-茚三酮反应的纸传感器可以快速轻松地检测脯氨酸，但其灵敏度较低。在这项研究中，我们开发了一种封闭的多层纸基微流体传感器，具有高灵敏度，可用于植物中脯氨酸的定量检测。多层纸基传感器使用简单的蜡印和折纸方法制造，并包含内部混合通道，使脯氨酸与茚三酮良好混合，增加脯氨酸 - 茚三酮反应性并提供准确和灵敏的脯氨酸检测。通过将茚三酮预加载到样品加载区域，实现了传感窗口的均匀着色，允许使用恒定反应时间对各种脯氨酸浓度进行定量分析。只有传感窗口和样品装载区暴露在外，以限制样品蒸发和外部环境的污染。制造的传感器的 LOD 为 23 μM，比之前提出的纸质传感器（657 μM）低约 29 倍。从经受干旱胁迫的拟南芥植物中提取样品用于脯氨酸检测。使用开发的纸传感器和分光光度法测量的脯氨酸浓度在 95% 的置信水平下没有统计学意义。因此，所开发的传感器可用于现场精确测量脯氨酸浓度，检测限低。