Integrated optical sensors for microfluidic free-flow electrophoresis

微芯片分析技术小型化的目标是在更小的尺度上更快地进行化学分析；为了节约资源，能够调查只有很少数量的生物和医学样本，并在需要时进行分析。微芯片上的电泳分离程序能够以高分辨率和灵敏度进行样品分析，但由于许多相关参数只能间接观察到，因此开发和读出非常复杂。在微流体分离过程中集成光学传感器旨在克服这些限制。在之前的项目期间，首次展示了平面光学化学传感器和传感器阵列在微流体自由流动电泳（micro-FFE）平台上的集成。环境参数，如pH值，现在可以在微ffe中进行空间解析和实时分析。在本项目中，利用芯片集成光学传感器研究了FFE中的两个重要参数，即等电聚焦（IEF）中的pH梯度和FFE （IEF）中焦耳加热产生的温差，并将结果用于改进和优化微分析分离程序。最初，这将在选定的蛋白质混合物上进行，然后将结果用于分析现实世界的样品。这将在细胞培养裂解物的蛋白质和细胞器上得到举例证明。为此，将为FFE开发检测方法，以便在不受干扰的情况下同时记录标记分析物和传感器层。这些是基于比值多光谱数据采集结合荧光寿命测量。在接下来的项目部分中，这些分析过程将被扩展。为了从芯片集成传感器层获得更多的信息，将研究微电泳中pH和温度的同时空间分辨双传感。将开发具有集成传感器层的微型ffe中原生未标记蛋白质的分析。在最后一部分，集成化学传感器的微ffe芯片将与微流控反应器耦合，以便使用集成光学化学传感器层在微芯片上进行化学合成和随后的电泳分析。

Miniaturization of analytical techniques on microchips aims for faster chemical analysis on smaller scales; in order to conserve resources, enable the investigation of biological and medical samples that are available only in very small amounts and perform analysis where it is needed. Electrophoretic separation procedures on microchips are capable of sample analysis with high resolution and sensitivity but are complex to develop and read out because many relevant parameters can only be observed indirectly. Integration of optical sensors in microfluidic separation procedures aims to overcome these constraints.In the previous project period the integration of planar optical chemical sensors and sensor arrays in microfluidic free flow electrophoresis (micro-FFE) platforms was demonstrated for the first time. Environmental parameters such as pH can now be analyzed spatially resolved and in real time in micro-FFE.In this project, two important parameters in FFE, the pH gradient in isoelectric focusing (IEF) and temperature differences produced by Joule heating in FFE (IEF) are investigated by chip-integrated optical sensors and the results are used for improvement and optimization of microanalytical separation procedures. Initially this will be performed on selected protein mixtures and the results are later adapted for analysis of real world samples. This will be exemplarily demonstrated on proteins and organelles from cell culture lysates. To this end, detection methods will be developed for FFE in order to provide for simultaneous recording of both labeled analytes and sensor layer without interferences. These are based on ratiometric multispectral data acquisition combined with fluorescence lifetime measurements.In further project sections these analytical procedures are then extended. In order to gain more information from chip-integrated sensor layers, simultaneous spatially resolved dual sensing of pH and temperature in micro electrophoresis will be investigated. The analysis of native, unlabeled proteins in micro-FFE with integrated sensor layers will be developed. In the final part micro-FFE chips with integrated chemical sensors will be coupled to a microfluidic reactor in order to enable both chemical synthesis and subsequent electrophoretic analysis on a microchip using integrated optical chemical sensor layers.