NMR-compatible BioMEMS platform for in vitro and in vivo protein structure and function characterization

生物价格的目标是解决现代分子生物学的一个关键挑战：开发一种强大的工具，允许在生活环境中对工作中的蛋白质进行核磁共振研究。我们的第一个目标是开发一个生物微机械环境平台，能够在平台内的电子寻址位置实现高空间和时间分辨率的按需蛋白质和细胞固定化。接下来，我们计划集成精心设计的核磁共振探测器，用于在先前定义的位置对固定目标进行高分辨率调查。微系统和生物工程的融合将使我们能够创建一个可电寻址的调查地点网络，并重建生活环境，以便在最后阶段，集成微核磁共振探测器将解决高通量蛋白质核磁共振研究的问题。我们已经确定了几个需要解决的研究目标，以便结果将成为核磁共振和生物系统研究界的有用工具。O1：为了核磁共振蛋白质研究的目的，复制蛋白质的自然环境。与确保复制蛋白质运作的自然环境的生活环境有关的目标涉及体外和体内(细胞内)研究。O2：在体外研究的核磁共振检测体积内以高空间分辨率按需加载和固定化蛋白质。为了能够执行统计相关和/或组合测量数据集，从核磁共振灵敏度和微核磁共振检测体积内的场均匀度的角度来看，需要快速、自动地将蛋白质加载和定位在最有利的位置。用于细胞内蛋白质核磁共振的微流控设备必须提供足够的氧气，供应给测量地点的细胞培养介质，累积的废物和代谢物及时被疏散。O4：同时检测多个与蛋白质相关的核物种进行多维、异核测量-开发能够瞄准多达3个不同Larmor频率的小型化核磁共振检测器，以便同一检测器可以同时用于从多个感兴趣的核捕获信息。O5：通过小型化提高检测器的灵敏度，解决长时间采集问题。另一个目标是探测器级别的材料工程，目的是获得零敏感度、低电阻率的传感器材料(例如导线)，以最大限度地减少B0场失真、最大信号拾取和低噪声。开发一个能够在广泛光谱范围内询问多个核物种的平台将为广泛的应用开辟道路，从代谢组学和蛋白质药物筛选，到低成本的靶向应用，如护理点调查或低场强核磁共振。

The BIO-PRICE goal is to address a key challenge of modern molecular biology: development of a robust tool to allow NMR investigation of proteins at work in a living environment. Our first aim is to develop a BioMEMS environmental platform enabling on-demand protein and cell immobilization with high spatial and temporal resolution at electro-addressable locations inside the platform. Next, we plan to integrate carefully designed NMR µ-detectors for high-resolution investigation of the immobilized target at the previously defined locations. Merging microsystem- and bio-engineering will enable us to create a network of electro-addressable investigation sites and to reconstitute the living environment so that, in the final stage, integration of µNMR detectors will address the issue of high-throughput protein NMR investigation. We have identified several research objectives which need to be addressed so that the results will constitute a useful tool for both NMR and biosystems research communities.O1: Replicate the natural environment of proteins for the purpose of NMR protein investigation. The objectives pertaining to ensuring a living environment, which replicates the natural environment where proteins operate, address both in vitro and in vivo (in-cell) investigation.O2: On-demand protein loading and immobilization with high spatial resolution inside the NMR detection volume for in vitro investigation. In order to be able to perform statistically relevant and/or combinatorial measurement datasets, fast and automatic loading and positioning of the protein at the most favorable position from the point of view of NMR sensitivity and field uniformity inside the µNMR detection volume is required.O3: Develop a dedicated platform for in-cell protein NMR. Microfluidic devices for in-cell protein NMR must provide sufficient aeration, cell-nurturing media supplied to the measurement site, accumulated waste and metabolites timely evacuated.O4: Simultaneous detection of multiple protein-relevant nuclear species for multi-dimensional, hetero-nuclear measurements - development of miniaturized NMR detectors capable of targeting up to 3 different Larmor frequencies, so that the same detector can be used to capture information from multiple nuclei of interest at the same time.O5: Address long acquisition times by increasing the detector sensitivity through miniaturization. Another objective is material engineering at detector level towards obtaining zero-susceptibility, low resistivity sensor material (e.g., wire) for minimum B0-field distortion, maximum signal pickup and low noise.O6: Enable high-throughput, parallel protein NMR investigation. The development of a platform capable of interrogating multiple nuclear species in a broad spectral range would open the way for a wide range of applications ranging from metabolomics and protein-medical drug screening, to low-cost targeted applications such as point of care investigation or low field NMR.