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首页> 外文期刊>Planetary and space science >Evaluation of cell lysis procedures and use of a micro fluidic system for an automated DNA-based cell identification in interplanetary missions
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Evaluation of cell lysis procedures and use of a micro fluidic system for an automated DNA-based cell identification in interplanetary missions

机译：评估细胞裂解程序并使用微流体系统进行星际任务中基于DNA的自动细胞识别

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A Modular Assay System for Solar System Exploration (MASSE) is being developed to include sample handling, pre-treatment, separation and analysis of biological target compounds by both DNA and protein microarrays. To better design sensitive and accurate initial upstream sample handling of the MASSE instrument, experiments investigating the sensitivity and potential extraction bias of commercially available DNA extraction kits between classes of environmentally relevant prokaryotes such as gram-negative bacteria (Escherichia coli), gram-positive bacteria (Bacillus megatarium), and Archaea (Haloarcula marismortui) were performed. For extractions of both planktonic cultures and spiked Mars simulated regolith, FTA~® paper demonstrated the highest sensitivity, with detection as low as ～1 x 10~1 cells and ～3.3 x 10~2 cells, respectively. In addition to the highest sensitivity, custom modified application of FT A~® paper extraction protocol is the simplest in terms of incorporation into MASSE and displayed little bias in sensitivity with respect to prokaryotic cell type. The implementation of FTA paper for environmental microbiology investigations appears to be a viable and effective option potentially negating the need for other pre-concentration steps such as filtration and negating concerns regarding extraction efficiency of cells. In addition to investigations on useful technology for upstream sample handling in MASSE, we have also evaluated the potential for μTAS to be employed in the MASSE instrument by employing proprietary lab-on-a-chip development technology to investigate the potential for microfluidic cell lysis of different prokaryotic cells employing both chemical and biological lysis agents. Real-time bright-field microscopy and quantitative PMT detection indicated that that gram positive, gram negative and archaeal cells were effectively lyzed in a few seconds using the microfluidic chip protocol developed. This included employing a lysis buffer with components including lysozyme, Protease, Proteinase K, Tween-20 and TritonX-100. The effectiveness of antibiotics and other chemical lysis agents were also screened and demonstrated partial effectiveness on all three cell types. This work demonstrates a step wise approach to evaluating the efficacy and sensitivity of commercial macro-scale technology and state-of-the-art developmental microfluidic technology under consideration for incorporation into the remotely operated MASSE instrument currently under development at the Carnegie Institution of Washington.
机译：正在开发一种用于太阳系探测的模块化测定系统（MASSE），以包括通过DNA和蛋白质微阵列对生物目标化合物进行样品处理，预处理，分离和分析。为了更好地设计MASSE仪器的灵敏和准确的初始上游样品处理，实验研究了市场上可买到的DNA提取试剂盒在环境相关的原核生物（例如革兰氏阴性细菌（大肠杆菌），革兰氏阳性细菌）之间的敏感性和潜在的提取偏差。 （巨型芽孢杆菌）和古细菌（Haloarcula marismortui）进行了。对于提取浮游生物和加标的火星模拟长石来说，FTA〜®纸显示出最高的灵敏度，分别低至〜1 x 10〜1个细胞和〜3.3 x 10〜2个细胞。除了最高的敏感性外，就掺入MASSE而言，自定义的FT A〜®纸质提取方案的修改应用是最简单的，并且对原核细胞类型的敏感性几乎没有偏差。将FTA论文用于环境微生物学研究似乎是一种可行且有效的选择，可能会消除对其他预浓缩步骤（如过滤）的需求，并消除了对细胞提取效率的担忧。除了研究在MASSE中用于上游样品处理的有用技术外，我们还通过采用专有的芯片实验室开发技术来研究MTASE的微流体细胞裂解潜力，从而评估了μTAS在MASSE仪器中使用的潜力。同时使用化学和生物裂解剂的不同原核细胞。实时明场显微镜和定量PMT检测表明，使用开发的微流控芯片方案可在数秒内有效溶解革兰氏阳性，革兰氏阴性和古细菌。这包括使用裂解缓冲液，其成分包括溶菌酶，蛋白酶，蛋白酶K，吐温20和TritonX-100。还筛选了抗生素和其他化学裂解剂的有效性，并证明了对所有三种细胞类型的部分有效性。这项工作展示了一种逐步评估方法，用于评估正在考虑将其纳入华盛顿卡内基研究所正在开发的远程操作的MASSE仪器中的商业宏观技术和最新发展的微流体技术的有效性和敏感性。

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