Dhawan Mohit D, Wise Frank, Baeumner Antje J
Dept. of Biological and Environmental Engineering, Cornell University, Ithaca, NY, USA.
Anal Bioanal Chem. 2002 Oct;374(3):421-6. doi: 10.1007/s00216-002-1489-2. Epub 2002 Sep 7.
A novel cell lysis system was developed that is based on laser-induced disruption of bacterial and yeast cells. It will find application as a rapid, efficient and clean sample preparation step in bioanalytical detection systems. Using E. coli as our model analyte, we optimized cell lysis with respect to optimal laser wavelength, lowest energy input requirements, RNA release from the cells, and potential protein damage. The optimized system was finally applied to the lysis of four additional microorganisms. All experiments were carried out with about 2000 cells per sample or less. Initially, lysis was determined by the detection of cell survival after laser treatment using standard microbiological techniques, (i.e., cells were grown on nutrient agar plates). Then, actual release of mRNA from the cells was proven. Wavelengths investigated ranged from 500 nm to 1550 nm. An average power of 100 mW for the lasers was shown to be sufficient to obtain cell lysis at wavelengths above 1000 nm, with optimal wavelengths between 1250 nm and 1550 nm. Since water absorbs energy at those wavelengths, it is assumed that laser exposure results in an instantaneous increase of the cell temperature, which causes rupture of the cell membrane. Second, damage to protein solutions treated under optimized laser-lysis conditions was also studied. Using a pure solution of horseradish peroxidase as a model protein, no loss in enzyme activity was observed. Thus, it was concluded that damage to intracellular proteins is unlikely. Third, RNA release was tested using an E. coli specific RNA biosensor. Release of RNA was not detected from untreated cells, but laser-treated E. coli cells displayed significant RNA release due to laser-induced cell lysis. Finally, lysis of M. luteus, B. subtilis, B. cereus, and S. cerevisiae were investigated under optimized conditions. In all cases, laser-induced lysis of the cells was confirmed by determination of cell survival. Hence, laser-induced cell lysis is an efficient procedure that can be used for sample preparation, without damage to macromolecules, in bioanalytical detection systems for microorganisms. Miniaturized lasers and miniaturized cell-lysis chambers will create a simple, field-usable cell lysis system and allow the application of laser-induced cell lysis in micro Total Analysis Systems.
开发了一种基于激光诱导破坏细菌和酵母细胞的新型细胞裂解系统。它将作为生物分析检测系统中快速、高效且清洁的样品制备步骤得到应用。以大肠杆菌作为我们的模型分析物，我们针对最佳激光波长、最低能量输入要求、细胞中的RNA释放以及潜在的蛋白质损伤对细胞裂解进行了优化。最终将优化后的系统应用于另外四种微生物的裂解。所有实验均在每个样品约2000个细胞或更少的条件下进行。最初，通过使用标准微生物技术检测激光处理后的细胞存活率来确定裂解情况（即细胞在营养琼脂平板上生长）。然后，证明了细胞中mRNA的实际释放。研究的波长范围为500纳米至1550纳米。结果表明，对于波长高于1000纳米的情况，激光平均功率为100毫瓦足以实现细胞裂解，最佳波长在1250纳米至1550纳米之间。由于水在这些波长下吸收能量，推测激光照射会导致细胞温度瞬间升高，从而导致细胞膜破裂。其次，还研究了在优化的激光裂解条件下处理的蛋白质溶液的损伤情况。以辣根过氧化物酶的纯溶液作为模型蛋白质，未观察到酶活性损失。因此，得出细胞内蛋白质不太可能受损的结论。第三，使用大肠杆菌特异性RNA生物传感器测试了RNA释放情况。未处理的细胞未检测到RNA释放，但激光处理的大肠杆菌细胞由于激光诱导的细胞裂解而显示出显著的RNA释放。最后，在优化条件下研究了藤黄微球菌、枯草芽孢杆菌、蜡样芽孢杆菌和酿酒酵母的裂解情况。在所有情况下，通过测定细胞存活率证实了激光诱导的细胞裂解。因此，激光诱导细胞裂解是一种有效的方法，可用于生物分析微生物检测系统中的样品制备，且不会对大分子造成损伤。小型化激光和小型化细胞裂解室将创建一个简单的、可现场使用的细胞裂解系统，并使激光诱导细胞裂解能够应用于微型全分析系统。
