Ebrahimi Aida, Alam Muhammad Ashraful
School of Electrical and Computer Engineering, Purdue University, West Lafayette, IN 47907; Birck Nanotechnology Center, Purdue University, West Lafayette, IN 47907.
School of Electrical and Computer Engineering, Purdue University, West Lafayette, IN 47907; Birck Nanotechnology Center, Purdue University, West Lafayette, IN 47907
Proc Natl Acad Sci U S A. 2016 Jun 28;113(26):7059-64. doi: 10.1073/pnas.1606097113. Epub 2016 Jun 14.
Bacteria cells use osmoregulatory proteins as emergency valves to respond to changes in the osmotic pressure of their external environment. The existence of these emergency valves has been known since the 1960s, but they have never been used as the basis of a viability assay to tell dead bacteria cells apart from live ones. In this paper, we show that osmoregulation provides a much faster, label-free assessment of cell viability compared with traditional approaches that rely on cell multiplication (growth) to reach a detectable threshold. The cells are confined in an evaporating droplet that serves as a dynamic microenvironment. Evaporation-induced increase in ionic concentration is reflected in a proportional increase of the droplet's osmotic pressure, which in turn, stimulates the osmoregulatory response from the cells. By monitoring the time-varying electrical conductance of evaporating droplets, bacterial cells are identified within a few minutes compared with several hours in growth-based methods. To show the versatility of the proposed method, we show detection of WT and genetically modified nonhalotolerant cells (Salmonella typhimurium) and dead vs. live differentiation of nonhalotolerant (such as Escherichia coli DH5α) and halotolerant cells (such as Staphylococcus epidermidis). Unlike the growth-based techniques, the assay time of the proposed method is independent of cell concentration or the bacteria type. The proposed label-free approach paves the road toward realization of a new class of real time, array-formatted electrical sensors compatible with droplet microfluidics for laboratory on a chip applications.
细菌细胞利用渗透调节蛋白作为应急阀门，以应对其外部环境渗透压的变化。自20世纪60年代以来，人们就已经知道这些应急阀门的存在，但它们从未被用作区分死细菌细胞和活细菌细胞的活力测定基础。在本文中，我们表明，与依靠细胞增殖（生长）达到可检测阈值的传统方法相比，渗透调节提供了一种更快的、无需标记的细胞活力评估方法。细胞被限制在一个蒸发液滴中，该液滴充当动态微环境。蒸发引起的离子浓度增加反映在液滴渗透压的成比例增加上，这反过来又刺激细胞的渗透调节反应。通过监测蒸发液滴随时间变化的电导率，与基于生长的方法需要数小时相比，几分钟内就能识别出细菌细胞。为了展示所提出方法的通用性，我们展示了对野生型和基因改造的非耐盐细胞（鼠伤寒沙门氏菌）的检测，以及对非耐盐细胞（如大肠杆菌DH5α）和耐盐细胞（如表皮葡萄球菌）的死活区分。与基于生长的技术不同，所提出方法的测定时间与细胞浓度或细菌类型无关。所提出的无需标记的方法为实现一类新型的实时、阵列式电传感器铺平了道路，这类传感器与用于芯片实验室应用的液滴微流控技术兼容。
