A quartz crystal microbalance cell biosensor: detection of microtubule alterations in living cells at nM nocodazole concentrations
A quartz crystal microbalance cell biosensor: detection of microtubule alterations in living cells at nM nocodazole concentrations
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DOI：
10.1016/s0956-5663(01)00219-6
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发表时间：
2001-12-01
影响因子：
12.6
通讯作者：
Braunhut, SJ
中科院分区：
文献类型：
--
作者：
Marx, KA;Zhou, TA;Braunhut, SJ
采用石英晶体微天平（QCM），以活内皮细胞（EC）为生物信号转导元件，构建了压电生物传感器。在含有血清的生长培养基下，EC粘附于经化学处理的金QCM表面。在加入细胞后24小时，构建校准曲线，将观察到的稳态Δ f和Δ R位移值与需要胰蛋白酶消化的电子计数细胞的数量从表面去除相关。然后，我们利用该EC QCM生物传感器检测[诺考达唑]对稳态Δ f和Δ R位移值的影响。诺考达唑，一种已知的微管结合药物，改变活细胞的细胞骨架特性。在这些研究中使用的剂量（0.11-15 μ M）下，诺考达唑以剂量依赖性方式引起活细胞中微管的解聚。这导致单层良好分布的EC逐渐占据更小的面积，失去细胞与细胞的接触，在细胞周边显示肌动蛋白应力纤维并获得圆形细胞形状。我们观察到在加入诺考达唑后的4小时孵育期内，负Δ f位移值和正Δ R位移值的幅度以剂量依赖性方式显著增加，过渡中点为900 nM。固定在金QCM表面上并对肌动蛋白染色的EC的荧光显微镜检查表明，EC的形状和细胞骨架受到低至330 nM的诺考达唑的影响。这些结果表明，EC QCM生物传感器可用于研究EC附着和检测EC细胞骨架的变化。我们建议的潜力，这种细胞生物传感器的真实的时间识别或筛选的所有类别的生物活性药物或生物大分子，影响细胞附着，无论其分子作用机制。(C)2001 Elsevier Science B. V.保留所有权利。
The quartz crystal microbalance (QCM) was used to create a piezoelectric biosensor utilizing living endothelial cells (ECs) as the biological signal transduction element. ECs adhere to the hydrophilically treated gold QCM surface under growth media containing serum. At 24 h following cell addition, calibration curves were constructed relating the steady state Deltaf and DeltaR shift values observed to the numbers of electronically counted cells requiring trypsinization to be removed from the surface. We then utilized this EC QCM biosensor for the detection of the effect of [nocodazole] on the steady state Deltaf and DeltaR shift values. Nocodazole, a known microtubule binding drug, alters the cytoskeletal properties of living cells. At the doses used in these studies (0.11-15 muM), nocodazole, in a dose dependent fashion, causes the depolymerization of microtubules in living cells. This leads a monolayer of well spread ECs to gradually occupy a smaller area, lose cell to cell contact, exhibit actin stress fibers at the cell periphery and acquire a rounded cell shape. We observed the negative Deltaf shift values and the positive DeltaR shift values to increase significantly in magnitude over a 4-h incubation period following nocodazole addition, in a dose dependent fashion, with a transition midpoint of 900 nM. Fluorescence microscopy of the ECs, fixed on the gold QCM surface and stained for actin, demonstrated that the shape and cytoskeleton of ECs were affected by as little as 330 nM nocodazole. These results indicate that the EC QCM biosensor can be used for the study of EC attachment and to detect EC cytoskeletal alterations. We suggest the potential of this cellular biosensor for the real time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment, regardless of their molecular mechanism of action. (C) 2001 Elsevier Science B.V. All rights reserved.