Pandian K, Ajanth Praveen M, Hoque S Z, Sudeepthi A, Sen A K
Micro Nano Bio-Fluidics Unit, Fluid Systems Laboratory, Department of Mechanical Engineering, Indian Institute of Technology Madras, Chennai 600036, India.
Biomicrofluidics. 2020 Nov 2;14(6):064101. doi: 10.1063/5.0026046. eCollection 2020 Nov.
Cell lysis is a critical step in genomics for the extraction of cellular components of downstream assays. Electrical lysis (EL) offers key advantages in terms of speed and non-interference. Here, we report a simple, chemical-free, and automated technique based on a microfluidic device with passivated interdigitated electrodes with DC fields for continuous EL of cancer cells. We show that the critical problems in EL, bubble formation and electrode erosion that occur at high electric fields, can be circumvented by passivating the electrodes with a thin layer (∼18 m) of polydimethylsiloxane. We present a numerical model for the prediction of the transmembrane potential (TMP) at different coating thicknesses and voltages to verify the critical TMP criterion for EL. Our simulations showed that the passivation layer results in a uniform electric field in the electrode region and offers a TMP in the range of 5-7 V at an applied voltage of 800 V, which is well above the critical TMP (∼1 V) required for EL. Experiments revealed that lysis efficiency increases with an increase in the electric field (E) and residence time (t): a minimum E ∼ 10 V/m and t∼ 1.0 s are required for efficient lysis. EL of cancer cells is demonstrated and characterized using immunochemical staining and compared with chemical lysis. The lysis efficiency is found to be ∼98% at E = 4 × 10 V/m and t= 0.72 s. The efficient recovery of genomic DNA via EL is demonstrated using agarose gel electrophoresis, proving the suitability of our method for integration with downstream on-chip assays.
细胞裂解是基因组学中用于提取下游分析所需细胞成分的关键步骤。电裂解(EL)在速度和无干扰方面具有关键优势。在此,我们报告了一种基于微流控装置的简单、无化学试剂且自动化的技术,该装置带有经钝化处理的叉指电极,利用直流电场对癌细胞进行连续电裂解。我们表明,通过用约18微米厚的聚二甲基硅氧烷薄层对电极进行钝化处理,可以规避在高电场下电裂解中出现的关键问题,即气泡形成和电极腐蚀。我们提出了一个数值模型,用于预测不同涂层厚度和电压下的跨膜电位(TMP),以验证电裂解的关键TMP标准。我们的模拟表明,钝化层在电极区域产生均匀电场,在施加800V电压时提供5 - 7V的跨膜电位,这远高于电裂解所需的临界TMP(约1V)。实验表明,裂解效率随着电场(E)和停留时间(t)的增加而提高:高效裂解所需的最小电场E约为10V/m,停留时间t约为1.0s。利用免疫化学染色对癌细胞的电裂解进行了演示和表征,并与化学裂解进行了比较。发现在电场E = 4×10V/m和停留时间t = 0.72s时,裂解效率约为98%。通过琼脂糖凝胶电泳证明了通过电裂解高效回收基因组DNA,证明了我们的方法适用于与下游芯片上分析集成。