CREATED USING THE RSC ARTICLE TEMPLATE (VER. 2.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 1 Integrated Microfluidic Cell Culture and Lysis on a Chip† J. Tanner Nevill, Ryan Cooper, Megan Dueck, David N. Breslauer, and Luke P. Lee ‡ * Receipt/Acceptance Data [DO NOT ALTER/DELETE THIS TEXT] Publication data [DO NOT ALTER/DELETE THIS TEXT] DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT] 5 We present an integrated microfluidic cell culture and lysis platform for automated cell analysis that improves on systems which require multiple reagents and manual procedures. Through the combination of previous technologies developed in our lab (namely, on-chip cell culture and electrochemical cell lysis) we have designed, fabricated, and characterized an integrated microfluidic platform capable of culturing HeLa, MCF-7, Jurkat, and CHO-K1 cells for up to five 10 days and subsequently lysing the cells without the need to add lysing reagents. On-demand lysis was accomplished by local hydroxide ion generation within microfluidic chambers, releasing both proteinacious (GFP) and genetic (Hoescht-stained DNA) material. Sample proteins exposed to the electrochemical lysis conditions were immunodetectable (p53) and are able to retain their enzymatic activity (HRP). 15 Introduction Scientific progress is often associated with the invention of a new experimental apparatus. New tools can increase the ease and efficiency of routine experiments as well as provide the means to make new discoveries by making possible novel 20 experiments. The development of Lab on Chip (LOC) devices is playing an important role in the progression of many different areas of research ranging from point of care diagnostics to the search for life on Mars [1, 2]. LOC devices hold promise to replace existing techniques with processes 25 that are not only more automated and consistent but also require less time and valuable reagents [3]. Cultured cells are used in a variety of contexts ranging from drug development to synthetic biology. Microfluidic devices for cell culture studies offer numerous advantages 30 over plate based cell culture and, because of this, are being increasingly used in laboratory settings [4, 5]. Microfluidic devices better mimic in vivo conditions by allowing for constant perfusion and 3D tissue-like structure [6-9]. Additionally, time and costs are reduced due to decreased 35 reagent volumes and automated handling. Higher surface to volume ratios can also offer improved and novel detection schemes [10]. Existing microfluidic systems for cell-based lysate studies require the addition of lysis buffers and subsequent washing 40 steps, increasing the complexity of such devices and reducing their ease of use [11-13]. We have developed an integrated microfluidic cell analysis system that allows for continuous perfusion cell culture with on-demand cell lysis. Lysis is achieved by applying a DC voltage to electrochemically 45 generate hydroxide inside the device. This lysis method differs from other electrical lysis techniques [14, 15]. Rather than relying on high electric fields to electroporate the cells, electrochemically generated hydroxide ions permantly disrupt the cellular membrane by cleaving fatty acid groups, thereby 50 releasing intracellular material. By combining two BASICs (Biological Application Specific Integrated Circuits) previously developed in our lab, we introduce an integrated cell analysis package that minimizes the need for external reagents and manual 55 procedures [7, 9, 16]. Here we demonstrate the practical use of this device by examining the culture and lysis of 4 different cell lines (HeLa, Jurkat, CHO-K1, and MCF-7). Additionally, we investigate the effects of this lysis technique on two biological molecules, Horseradish Peroxidase (HRP) and p53. 60 HRP is an enzyme (derived from the plant of the same name) that is oft-used in molecular biology. P53, the 1993 ‘molecule of the year’, is a transcription factor that plays a central role in many cancer mechanisms [17]. We show that the immunodetection of p53 is not compromised by the lysis 65 procedure within the device. The enzymatic activity of HRP is diminished as applied DC voltage increases. However, we also show that it is possible to lyse cells using a voltage with minimal effect on HRP enzymatic activity. Given the many applications that require a combination of cell culture and 70 lysis, we believe this integration of microfluidic devices is a valuable advancement in the field of biological research and diagnostics. Materials and Methods Chip Design 75 The cell chambers were designed to have four fluid-permeable cell holding structures with 11nl of volume each (Fig 1): this equates to roughly 12,500 cells per trap and 50,000 cells per chamber. Each device has six chambers (Fig. 1a), and each chamber is individually addressable via polymer tubing 80 connections. Independent lysing electrodes are placed on either side of the trapping region. * Biomolecular Nanotechnology Center, Berkeley Sensor & Actuator Center, Department of Bioengineering, University of California, Berkeley UCSF/UCB Joint Graduate Group in Bioengineering E-mail: lplee@berkeley.com † Electronic Supplementary Information (ESI) available: Additional images and movies. See http://dx.doi.org/10.1039/b000000x/